Environmental Biotechnology

Basic Concepts and Applications

Environmental Biotechnology
Basic Concepts and Applications

Viswanath Buddolla

α
Alpha Science International Ltd.
Oxford, U.K.
Environmental Biotechnology:
Basic Concepts and Applications
544 pgs.  |  50 figs.

Viswanath Buddolla
Department of Bionanotechnology
Gachon University
Republic of Korea

Copyright © 2017
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 Preface

Biotechnology is the integration of natural sciences and engineering in order

to achieve the application of organisms, cells, parts thereof and molecular
analogues for products and services. It is versatile and has been assessed
a key area which has greatly impacted various technologies based on the
application of biological processes in manufacturing, agriculture, food
processing, medicine, environmental protection, resource conservation. This
new wave of technological changes has determined dramatic improvements
in various sectors since it can provide entirely novel opportunities for
sustainable production of existing and new products and services. In
addition, environmental concerns help drive the use of biotechnology not
only for pollution control, but prevent pollution and minimize waste in the
first place, as well as for environmentally friendly production of chemical
and biomonitoring. This book describes the state-of-the-art and possibilities
of environmental biotechnology and explain its various areas together with
their related issues and implications. Considering the number of problems
that define and concretize the field of environmental biotechnology, the role of
some bioprocesses and biosystems for environmental protection, control and
health, based on the utilization of living organisms, are analyzed.
Environmental remediation, pollution prevention, detection and
monitoring are evaluated considering the achievements, as well as the
perspectives in the development of biotechnology. Various relevant topics have
been chosen to illustrate each of the main areas of environmental biotechnology:
wastewater treatment, soil treatment, solid waste treatment, and waste gas
treatment, dealing with both the microbiological and process engineering
aspects. The distinct role of environmental biotechnology in the future is
emphasized considering the opportunities to contribute with new solutions
and directions in remediation of contaminated environments, minimizing
vi Preface

future waste release and creating pollution prevention alternatives. To take

advantage of these opportunities, innovative new strategies, which advance
the use of molecular biological methods and genetic engineering technology,
are examined. These methods would improve the understanding of existing
biological processes in order to increase their efficiency, productivity, and
flexibility. Examples of the development and implementation of such strategies,
are included. In addition, the contribution of environmental biotechnology to
the progress of a more sustainable society is revealed in this book.

Viswanath Buddolla
 Contents

Preface........................................................................................................................... v

1. Introduction to Environmental Biotechnology............................1.1—1.37

1.1 Biotechnology for Environment...................................................... 1.3
1.2 Biotechnology, Agriculture and Environmental Pollution.......... 1.5
1.3 Challenges Imposed on the Environment by Human
Activities............................................................................................. 1.6
1.4 Contributions of Biotechnology to Improve Agricultural
Productivity........................................................................................1.7
1.4.1 Biotechnology in reducing the use of chemical
pesticides, herbicides and fertilizers.................................1.9
1.4.2 Biofertilizers.......................................................................... 1.9
1.5 Biotechnology and Livestock Production in the
Improvement of Environmental Conditions...............................1.11
1.6 Biotechnology and the Removal of Toxic Chemicals
from the Environment..................................................................... 1.11
1.7 Biotechnology and the Removal of Heavy Metal
Pollution from the Environment....................................................1.12
1.8 Biotechnology and Desulphurization of Fossil Fuels................. 1.13
1.9 Biotechnology and Ecosystem Modeling..................................... 1.13
1.10 Microbial Biotechnology in the Monitoring of
Environmental Pollution.................................................................1.13
1.11 Microbial Biotechnology in the Bioassay of Environ-
mental Toxicity.................................................................................1.14
1.12 Biotechnology and Control of Oil Spillage..................................1.14
viii Contents

1.13 Bioremediation and their Importance in Environment

Protection..........................................................................................1.15
1.13.1 Factors affecting biodegradation.....................................1.15
1.13.2 In situ bioremediation....................................................... 1.16
1.13.2.1 Intrinsic bioremediation.................................... 1.16
1.13.2.2 Engineered in situ bioremediation................... 1.16
1.13.3 Ex-situ bioremediation...................................................... 1.17
1.13.3.1 Solid phase treatment.........................................1.17
1.13.3.2 Slurry phase treatment ......................................1.17
1.13.4 Use of genetic engineering and genetic mani-
pulations for more efficient bioremediation.................. 1.18
1.13.5 Biotechnology to reduce atmospheric Carbon
dioxide (CO2) ..................................................................... 1.19
1.13.6 Treatment of sewage using microorganisms.................1.20
1.13.7 Treatment of industrial effluents using
biotechnology.....................................................................1.21
1.13.8 Use of biotechnology for toxic site reclamation............ 1.22
1.13.9 Use of biotechnology in the removal of oil and
grease deposits................................................................... 1.23
1.13.10 Tannery effluents and their treatment............................ 1.24
1.13.11 Bioremediation of Radioactive Contaminants...............1.25
1.14 Use of Biosensors to Detect Environmental Pollutants..............1.26
1.14.1 Principle of a biosensor..................................................... 1.27
1.14.2 Use of selected and engineered microbes for
removal and recovery of strategic and precious
metals from contaminated degraded lands................... 1.28
1.15 Biotechnology and the Saving of Resources and Energy...........1.29
1.16 Fears and Concerns About Biotechnology Approach in
Achieving a Safe Environment and Agriculture......................... 1.30
1.17 Fears and Concerns about Environmental Impact of
Bioremediation................................................................................. 1.31
1.18 Role of Biotechnology in Restoration of Degraded Lands.........1.31
1.18.1 Use of micropropagation and Mycorrhiza for
reforestation........................................................................1.31
1.18.2 Improvement of soil infertility through the
use of nitrogen fixing bacteria, Rhizobium in
association with leguminous trees and Frankia
in association with non-leguminous species.................1.32
Contents ix

1.18.3 Development of plants tolerant to abiotic stress

which can be grown on degraded lands........................1.32
1.19 Biomimicry and Biotechnology.....................................................1.33
1.20 Biotechnology in the Conservation of Biodiversity.................... 1.34
1.21 Basic Tools and Methodologies Associated with
Environmental Biotechnology....................................................... 1.35
1.22 Developing Environmentally Sound Biotechnologies
in India ..............................................................................................1.36

2. Environmental Microbiology—Soil.............................................. 2.1—2.71

2.1 Soil and Soil Microorganisms.......................................................... 2.2
2.1.1 The characteristics of soil microorganisms......................2.2
2.1.1.1 Viruses....................................................................2.3
2.1.1.2 Bacteria................................................................... 2.3
2.1.1.3 Actinomycetes....................................................... 2.5
2.1.1.4 Fungi.......................................................................2.5
2.1.1.5 Soil phytoedaphon...............................................2.6
2.1.1.6 Fauna of soil.......................................................... 2.7
2.1.2 The Number of Soil Microorganisms................................ 2.9
2.1.2.1 Viruses....................................................................2.9
2.1.2.2 Bacteria................................................................... 2.9
2.1.2.3 Fungi.......................................................................2.9
2.1.2.4 Algae.....................................................................2.10
2.1.2.5 Soil fauna............................................................. 2.10
2.1.3 Activity of Microorganisms in soil..................................2.10
2.1.3.1 Cellulose decomposition...................................2.11
2.1.3.2 Lignin decomposition........................................2.11
2.1.3.3 Synthesis and humus decomposition.............. 2.12
2.1.3.3.1 The synthesis of humus
(Humification)..................................... 2.12
2.1.3.3.2 Decomposition of humus..................2.13
2.1.3.4 Atmospheric nitrogen fixation..........................2.14
2.1.3.5 Free-living N2 assimilators (non-
symbiotic nitrogen fixation).............................. 2.15
2.1.3.6 Ammonification..................................................2.15
2.1.3.7 Nitrification.........................................................2.15
2.1.3.8 Denitrification ....................................................2.16
2.1.4 Interactions among Soil Microorganisms....................... 2.16
x Contents

2.1.4.1 Beneficial Associations/Interactions................. 2.17

2.1.4.1.1 Mutualism (Symbiosis)......................2.17
2.1.4.1.2 Commensalisms................................. 2.17
2.1.4.1.3 Proto-cooperation............................... 2.17
2.1.4.2 Detrimental (Harmful) Associations/
Interactions............................................................. 18
2.1.4.2.1 Antagonism......................................... 2.18
2.1.4.2.2 Ammensalism..................................... 2.18
2.1.4.2.3 Ammensalism Competition..............2.18
2.1.4.2.4 Parasitism............................................ 2.19
2.1.4.2.5 Predation............................................. 2.19
2.1.5 Mutual interaction of plants and microorganisms.......2.19
2.1.5.1 The symbiosis of microbes and plants –
bacterrhiza...........................................................2.19
2.1.5.1.1 Rhizosphere......................................... 2.20
2.1.5.2 The symbiosis of fungi and plants................... 2.21
2.1.5.2.1 Ectotrophic mycorrhiza..................... 2.21
2.1.5.2.2 Endotrophic mycorrhiza...................2.21
2.1.6 Soil Bioremediation...........................................................2.22
2.1.6.1 Microorganisms used in remediation
technologies......................................................... 2.22
2.1.6.2 Stimulation of bioremediation.......................... 2.24
2.1.6.3 Classification of bioremediation methods......2.24
2.1.6.3.1 In situ methods................................... 2.25
2.1.6.3.2 Ex situ methods.................................. 2.28
2.1.7 Role of Soil Microorganisms in Biodegradation
of Pesticides and Herbicides............................................2.30
2.1.7.1 Effects of pesticides............................................2.30
2.1.7.2 Persistence of pesticides in soil.........................2.31
2.1.7.3 Biodegradation of Pesticides in Soil.................2.31
2.1.7.4 Criteria for Bioremediation/Biodegradation..2.32
2.1.7.5 Strategies for Bioremediation...........................2.33
2.1.8 Microbial Ecology of Petroleum Contaminant
Plumes ................................................................................ 2.33
2.1.9 Molecular Microbial Ecology...........................................2.34
2.1.10 Soil Microorganisms as Biofertilizers.............................2.36
2.1.10.1 Role of Biofertilizers in soil fertility and
Agriculture..........................................................2.36
Contents xi

2.1.10.2 Quality Control Measures (as per ISI

Specifications)...................................................... 2.38
2.1.11 Factors Affecting Distribution, Activity and
Population of Soil Microorganisms.................................2.38
2.1.12 Rhizosphere Concept and It’s Historical
Background......................................................................... 2.42
2.1.12.1 Microorganisms in the Rhizosphere and
Rhizosphere Effect.............................................. 2.43
2.1.12.2 Factors affecting microbial flora of the
Rhizosphere/Rhizosphere Effect...................... 2.45
2.1.12.3 Alterations in Rhizosphere Microflora............2.47
2.1.12.4 Associative and Antagonistic activities in
the Rhizosphere..................................................2.48
2.1.12.5 Rhizosphere in relation to Plant Pathogens.... 2.49
2.1.13 Soil Microorganisms in Cycling of Elements or
Plant Nutrient..................................................................... 2.51
2.1.13.1 Nitrogen Cycle....................................................2.51
2.1.13.2 Sulphur Cycle/Sulphur Transformations........2.55
2.1.13.3 Phosphorus Cycle or Transformation..............2.57
2.1.13.4 Iron Cycle or Transformation............................ 2.58
2.1.14 Organic Matter................................................................... 2.59
2.1.14.1 Microbiology of decomposition of various
constituents in organic matter..........................2.62
2.1.15 Notable contributions made by several scientists
in the field of soil microbiology ...................................... 2.64
2.1.16 Notable contributions made by Indian scientists
in the field of soil microbiology....................................... 2.69

3. Environmental Microbiology—Water and Air............................3.1—3.42

3.1 Water and Water Microorganisms .................................................. 3.1
3.1.1 Types of microorganisms in water.................................... 3.2
3.1.1.1 Groups of water organisms................................. 3.2
3.1.2 Factors limiting growth of microorganisms in water..... 3.4
3.1.3 Characterization of water microorganisms...................... 3.5
3.1.4 Sources and types of pollutants....................................... 3.11
3.1.5 Self-purification of surfacewaters.................................... 3.11
3.1.6 Water-transmitted pathogenic microorganisms............3.14
3.1.7 Sanitary quality of water................................................... 3.17
3.1.8 Wastewater treatment....................................................... 3.21
xii Contents

3.1.9 Biological methods of wastewater treatment ................3.25

3.1.10 Artificial methods of wastewater treatment..................3.28
3.1.11 Methods of chemical wastewater treatment.................. 3.31
3.1.12 Nucleic Acid - Based Techniques for Analyzing
the Diversity, Structure, and Dynamics
of Microbial Communities in Wastewater
Treatment............................................................................3.33
3.2 Air As An Environment of Microorganisms................................3.39
3.2.1 Adaptation of microorganisms to the air
environment.......................................................................3.39

4. Environmental Microbiology—Methods and Applications.....4.1—4.56

4.1 Microorganisms-Metal Transformations........................................ 4.1
4.2 Microorganisms in Environmental Monitoring............................4.6
4.3 Applications of the Polymerase Chain Reaction in
Environmental Microbiology......................................................... 4.15
4.4 Pesticides and Other Pollutants Degradation by
Microorganisms and Genetically Engineered Microbes............4.28
4.5 Degradation of Oil by Microorganisms for the
Production of Useful Products.......................................................4.34
4.6 Degradation of Plastics by Microorganisms for
Production of Useful Products.......................................................4.37
4.7 Recovery of Minerals By Microbes................................................4.38
4.8 Bioindicators of Hazardous Pollutants.........................................4.46

5. Beneficial and Effective Microorganisms for a Sustainable

Agriculture and Environment......................................................... 5.1—5.18
5.1 The Concept of Effective Microorganisms: Their Role
and Application..................................................................................5.3
5.2 Utilization of Beneficial Microorganisms in Agriculture............. 5.3
5.3 Beneficial and Effective Microorganisms for a
Sustainable Agriculture..................................................................... 5.5
5.4 Beneficial Microorganisms for Soil Quality and a more
Sustainable Agriculture..................................................................... 5.6
5.5 Controlling The Soil Microflora: Principles and Strategies.........5.7
5.6 Controlling The Soil Microflora for Optimum Crop
Production and Protection................................................................ 5.9
5.7 Application of Beneficial And Effective Microorganisms..........5.11
5.8 Classification of Soils Based on Their Microbiological
Properties..........................................................................................5.12
Contents xiii

5.8.1 Functions of Microorganisms: Putrefaction,

Fermentation, and Synthesis............................................5.13
5.8.2 Relationships Between Putrefaction, Fermentation,
and Synthesis...................................................................... 5.14
5.9 Classification of Soils Based on the Functions of
Microorganisms...............................................................................5.15

6. Phytoremediation.............................................................................. 6.1—6.13
6.1 Principal Mechanism of Phytoremediation...................................6.2
6.2 Phytoremediation Processes ...........................................................6.2
6.2.1  Rhizofiltration.......................................................................6.3
6.2.2  Phytostabilization................................................................ 6.3
6.2.3  Phytoextraction.................................................................... 6.3
6.2.4  Phytovolatilisation...............................................................6.4
6.2.5  Phytotransformation or Phytostimulation or
Rhizodegradation ............................................................... 6.4
6.3 Phytoremediaiton of Organic Pollutants........................................ 6.5
6.4 Plants’ Response to Heavy Metals...................................................6.7
6.4.1  Metal excluders.................................................................... 6.8
6.4.2  Metal indicators...................................................................6.8
6.4.3  Metal accumulator plant species....................................... 6.8
6.5 Hydraulic Control of Pollutants .....................................................6.9
6.5.1  Riparian corridors................................................................ 6.9
6.5.2  Vegetative cover...................................................................6.9
6.6 Advantages and Disadvantages of Phytoremediation............... 6.10
6.7 Phytoremediation & Biotechnology.............................................. 6.10
6.7.1 Risk Assessment................................................................. 6.11
6.7.2 Future of Phytoremediation.............................................6.12

7. Solid Waste Disposal and Management.......................................7.1—7.31

7.1 Types of Solid Waste..........................................................................7.2
7.1.1 Municipal solid waste......................................................... 7.2
7.1.2 Industrial solid waste ......................................................... 7.3
7.1.3 Biomedical Waste .................................................................7.4
7.2 Solid Waste Disposal and Management: .......................................7.5
7.4 Biological Reprocessing of Solid Waste Disposal ....................... 7.10
7.4.1 Composting........................................................................ 7.11
7.5 Biotechnological Methods of Solid Waste (Agriculture,
Domestic and Industrial) Degradation.........................................7.13
xiv Contents

7.6 Importance, Health Impacts and Awareness of

Waste Management.........................................................................7.14
7.7 Health Impacts of Solid Waste....................................................... 7.15
7.8 Key Concepts in Municipal Waste Reduction............................. 7.17
7.9 Wastewater........................................................................................ 7.18
7.9.1 Wastewater treatment and Management....................... 7.19
7.9.2 Water Purification by Waterweeds and
Membrane Filters...............................................................7.25
7.9.3 Indicator Organisms.......................................................... 7.28
7.9.4 Reclaim of Treated Wastewater........................................7.29
7.9.5 Uses and benefits of reclaimed water.............................7.30

8. Biological Methods of Pest Management.....................................8.1—8.27

8.1 Advantages of Microbial Insecticides.............................................8.9
8.2 Disadvantages of Microbial Insecticides...................................... 8.10
8.3 Insect Growth Regulators...............................................................8.10
8.4 Use of Pheromones for Pest Management...................................8.14
8.5 Pheromones: Insect Pest Management......................................... 8.15
8.6  Biological Control of Weeds...........................................................8.16
8.6.1 The process...........................................................................8.17
8.7 Chromosomal Manipulation .........................................................8.19
8.8 Sterile Male Technology.................................................................. 8.20
8.9 Environmental Epidemiological Surveys as Indices of
Health Hazards for Environmental Pollution.............................8.21
8.10 Toxicity...............................................................................................8.24
8.10.1 Types of toxicity................................................................. 8.25
8.10.2 Factors influencing toxicity..............................................8.25
8.11 Measuring Toxicity...........................................................................8.25
8.11.1 Toxic..................................................................................... 8.27
8.11.2 Highly toxic........................................................................8.27

9. Algae-Biotechnology........................................................................9.1—9.32
9.1 Algae as A Source of Food and Feed...............................................9.4
9.1.1 Microalgae nutritional composition.................................. 9.5
9.2 Mass Cultivation of Commercially Valuble Marine
Microalgae for Agar Agar, Alginates ............................................. 9.9
9.2.1 Other applications.............................................................9.18
Contents xv

9.3 Commercial Application of Microalgae and their Products......9.20

9.3.1 Current commercial uses of algae...................................9.21
9.3.2 Future Development of Microalgal Applications.........9.26
9.4 Mass Cultivation of Microalgae as a Source of Protein
and Feed............................................................................................ 9.28
9.5 Microalgae as a Source of Feed......................................................9.29

10. Concepts and Scope of Plant Biotechnology...........................10.1—10.23

10.1 Applications of Genetic Engineering Technology for
Crop Improvement.......................................................................... 10.2
10.2 Production of Transgenic Plants with Improved Yields
and Nutritional Quality.................................................................. 10.5
10.3 Transgenic Plants for The Production of Viral Antigens.......... 10.10
10.3.1 Edible Vaccines.................................................................10.13
10.4 Biotechnology in Agriculture—Merits and Demerits .............10.15
10.4.1 Transgenic Crops in the U.S. Market............................ 10.17
10.4.2 Possible Risks Associated with using Transgenic
Crops in Agriculture .......................................................10.19
10.4.3 Health-related Issues ...................................................... 10.19
10.4.4 Environmental and Ecological Issues .......................... 10.20
10.4.5 Social Issues .....................................................................10.21

11. Animal Biotechnology..................................................................11.1—11.23

11.1 History of Animal Cell Culture .....................................................11.1
11.2 Animal Cell Culture ........................................................................ 11.2
11.3 Requirements for Animal Cell Culture ........................................11.4
11.3.1 Synthetic Media.................................................................. 11.5
11.4 Cell-Based Therapy.......................................................................... 11.6
11.5 Applications of Animal Cell Culture.............................................11.6
11.5.1 Somatic Cell Fusion........................................................... 11.6
11.5.2 Blood Factor VIII ...............................................................11.7
11.5.3 Erythropoietin (EPO) ........................................................11.8
11.5.4 The production of Monoclonal Antibodies using
Hybridoma Technology....................................................11.8
11.6 Scale-Up of Animal Cell Culture .................................................. 11.9
11.6.1 Roller Bottles ....................................................................11.10
11.6.2 Micro Carrier Beads.........................................................11.11
11.6.3 Spinner cultures...............................................................11.11
xvi Contents

11.7 Types of Cell Cultures....................................................................11.11

11.8 Characterization of Cell Lines...................................................... 11.13
11.9 Stem Cell Technology....................................................................11.14
11.9.1 Genetic Engineering of Animal Cells and
their Applications ...........................................................11.16
11.9.2 Manipulation of Gene Expression in Eukaryotes ...... 11.16
11.9.3 Collection and purification process of
Recombinant proteins ....................................................11.17
11.9.4 Organ culture and Histotypic cultures ........................11.17
11.9.5 Organ culture ..................................................................11.18
11.9.6 Techniques and Procedure for organ culture ............. 11.18
11.9.7 The advantages of organ culture ..................................11.19
11.9.8 Limitations of organ culture .......................................... 11.19
11.9.9 Histotypic cultures .........................................................11.19
11.9.10 Organotypic cultures ......................................................11.20
11.10 Cell and Tissue Engineering........................................................11.20
11.10.1 Design and Engineering of Tissues ..............................11.20

12. Biotechnology of Aquaculture....................................................12.1—12.27

12.1 Production of Transgenic Fish....................................................... 12.6
12.2 Pearl Oyster Culture........................................................................ 12.9
12.2.1 Early Development and Larval Rearing....................... 12.11
12.3 Shrimp Farming.............................................................................12.13
12.3.1 Shrimp Nutritional Value Details.................................. 12.17
12.4 Growth and Reproduction of Edible Crustaceans.................... 12.18
12.5 Neuroendocrine Principles Involved in the Regulation of
Growth, Reproduction and Metabolism of Prawns and
Crabs (Edible Crustaceans).......................................................... 12.22
12.5.1 Inhibitory Principles........................................................12.23
12.5.2 Stimulatory principles.....................................................12.25
12.5.3 Inhibitory principles........................................................12.25

13. Nutrient Film Culture Techniques............................................. 13.1—13.18

13.1 Plant Diseases................................................................................... 13.2
13.2 Phytodiagnostics Based on Immunological and
Molecular Techniques.....................................................................13.4
13.3 Molecular or Nucleic acid-based techniques............................... 13.6
13.3.1 Polymerase Chain Reaction (PCR)................................... 13.7
Contents xvii

13.3.2 Real-time PCR..................................................................... 13.8

13.3.3 Rolling Circle Amplification (RCA)............................... 13.11
13.4 Antagonistic Fungi........................................................................13.14
13.5 Antagonistic Bacteria..................................................................... 13.16
13.6 Antifeedants................................................................................... 13.16
13.7 Insecticidal Activities of The Compounds of Botanics
(Botanical Insecticdes)...................................................................13.17
13.8 Predators......................................................................................... 13.18

14. Biotechnology: Industrial Sustainability.................................14.1—14.15

14.1 Industrial Sustainability..................................................................14.1
14.2 Technology, Cleaner Production and Sustainability...................14.3
14.3 Learning from Nature: Biomimicry and Biotechnology............14.5
14.4 Bio-Safety..........................................................................................14.6

15. Ethical Issues in Environmental Biotechnology.....................15.1—15.12

15.1 Release of Genetically Modified Organisms (GMOS).................15.4
15.1.1 Effects on the Environment..............................................15.5
15.1.2 Effects on Human Health................................................. 15.5
15.1.3 Environmental Biotechnology—Biosafety
Management.......................................................................15.9
15.1.4 Environmental Biotechnology & Intellectual
Property Rights (IPRs).....................................................15.11

Appendix Useful Terms and their Meanings of Environmental

Biotechnology........................................................................... A.1—A.68

Index .................................................................................................................. I.1—I.3

 CHAPTER

1 Introduction to
Environmental Biotechnology

The word “environment” is derived from the French word “environ”. The
meaning of the French word is somewhat related to “encompass”, “encircle”,
etc. It is believed to have been introduced into the subject by biologist Jacob
Van Erkulin in the early 1900s and the term “environment” is defined as
our surroundings, which includes the abiotic component (the non-living)
and the biotic component (the living) around us. The abiotic environment
includes water, air and soil while the biotic environment consists of all living
organisms—plants, animals and micro-organisms. The environment is a very
important component necessary for the existence of both human and other
biotic organisms. Contrary to its name, biotechnology is not a single technology.
Rather, it is a group of technologies that share two (common) characteristics
working with living cells and their molecules and having a wide range of
practical uses that can improve our lives. Biotechnology can be broadly defined
as “using organisms or their products for commercial purposes.” Production
may be carried out by using intact organisms of bacteria, fungi and other
microbes, or by using natural substances created by the organisms, such as
enzymes. Environmental biotechnology is “the integration of natural sciences
and engineering in order to achieve the application of organisms, cells, parts
thereof and molecular analogues for the protection and restoration of the
quality of our environment”. The International Society for Environmental
Biotechnology defines Environmental Biotechnology as the development,
use and regulation of biological systems for remediation of contaminated
environments (land, air and water) and for environment-friendly processes
(green manufacturing technologies and sustainable development). It can also
be described as ‘‘the optimal use of nature, in the form of plants, animals,
bacteria, fungi and algae, to produce renewable energy, food and nutrients in
a synergistic integrated cycle of profit-making processes where the waste of
each process becomes the feedstock for another process’’.
The prime target of this science is the abatement of pollution through
bioremedation/biotreatment or supporting as resources for human use
1.2 Environmental Biotechnology

in non-polluting ways. It can also help in cleaner production of existing

products. On the whole, it encompasses aspects of natural resources
management, the treatment of waste and control of pollution. Thus, the major
areas of understanding are environmental pollution abatement through
biodegradation, biotransformation, bioaccumulation of toxicity like organics,
metals, oil, and hydrocarbons, dyes, detergents, etc.; Energy management
through production of non-conventional non-polluting energy like biodiesel
methanol, biogas, biohydrogen etc.; Agricultural application of biofertilizer,
biopesticides, or bioorganics of multiple users. Recovery of resources from
toxic or non-toxic wastes through biotechnological approach; Biosensor
approach of pollution monitoring and several other allied issues.
Environmental Biotechnologies are competing with great success against
traditional technologies recent days and are providing solutions to acute
problems through the so called ‘end of pipe’ treatment technologies and
bioremediation. Environmental biotechnology can also provide a natural way
of addressing mainly environmental problems ranging from the identification
of biohazards to bioremediation techniques for industrial, agricultural and
domestic ends like municipal effluents and residues. Thus, environmental use
of biotechnology includes the development, biosecurity use, and regulation of
biological systems for remediation of contaminated environments like land,
water, air as well as for use in environmentally sound processes leading to
clean technologies and sustainable development. Virtually, all types of human
activities generate wastes and this places a heavy burden on the environment.
So far, we have relied more upon the physical and chemical methods of
pollution control. However, micro-organisms are likely to prove as more
suitable tools for pollution control due to their versatility and adaptability.
In the field of environmental management, biotechnology have helped in
environmental monitoring, degradation of wastes, substitution of non-
renewable resource base with a renewable one and production of eco-friendly
products and processes.
The application of biotechnologies is frought with risks that are difficult
to define, much less to assess. The introduction of genetically engineered
microbes can create new ecological niches and bring about large-scale
transformations in the structure and function of the ecosystem as a whole.
There are many unknown risks and hazards associated with the accidental or
deliberate release of self-propagating, genetically engineered novel forms of
life into the biosphere. In the event of disastrous consequences, it is likely that
people will be harmed who had little gain from the use of biotechnology. In
recent years, considerable development in terms of R&D and its application
on “Environmental Biotechnology” is made round the world. The major areas
of application are microbial biodegradation of pollutants, development of
appropriate technology for biofertilizers and biopesticides production and
its use in the field of agriculture and horticulture; production of single cell
protein, etc. Considerable discussion occurred in recognition and support
Introduction to Environmental Biotechnology 1.3

of the tremendous potential that environmental biotechnology offered

particularly in the development of the next generation of pollution prevention,
pollution abatement, and sustainable development of green technologies. The
emergence and acceptance of the concept of sustainable development warrants
that the scope of environmental biotechnology be enlarged to address issues
like environmental monitoring, restoration of environmental quality, resource/
residue/waste-recovery/utilization/treatment, and substitution of the non-
renewable resource base with renewable resources.

1.1 BIOTECHNOLOGY FOR ENVIRONMENT

Industrial growth, economic development, consumerisation indicate a
country’s progress and life standard of its individuals. Industrial growth in the
20th century has brought along with it new problems, too. Water pollution,
air pollution, land pollution, noise pollution, radioactive pollution, solid
wastes, depletion of resources, scarcity of good quality water, proliferating
health hazards, are all results or consequences of stupendous industrial
activities with less attention to their negative impacts on the humans and the
environment. Nature’s built-in mechanisms and self-regulation ability has
been thrown out of gear by the quantity and complexity of wastes generated
by the modern society. In economic terms, we only consider material costs
and energy costs involved and fail to pay attention to the costs involved in
the form of loss of environmental quality. Urbanization, wrong agricultural
practices, etc.; are responsible for pollution. As technological progress has
followed the industrial revolution, solving of environmental problem must
follow technological progress. Industrial processes and products thereof,
both must become environmentally friendly and least damaging. Pollution
may be defined as an undesirable change in physical, chemical or biological
characteristics of air, water and land that can harm human life, the lives of
desirable species, our industrial processes, living conditions and cultural
assets; or waste our raw material resources. Environment protection means
limiting the impairment of environment and it includes conservations of
resources. ‘Environment protection’ has three main objectives:
• To prevent damage and discomfort;
• To improve productivity and pleasure; and
• To maintain balances of the ecosystem.
Environment protection efforts will pay us back in terms of money,
economy, productivity, social justice, cleaner surrounding and sound health.
Environmental problems differ only a little with respect to a country; so
the problems faced elsewhere and controls applied are equally applicable.
Awareness, participation and action in a concerted manner by all who are
connected will be needed to solve the pollution problems. ‘Environmental
Biotechnology’ involves specific applications of biotechnology to the
management of environment and related socio-economic and developmental
1.4 Environmental Biotechnology

issues, keeping in view the concept of sustainable development. ‘Environmental

biotechnology encompasses issues like:
• Environmental monitoring;
• Restoration of environmental quality;
• Resource/residue/waste-recovery /utilization/treatment by application
of r-DNA technology;
• Substitution of non-renewable resources by renewable ones;
• Strain improvement for degradation of highly-toxic pollutants with the
production of chemicals;
• Global changes;
• Biological diversity; and
• Risk management.
Industrial pollution management is thus one amongst the many issues
that environmental biotechnology addresses to. For a long time, the point of
discussion in environmental pollution as an issue has been symptoms rather
than causes of pollution. This naturally influenced our thinking, and emphasis
was given on measurement and removal (treatment technologies). Then slowly
attention was directed towards the Environment Impact Assessment (EIA)
and this could add to the better planning and possibility of better control.
And today—we have started to attack the root cause of pollution. We have
started discussing of what is described as ‘clean technologies’ which believe in
process development and modifications to minimize pollution.
The points useful in effective pollution management are:
(a) In-process treatment.
(b) End-of-pipe treatment.
(c) Remediation of polluted sites.
(d) Modification of existing processes.
(e) Introduction of new processes and products.
Though many technologies have been available for clean-up purpose,
only a few of them have been proved to be of routine application value. In
1989, the US Environmental Protection Agency published a broad assessment
of international technologies (particularly those from Europe, Canada and
Japan) for remediation of superfund sites. The criteria used by them for
assessment apply in general also while evaluating different technologies for
pollution abatement purpose. The criteria are:
(i) Function—object and applicability of technology;
(ii) Description—details of operating principles and design features;
(iii) Performance—demonstration of effectiveness;
(iv) Limitations;
(v) Economics; and
(vi) Status of research, development and availability.
Introduction to Environmental Biotechnology 1.5

In addition to the above mentioned points, the choice of technology is

also influenced by the social, political, and geographical considerations. The
criteria apply to the assessment of technologies in other environmental clean-
up work, too.

1.2 BIOTECHNOLOGY, AGRICULTURE AND ENVIRONMENTAL

POLLUTION
Agriculture is the use of natural resources base for the improvement and
increase in production of crops, livestock, fish and trees. In Agricultural
biotechnology, improvement is accelerated and production is increased
using updated knowledge of living organisms including the genetic code.
These include well-established conventional techniques as in biological pest
control, fermentation, and production of vaccines and biofertilizers as well
as modern techniques like tissue culture, genetic engineering (also called
genetic modification), recombinant DNA technology (rDNA) and crop and
animal transformation as a result of transgenesis. The importance of these new
technologies like biotechnology in food security, environmental sustainability
and economic development was captured at the United Nations General
Assembly in 2005.
Global industrial explosion which is intended to cater to the needs of
the world’s increasing population is always associated with environmental
pollution. Pollution occurs as a result of improper management of industrial
by-products, their accumulation in the environment beyond acceptable limits
and therefore, causes hazard and/or nuisance to man. The industrial by-
products that are pollutants may be either organic or inorganic compounds.
Man’s environment is composed of abiotic and biotic components. Developing
countries are faced with the challenge of rapidly increasing agricultural
productivity to help feed their growing populations without depleting
the natural resource base. In many countries, agriculture is still subsistent
and primitive and this raises concerns on food security, deforestation,
rapid population growth, environmental protection, poor soils, stressed
environments, unfavorable climatic conditions and improved crops and
livestock. For instance, an environment in which pollution of a particular type
is maximum. The effluents of a starch industry mixing up with a local water
body like a lake or pond. These cause huge deposits of starch which are not
easily degraded by micro-organisms except for a few exceptions. Through
genetic engineering, a few micro-organisms were isolated from the polluted
site and scanned for any significant changes in their genome like evolutions or
mutations. The modified genes were then identified because the isolate would
have adapted itself to utilize/degrade the starch better than other microbes of
the same genus. As a result, the resultant genes are cloned onto industrially
significant micro-organisms, which are used for economically significant
processes like fermentation, and it can also be applied in pharmaceutical
industries.
1.6 Environmental Biotechnology

Another case study is the incidence of oil spills in the oceans, which
require clean-up; microbes isolated from oil rich environments like oil transfer
pipelines; oil wells, etc. have been discovered to have the potential to degrade
or use it as an energy source and thus serve as a remedy to oil spills. Still
another case study is the case of microbes isolated from pesticide-rich soils.
These microbes have the potential to utilize the pesticides as a source of energy
and so when mixed along with biofertilizers, they would serve as a good
insurance against increased pesticide-toxicity levels in agricultural processes.
However, there are counter arguments that the newly introduced
micro-organisms used for clean-up of oil spillage could create an imbalance in
the natural environment concerned. There are also concerns that the mutual
harmony in which the organisms existing in that particular environment may
be altered and extreme caution should be taken so as not to disturb the mutual
relationships that are already established in that environment to which these
newly discovered and cloned micro-organisms are introduced. This leads
to a suggestion that the positive and negative environmental consequences
of environmental and agricultural biotechnology needs to be promptly
addressed.

1.3 CHALLENGES IMPOSED ON THE ENVIRONMENT BY HUMAN

ACTIVITIES
Human activities constitute one of the major means of introduction of heavy
metals into the environment. One of the major developmental challenges
facing this decade is how to achieve a cost-effective and environmentally-
sound strategies to deal with the global waste crisis facing both the developed
and developing countries. The crisis has threatened the assimilative and
carrying capacity of the earth, our life supporting system. Although the
nutrient content of wastes makes them attractive as fertilizers, land application
of many industrial wastes and sewage is constrained by the presence of heavy
metals, hazardous organic chemicals, salts, and extreme pH.
Heavy metal pollution of the environment, even at low levels, and their
resulting long-term cumulative health effects are among the leading health
concerns all over the world. For example, the bioaccumulation of lead (Pb) in
human body interferes with the functioning of mitochondria, thereby impairing
respiration, and also causes constipation, swelling of the brain, paralysis and
eventual death. This problem is even more pronounced in the developing
countries where research efforts towards monitoring the environment have
not been given the desired attention by the stakeholders. Heavy metals
concentration in the environment cannot be attributed to geological factors
alone, as human activities do modify considerably the mineral composition
of soils, crops and water. The recent population and industrial growth has led
to increasing production of domestic, municipal and industrial wastes, which
are indiscriminately dumped in landfill and water bodies without treatment.
Introduction to Environmental Biotechnology 1.7

Municipal waste contains such heavy metals as Arsenic (As), Cadmium (Cd),
Copper (Cu), Iron (Fe), Gold (Av), Manganese (Mn), Lead (Pb), Nickel (Ni)
and Zinc (Zn) which end up in the soil as the sink when they are leached out
from the dumpsites. Soil is a vital resource for sustaining two human needs of
quality food supply and quality environment. Plants grown on a land polluted
with municipal, domestic or industrial wastes can absorb heavy metals in the
form of mobile ions present in the soil solution through their roots or through
foliar absorption. These absorbed metals get bioaccumulated in the roots,
stems, fruits, grains and leaves of plants. Plants are known to take up and
accumulate heavy metals from contaminated soils. The consumption of such
plants could particularly be hazardous because the accumulated metals in
edible plants may end up in human food chain with the attendant adverse
effects on human and animal health. A promising cost-effective plant-based
technology for the clean-up of heavy metal pollution is phytoremediation.
Phytoremediation has attracted attention in recent years because of the low
cost of implementation and is particularly attractive in the tropics, where
normal climatic conditions favour plant growth and microbial activity. Plants
that sprout and grow in metal-laden soils are tolerant to metal pollution in
soil and are ‘candidates’ for remediation strategies and management for heavy
metals contaminated soils.

1.4 CONTRIBUTIONS OF BIOTECHNOLOGY TO IMPROVE

AGRICULTURAL PRODUCTIVITY
Biotechnology is regarded as a means to the rapid increase in agricultural
production through addressing the production constraints of small-scale or
resource-poor farmers who contribute more than 70% of the food produced
in developing countries. Biotechnology is applicable to all areas and fields
of human endeavours. Agricultural biotechnology has the potential to
address some of the problems of developing countries like food insecurity;
unfavourable environmental and climatic conditions, etc. and improve
agricultural productivity. Agricultural biotechnology has provided animal
agriculture with safer, more efficacious vaccines against pseudo rabies, enteric
collibacilosis and foot and mouth disease (FMD).
Disease detection in crops and animals are more efficiently and rapidly
done using DNA probes. Biotechnology acts as a key tool to breakthrough
in medical and veterinary research. Crops are now routinely genetically
modified for insect and pest resistance, delayed ripening, herbicide tolerance
and maximal production under stressed environments. Molecular mapping of
crops and farm animals has markedly cut down breeding time and enhanced
man’s understanding and manipulation of genes. Nutrition is one of the most
serious limitations to livestock production in developing countries. Plants
generally contain anti-nutrients acquired from fertilizers, pesticides and
several naturally occurring chemicals. Some of these chemicals are known as
1.8 Environmental Biotechnology

secondary metabolites and they have been shown to be highly biologically

active. Examples of these secondary plant metabolites are saponins, tannins,
flavonoids, alkaloids, oxalates, phytates, trypsin (protease) inhibitors,
cyanogenic glycosides, etc. Some of these chemicals have been shown to be
deleterious to health or evidently advantageous to human and animal health
if consumed at appropriate amounts. These anti-nutritional factors affect the
overall nutritional value of human foods and animal feeds. Some of these
plant components have the potential to precipitate adverse effects on the
productivity of farm livestock. Conventional plant breeding methods has been
used to reduce and in some cases, eliminate such anti-nutritive factors (ANF).
An example is the introduction of cultivars of oilseed rape, which are low in or
free from erucic acid and glucosinolates. A combination of genetic engineering
and conventional plant breeding methods could lead to substantial reduction
or removal of the major anti-nutritive factors in plant species of importance in
animal feeds.
Transgenic rumen microbes could also play a role in the detoxification of
plant poisons or inactivation of anti-nutritional factors. Successful introduction
of a caprine rumen inoculum obtained in Hawaii into the bovine rumen
in Australia detoxified 3 hydroxy 4(IH) pyridine (3,4 DHP), a breakdown
product of the non-protein amino acid mimosine found in Leucaena forage.
In animal production, biotechnology techniques applied include gene cloning,
embryo transfer, artificial insemination, milk modification, etc. In animal
health, biotechnology techniques are used for the fast and accurate diagnosis
and treatment of diseases. Gene therapy, vaccine production, production
of recombinant pharmaceuticals, etc. are examples. Biotechnology can help
promote sustainable and safe agriculture and environment, respectively,
globally in two ways:
1. By increasing food production within existing land area under plough,
making it unnecessary to use marginal land or environmentally-
sensitive methods and areas. This leads to conservation of bioresources
thereby avoiding soil erosion.
2. Using environment-friendly crops such as insect-resistant, herbicide
tolerant species, as well as crops that can fix nitrogen leading to
purification of the environment.
Consequently, less chemicals like pesticides, herbicides and synthetic
nitrogen fertilizers are used. Agricultural biotechnology has long been a
source of innovation in the production and processing of agricultural products
and has profound impact on the livestock sector. Globally, if hunger and
malnutrition is to be reduced drastically, agriculture must be tailored to meet
the future demands of increased population. The increase in human population
increases the demand for land, space and available resources and primitive
agricultural practices cause desertification, environmental pollution and
produces resultant effects on climate, ecosystems, biogeochemical cycles and
Introduction to Environmental Biotechnology 1.9

human health. Sustainable agricultural practices targeted towards improved

agricultural productivity, under clean, safe and environment-friendly
conditions must be introduced into the global agricultural system in order to
reduce the adverse effects of environmental pollution on human health and
the climate (global warming). The new techniques of biotechnology provide
innovations that complement the weaknesses of conventional agricultural
practices and should be adopted for increased food production.
Many scientists would agree that biotechnology is an important contributor to
a sustainable agriculture system because it can produce more food with a lesser
environmental impact as compared to conventional agriculture.

1.4.1 Biotechnology in reducing the use of chemical pesticides,

herbicides and fertilizers
A lot of debate is going on the overuse of chemical herbicides, pesticides and
fertilizers. They become an environmental hazard because they undergo
degradation by microorganisms and ultraviolet light which releases toxic
chemicals in the environment. Using biotechnology, bacterial pesticides and
viral pesticides are being developed which will help in reducing the use of
chemical pesticides. Several companies in USA like Monsanto, Mycogen,
Ecogen, Repligen, Zoecon, etc. are actively involved in the development of
biological pesticides. The trials are going on to use the genetically engineered
live soil bacteria for coating seeds before planting. Another method being
tried is to kill the recombinant bacteria and apply them to the leaves of crop
plants. Both these approaches protect the toxin from degradation by microbes
or ultraviolet rays once applied to the crop plants.
The company Ecogen Inc. was involved in developing biological pesticides
against the two major crop pests, budworm and ballworm, by transferring
a gene from Bacillus thuringiensis (Bt), into either a naturally occurring soil
bacterium or in to a strain of Pseudomonas. Bt insecticides are already being
marketed for past few years and in future these will be modified using
genetic engineering and will be used against a variety of insects. Genetically
engineered insect resistant plants have been successfully produced which will
further help in reducing the use of insecticides in the future. The experiments
are going on to develop environmentally safe herbicides. In order to use these
herbicides for crop protection programme, genetically engineered herbicide
resistant plants have been produced in a variety of crop plants. This will
ensure the use of environmentally safer herbicides.

1.4.2 Biofertilizers
Biofertilizers are also being used in place of chemical fertilizers to further
reduce the environmental hazards caused due to chemical fertilizers. The
term biofertilizers is used to refer to the nutrient inputs of biological origin to
1.10 Environmental Biotechnology

support plant growth which is generally achieved by the addition of microbial

inoculants as a source of biofertlizers. Biofertilizers broadly includes the
following categories:
• Symbiotic nitrogen fixers: The diazotrophic microorganisms are the
symbiotic nitrogen fixers that serve as biofertilizers e.g. Rhizobium sp.,
Bradyrhizopium sp.
• Asymbiotic nitrogen fixers: The asymbiotic nitrogen fixing bacteria
can directly convert the gaseous nitrogen to nitrogen rich compounds.
On the death of these nitrogen fixers, the soil becomes enriched with
nitrogenous compounds thereby serving as biofertilizers e.g. Azobacter
sp., Azospirillum sp.
• The blue green algae, multiply in the water logging conditions and fixes
the nitrogen. They accumulate the biomass which helps in improving
the physical properties of the soil. This is useful for reclamation of
alkaline soils besides providing partial tolerance to pesticides. The
most common blue green algae are Azobacter sp. and Azospirillum sp.
Azolla, which is an aquatic fern contains an endophytic cynobacterium
Anabaena azollae in the leaf cavities providing symbiotic relationship.
Azolla with Anaebaena is useful as biofertilizer.
• Phosphate solubilizing bacteria: Some bacteria like Thiobacillus, Bacillus
are capable of converting non-available inorganic phosphorus present
in the soil to organic or inorganic form of phosphate. These bacteria
can also produce siderophores, which chelates with iron, and makes it
unavailable to pathogenic bacteria. Siderophores are iron-binding low
molecular weight (400–1,000 Daltons) peptides synthesized by some
soil bacteria.
• Organic fertilizers: Certain types of organic wastes are used as fertilizers
e.g. animal dung (cow dung, elephant dung, etc.), urine, urban garbage,
sewage, crop residues and oil cakes. All these wastes can be converted
into organic manures.
Advantages of using biofertilizers
• Biofertilizers improve the tolerance of plants against toxic heavy
metals.
• It is possible to reclaim salinity or alkalinity of soil by using biofertilizers.
• Use of biofertilizers helps in controlling environmental pollution.
• Fertility of soil is increased year after year.
• Low cost and easy to produce.
• Biofertilizers increase the physico-chemical properties of the soil, soil
texture and water holding capacity.
Some of the limitations encountered while using the biofertilizers are
that they alone cannot meet the total needs of the plants for nutrient supply
Introduction to Environmental Biotechnology 1.11

and also they do not produce the spectacular results as observed in synthetic
fertilizers. It is important to evolve an approach which can maximize the use of
biofertilizers and reduce the dependency on the chemical fertilizers in the near
future without affecting the crop productivity. This will help us to solve the
environment related problems caused due to overuse of chemical fertilizers.

1.5 BIOTECHNOLOGY AND LIVESTOCK PRODUCTION IN THE

IMPROVEMENT OF ENVIRONMENTAL CONDITIONS
Livestock recycle nutrients on the farm, produce valuable output from land
that is not suitable for sustained crop production and provide energy and
capital for successful farm operations. Livestock can also help maintain
soil fertility in soils lacking adequate organic content or nutrients. Adding
animal manure to the soil increases the nutrient retention capacity (or cation-
exchange capacity), improves the soil’s physical condition by increasing its
water-holding capacity and improves soil structure. Animal manure provides
favourable conditions for microflora and fauna in soils. Grazing animals
improve soil cover by dispersing seeds, controlling shrub growth, breaking
up soil crusts and removing biomass that otherwise might be fuel for bush
fires. These activities stimulate grass tilling and improve seed germination
and thus improve land quality and vegetation growth. Livestock production
also enables farmers to allocate plant nutrients across time and space by way
of grazing to produce manure, and in the land land that cannot sustain crop
production. This makes other land more productive. Grazing livestock can
also accelerate transformation of nutrients in crop by-products to fertilizer,
thus speeding up the process of land recovery between crops. As disease
constraints are also removed, large breeds of livestock can be integrated into
crop operations for providing farm power and manure. Biotechnology has
enhanced increased animal production through Artificial insemination (AI)
and also improved animal health and disease control through the production
of DNA recombinant vaccines.
Crops improved through biotechnology are producing higher yields worldwide to
help feed a hungry and growing world.

1.6 BIOTECHNOLOGY AND THE REMOVAL OF TOXIC CHEMICALS

FROM THE ENVIRONMENT
Microorganisms have broadened the environments they live in by evolving
enzymes that allow them to metabolize numerous man-made chemicals
(that is, xenobiotics). Bioremediation is the use of microorganisms or
microbial processes to detoxify and degrade environmental contaminants.
Microorganisms have been used for the routine treatment and transformation
of waste products for several decades. The fixed-film and activated sludge
treatment systems depend on the metabolic activities of microorganisms which
degrade the wastes entering the treatment facility. Specialized waste treatment
1.12 Environmental Biotechnology

plant containing selected and acclimated microbial populations are often used
to treat industrial effluents. The innovation in bioremediation has been applied
to the remediation of soils, groundwater and similar environmental media.
Bioremediation techniques depend on having the right microorganisms
in the right place with the right environmental condition for degradation to
occur. The right microorganisms are those bacteria and fungi which possess the
physiological and metabolic capability to degrade the contaminants. Already,
bacteria with natural abilities to digest certain chemicals are being used to
clean up industrial sites. By means of genetic engineering, biotechnology has
brought about the rapid production of bacteria.

1.7 BIOTECHNOLOGY AND THE REMOVAL OF HEAVY METAL

POLLUTION FROM THE ENVIRONMENT
Basically, the heavy metals of environmental interest include mercury,
vanadium, nickel, cobalt, lead, cadmium, chromium, tin, etc. Some harmful
compounds that cause serious environmental pollutions and disaster like
Dichlorodiphenyltrichloroethane (DDT) and lead (Pb) could be safely
removed by means of genetic engineering of bacteria manufactured for
that purpose. The ability of microorganisms to accumulate metals and
their potential use in the decontamination of environments impacted by
toxic metals has been reported by many scientists. Microorganisms remove
toxic metals by various mechanisms such as adsorption to cell surfaces,
complexation of exopolysaccharides, intracellular accumulation, biosynthesis
of metallothionins and other proteins that trap metals and transform them to
volatile compounds. Micrococcus luteus and Azotobacter sp. have been shown to
immobilize large quantities of lead from sites containing high concentrations
of lead salts, without a detectable effect on viability. Uranium, copper and
cobalt could be removed by polyacrylamide immobilized cells of Streptomyces
albus. Microbial processes can also mediate the precipitation of metals from
aqueous solutions. Certain bacteria extracellular products may interact with
free or absorbed metal cations forming insoluble metal precipitates.
The major mechanism involved in such precipitation is through the
formation of hydrogen sulphide and the immobilization of metal cations as
metal sulphides. Certain fungi that produce oxalic acid (oxalates) facilitate
the immobilization of metals such as metal oxalate crystals. Microbes can
also catalyze a range of metal transformations, which are useful for waste
treatment. These transformations include oxidation, reduction and alkylation
reactions. Bacteria, fungi, algae or protozoa, in the oxidation reactions,
can deposit ferrous and manganese ions. Geobacter metallireducens remove
uranium, a radioactive waste, from drainage waters in mining operations and
from contaminated ground waters.
Introduction to Environmental Biotechnology 1.13

1.8 BIOTECHNOLOGY AND DESULPHURIZATION OF FOSSIL

FUELS
The removal of inorganic sulphur from coal is mediated by microbial oxidation
of sulphur.
The direct oxidation of inorganic sulphur by Thiobacillus sp. is a membrane-
bound reaction and requires direct contact of the substrate with the bacterium.
As a result of this, the attachment of the culture to coal particle is the absolute
requirement. Mixed and pure cultures of a variety of microorganisms
(heterotrophic bacteria) can be used to remove organic sulphur from coal
and oil. However, sulphur removal has also been reported under anaerobic
microbial action.

1.9 BIOTECHNOLOGY AND ECOSYSTEM MODELING

An ecosystem consists of producers, consumers, decomposers and
detritivores and their physical environment, all interacting through energy
flow and materials recycling. A food web is a network of crossing, interlinked
food chains involving primary producers, consumers and decomposers.
Disturbances to one part of an ecosystem can have unexpected effects on
other, seemingly unrelated parts. Ecosystem modeling is an approach to
predict unforeseen effects. By this method, researchers identify crucial bits
of information about different ecosystem components. They use computer
programs and models to combine the information and then use the resulting
data to predict the outcome of the next disturbance. Biotechnology techniques
like bioinformatics are useful in ecosystem modeling. Bioinformatics deals in
gene database management, gene mapping, coding, sequence alignment, etc.

1.10 MICROBIAL BIOTECHNOLOGY IN THE MONITORING OF

ENVIRONMENTAL POLLUTION
A number of microbial parameters are used for the detection and monitoring
of pollutants, especially in water bodies. Some micro-organisms serve as
indicators of organic pollution while others serve as indicators of inorganic
pollution. Some of the parameters used for the monitoring of organic pollution
are heterotrophic bacteria, total and faecal coliforms and faecal streptococci.
Parameters used for monitoring inorganic pollution include nitrifying
bacteria, sulphur-oxidizing bacteria, sulphate-reducing bacteria (SRB), iron
bacteria, etc. The presence of faecal coliforms in numbers above the World
Health Organization (WHO) standard for portable water is indicative of faecal
contamination of human origin.
Heterotrophic microorganisms are organisms that derive their energy
from the oxidation of organic molecules. Their presence in large numbers
in aquatic systems is indicative of organic pollution. The presence of
biodegradable carbon sources supports the proliferation of heterotrophs in
1.14 Environmental Biotechnology

aquatic systems. In the case of xenobiotics, the few species that can degrade
them may produce by-products during metabolism that may support other
microbial species. Thus, a high heterotrophic microbial count is suggestive of
high level of organics in aquatic system while low count is suggestive of either
a low level of organic pollution or the presence of persistent organic matter
within the aquatic system.

1.11 MICROBIAL BIOTECHNOLOGY IN THE BIOASSAY OF

ENVIRONMENTAL TOXICITY
Toxic industrial wastes are a threat to both the biological waste treatment
systems and the environment of their ultimate disposal. As a result, bioassays
are very necessary to generate data that could be used for the prediction of
environmental effects of waste and regulation of discharges. Although fishes
have been the most popular test organisms, standard organisms for aquatic
bioassays also include phytoplanktons, zooplanktons, molluscs, insects and
crustaceans. The use of microbes (especially bacteria) as bioassay organism is
gaining wide acceptance and offers a number of advantages over the standard
organisms.
Bacteria are easily handled and require relatively small space for
culturing and/or testing, compared with other bioassays. Moreover, the short
life cycle means fast experimental results, thus enabling the laboratory to
process more samples. The simple and rapid bacteria bioassay techniques
include Nitrobacter assay, Microtox tests, the Toxi-chromotest and the
Ames/Salmonella test. The Nitrobacter bioassay relies on the quantification
of Nitrobacter activity determined by measuring the toxicant effect on the
rate of nitrite utilization. Photobacterium phosphoreum is the basis of the
Microtox assay. Toxichromotest is based on the inhibition of beta galactosidase
biosynthesis in E. coli or biosynthesis of enzymes, such as tryptophanse and
alpha-glucosidase, under the control of operons (other than the Lac operon)
by environmental pollutants.

1.12 BIOTECHNOLOGY AND CONTROL OF OIL SPILLAGE

Microorganisms can now be genetically engineered for use in oil recovery,
pollution control, mineral leaching and recovery. In the petroleum industry,
micro organisms can also be genetically engineered to produce chemicals
useful for enhanced oil recovery. Cleaning up oil spills could, in the
future, be left to genetically-engineered bacteria. In the mining industries,
microorganisms with the property of enhanced leaching ability could be
designed. Microorganisms can bind metals to their surfaces and concentrate
them internally. As a result of this, genetically improved strains can be used
to recover valuable metals or remove polluting metals from dilute solution
as in industrial waste. Research is already being carried out to improve the
naturally-occurring bacteria that can ‘eat oil’, for use following an oil spill. By
Introduction to Environmental Biotechnology 1.15

applying bacteria to oil covered beaches, the complex oil molecules would be
broken down into harmless sugars.
Many microorganisms can degrade various kinds of environmental
pollutants into relatively harmless materials before the death of the micro-
organisms. This property could also be used in overcoming the environmental
hazards of DDT, lead and other environmental pollutants like toxic wastes
globally. Strains of bacteria which can degrade fuel hydrocarbons have been
designed and the use of genetically engineered micro-organisms to clean up oil
spillages or treat sewages has been proposed and is undergoing production/
manufacturing.

1.13 BIOREMEDIATION AND THEIR IMPORTANCE IN ENVIRONMENT

PROTECTION
Bioremediation is defined as ‘the process of using microorganisms to remove
the environmental pollutants where microbes serve as scavengers’.
• The removal of organic wastes by microbes leads to environmental clean-
up. The other names/terms used for bioremediation are biotreatment,
bioreclamation, and biorestoration.
• The term “Xenobiotics” (xenos means foreign) refers to the unnatural,
foreign and synthetic chemicals, such as pesticides, herbicides,
refrigerants, solvents and other organic compounds.
• The microbial degradation of xenobiotics also helps in reducing the
environmental pollution. Pseudomonas which is a soil microorganism
effectively degrades xenobiotics.
• Different strains of Pseudomonas that are capable of detoxifying more than
100 organic compounds (e.g. phenols, biphenyls, organophosphates,
naphthalene, etc.) have been identified.
• Some other microbial strains are also known to have the capacity to
degrade xenobiotics such as Mycobacterium, Alcaligenes, Norcardia, etc.

1.13.1 Factors affecting biodegradation

The factors that affect the biodegradation are:
• the chemical nature of xenobiotics,
• the concentration and supply of nutrients,
• O2, temperature, pH, redox potential and
• the capability of the individual microorganism.
The chemical nature of xenobiotics is very important because it was
found out that the presence of halogens, e.g. in aromatic compounds, inhibits
biodegradation. The water-soluble compounds are more easily degradable
whereas the presence of cyclic ring structure and the length chains or branches
decrease the efficiency of biodegradation. The aliphatic compounds are more
easily degraded than the aromatic ones.
1.16 Environmental Biotechnology

Bio-stimulation: It is a process by which the microbial activity can be

enhanced by increased supply of nutrients or by addition of certain stimulating
agents like electron acceptors, surfactants, etc.
Bio-augmentation: It is possible to increase biodegradation through
manipulation of genes i.e. using genetically engineered microorganisms and
by using a range of microorganisms in biodegradation reaction.
Depending on the method followed to clean up the environment,
bioremediation is carried out in two ways:

1.13.2 In situ bioremediation

In situ bioremediation involves a direct approach for the microbial degradation
of xenobiotics at the site of pollution which could be soil, water etc. The
adequate amount of essential nutrients is supplied at the site which promotes
the microbial growth at the site itself. The in situ bioremediation is generally
used for clean up of oil spillages, beaches, etc. There are two types of in situ
bioremediation-
1. Intrinsic bioremediation
2. Engineered in situ bioremediation

1.13.2.1  Intrinsic bioremediation

The microorganisms which are used for biodegradation are tested for the
natural capability to bring about biodegradation. So, the inherent metabolic
ability of the microorganisms to degrade certain pollutants is the intrinsic
bioremediation. The ability of surface bacteria to degrade a given mixture
of pollutants in ground water is dependent on the type and concentration
of compounds, electron acceptor and the duration of bacteria exposed to
contamination. Therefore, the ability of indigenous bacteria degrading
contaminants can be determined in laboratory by using the techniques of
plate count and microcosm studies. The conditions of site that favour intrinsic
bioremediation are groundwater flow throughout the year, carbonate minerals
to buffer acidity produced during biodegradation, supply of electron acceptors
and nutrients for microbial growth, and absence of toxic compounds.

1.13.2.2  Engineered in situ bioremediation

When the bioremediation process is engineered to increase the metabolic
degradation efficiency (of pollutants), it is called engineered in situ
bioremediation. This is done by supplying sufficient amount of nutrients
and oxygen supply, adding electron acceptors, and maintaining optimal
temperature and pH. This is done to overcome the slow and limited
bioremediation capability of microorganisms.
 Advantages of in situ bioremediation
• The method ensures minimal exposure to public or site personnels.
• There is limited or minimal disruption to the site of bioremediation.
Introduction to Environmental Biotechnology 1.17

• Due to these factors, it is cost effective.

• The simultaneous treatment of contaminated soil and water is possible.
Disadvantages of in situ bioremediation
• The sites are directly exposed to environmental factors like temperature,
oxygen supply, etc.
• The seasonal variation of microbial activity exists.
• Problematic application of treatment additives like nutrients,
surfactants, oxygen, etc.
• It is a very tedious and time-consuming process.

1.13.3  Ex-situ bioremediation

In this, the waste and the toxic material is collected from the polluted sites
and the selected range of microorganisms carry out the bioremediation
at designated place. This process is an improved method over the in situ
bioremediation method. On the basis of phases of contaminated materials
under treatment, ex-situ bioremediation is classified into two :
• Solid phase system and
• Slurry phase system.

1.13.3.1  Solid phase treatment

• This system includes land treatment and soil piles comprising of organic
wastes like leaves, animal manures, agricultural wastes, domestic and
industrial wastes, sewage sludge, and municipal solid wastes.
• The traditional clean-up practice involves the informal processing of
the organic materials and production of composts which may be used
as soil amendment.
• Composting is a self-heating, substrate-dense, managed microbial
system which is used to treat large amount of contaminated solid
material.
• Composting can be done in open system i.e. land treatment and/or in
closed treatment system.
• The hazardous compounds reported to disappear through composting
include aliphatic and aromatic hydrocarbons and certain halogenated
compounds.
• The possible routes leading to the disappearance of hazardous
compounds include volatilization, assimilation, adsorption,
polymerization and leaching.

1.13.3.2  Slurry phase treatment

• This is a triphasic treatment system involving three major components—
water, suspended particulate matter and air.
1.18 Environmental Biotechnology

• Here, water serves as suspending medium where nutrients, trace

elements, pH adjustment chemicals and desorbed contaminants are
dissolved.
• Suspended particulate matter includes a biologically inert substratum
consisting of contaminants and biomass attached to soil matrix or free
in suspending medium.
• The contaminated solid materials, microorganisms and water
formulated into slurry are brought within a bioreactor i.e. fermenter.
• Biologically, there are three types of slurry-phase bioreactors: aerated
lagoons, low-shear airlift reactor, and fluidized-bed soil reactor.
• The first two types are in use of full-scale bioremediation, while the
third one is in developmental stage.
Advantages of ex-situ bioremediation
• As the time required is short, it is a more efficient process.
• It can be controlled in a much better way.
• The process can be improved by enrichment with desired and more
efficient microorganisms.
Disadvantages of ex-situ bioremediation
• The sites of pollution remain highly disturbed.
• Once the process is complete, the degraded waste disposal becomes a
major problem.
• It is a costly process.
Several types of reactions occur during the bioremediation/microbial
degradation
(a) Aerobic bioremediation- When the biodegradation requires oxygen
(O2) for the oxidation of organic compounds, it is called aerobic
bioremediation. Enzymes like monooxygenases and dioxygenases are
involved and act on aliphatic and aromatic compounds.
(b) Anaerobic bioremediation: This does not require oxygen. The
degradation process is slow but more cost-effective since continuous
supply of oxygen is not required.
(c) Sequential bioremediation: Some of the xenobiotic degradations require
both aerobic as well as anaerobic processes which very effectively
reduce the toxicity e.g. tetrachloromethane and tetrachloroethane
undergo sequential degradation.

1.13.4 Use of genetic engineering and genetic manipulations for more

efficient bioremediation
In recent years, efforts have been made to create genetically engineered
microorganisms (GEMs) to enhance bioremediation. This is done to overcome
Introduction to Environmental Biotechnology 1.19

some of the limitations and problems in bioremediation. These problems

are:
• Sometimes the growth of microorganisms gets inhibited or reduced by
the xenobiotics.
• No single naturally occurring microorganism has the capability of
degrading all the xenobiotics present in the environmental pollution.
• The microbial degradation is a very slow process.
• Sometimes, certain xenobiotics get adsorbed on to the particulate
matter of soil and thus become unavailable for microbial degradation.
As the majority of genes responsible for the synthesis of enzymes
with biodegradation capability are located on the plasmids, the genetic
manipulations of plasmids can lead to the creation of new strains of bacteria
with different degradative pathways. In 1970s, Chakrabarty and his team of co-
workers reported the development of a new strain of bacterium Pseudomonas
by manipulations of plasmid transfer which they named as “superbug”.
This superbug had the capability of degrading a number of hydrocarbons of
petroleum simultaneously such as camphor, octane, xylene, naphthalene, etc.
In 1980, United States granted the patent to this superbug making it the first
genetically engineered microorganism to be patented.
In certain cases, the process of plasmid transfer was used. E.g., the
bacterium containing CAM (camphor degrading) plasmid was conjugated
with another bacterium with OCT (octane degrading) plasmid. Due to non-
compatibility, these plasmids cannot co-exist in the same bacterium. However,
due to the presence of homologous regions of DNA, recombination occurs
between these two plasmids which results in a single CAM-OCT plasmid
giving the bacterium the capacity to degrade both camphor as well as octane.

1.13.5 Biotechnology to reduce atmospheric Carbon dioxide (CO2)

Carbon dioxide is the gas that is the main cause of greenhouse effect and rise
in the atmospheric temperature. During the past 100-150 years, the level of
CO2 has increased about 25% with an increase in the atmospheric temperature
by about 0.5% which is a clear indication that CO2 is closely linked with global
warming. There is a steady increase in the CO2 content due to continuous
addition of CO2 from various sources particularly from industrial processes.
It is very clear that the reduction in atmospheric CO2 concentration assumes
significance. Biotechnological methods have been used to reduce the
atmospheric CO2 content at two levels:
(a) Photosynthesis: Plants utilize CO2 during photosynthesis which reduces
the CO2 content in the atmosphere. The equation for photosynthesis is:
6CO2 + 6H2O → C6H12O6 + 6O2
The fast growing plants utilize the CO2 more efficiently for
photosynthesis. The techniques of micro-propagation and synthetic
1.20 Environmental Biotechnology

seeds should be used to increase the propagation of such fast growing

plants. Further, the CO2 utilization can be increased by enhancing the
rate of photosynthesis. The enzyme ribulose biphosphate carboxylase
(RUBP-case) is closely linked with CO2 fixation. The attempts are being
made to genetically manipulate this enzyme so that the photosynthetic
efficiency is increased.
Some microalgae like Chlorella pyrenodiosa, Spirulina maxima are known
to be more efficient than higher plants in utilizing atmospheric CO2
for photosynthesis and generate more O2 than the amount of CO2
consumed.
The growing of these microalgae near the industries and power plants
(where the CO2 emission into atmosphere is very high) will help in
the reduction of polluting effects of CO2. Using genetic engineering,
attempts are being made to develop new strains of these microalgae
that can tolerate high concentrations of CO2. A limited success has
already been reported in the mutants of Anacystis nidulans and Oocystis
sp.
(b) Biological Calcification: Certain deep-sea organisms like corals, green
and red algae store CO2 through a process of biological calcification. As
the CaCO3 gets precipitated, more and more atmospheric CO2 can be
utilized for its formation. The process of calcification is as follows:
H2O + CO2 → H2CO3
H 2CO3 + Ca2+ → CaCO3 + CO2 + H2O

1.13.6 Treatment of sewage using microorganisms

The sewage is defined as the wastewater resulting from the various human
activities, agriculture, and industries and mainly contains organic and inorganic
compounds, toxic substances, heavy metals, and pathogenic organisms. The
sewage is treated to get rid of these undesirable substances by subjecting the
organic matter to biodegradation by microorganisms. The biodegradation
involves the degradation of organic matter to smaller molecules (CO2, NH3,
PO4, etc.) and requires constant supply of oxygen. The process of supplying
oxygen is expensive, tedious, and requires a lot of expertise and manpower.
These problems are overcome by growing microalgae in the ponds and
tanks where sewage treatment is carried out. The algae release the O2 while
carrying out photosynthesis which ensures a continuous supply of oxygen for
biodegradation.
The algae are also capable of adsorbing certain heavy toxic metals due to
the negative charges on the algal cell surface which can take up the positively
charged metals. The algal treatment of sewage also supports fish growth as
algae is a good source of food for fishes. The algae used for sewage treatment
are Chlorella, Euglene, Chlamydomnas, Scenedesmus, Ulothrix, Thribonima, etc.
Introduction to Environmental Biotechnology 1.21

1.13.7 Treatment of industrial effluents using biotechnology

• The industrial effluents should be properly treated in order to control
the environmental pollution.
• The industrial effluent often contains toxic materials like suspended
solids and soluble organic compounds, heavy metals, cyanides, non-
biodegradable chemical and volatile materials like H2 S and SO2, etc.
• The soluble organic compounds undergo slow decomposition resulting
in oxygen depletion and production of noxious gases. The heavy metals
and other toxic organic materials such as chlorinated compounds in
effluents from paper industry have adverse effects on the aquatic flora
and fauna.
• The high levels of nitrogen and phosphorus causes eutrophication
which leads to undesirable algal growth and death of animals due to
lack of oxygen.
Biotechnological techniques are being used to address some of these
problems. The biological treatment of effluents has been in use for quite
some time in many countries around the world. However, some specific
problems which are related to conventional treatment are being solved using
biotechnology. Among the substances released in effluents are calcitrants which
cannot be degraded using conventional treatment methods. Biotechnology
helps in overcoming this problem.
• In USA, the company BioTechnica is using lignin degradation for the
treatment of substances like polychlorinated biphenyls (PCBs) and
dioxin.
• In Europe, ICI and Ciba-Geigy are working on enzymatic detoxification
(breaking down) of substances such as cyanides and also the by-
products from the synthesis of S-triazine herbicides.
Microbial transformation of certain substances such as dibenzofurans,
biarylketones halogenated bibenzodioxins, etc. are used to minimize the
problem of pollution due to toxic effluents.
• E.g., certain strains of Pseudomonas have been isolated which selectively
deoxygenate the 1,2 positions of substituted biarylethers and
biarylketones.
— Microbial degradation of chloro-, dichloro-, and carbontetra-
chloride, etc. is being also tried to deal with the problem.
— The paper and pulp industries release effluents which contain
chromophoric compounds and chlorogenated organic materials like
chlorolignins, chlorosyringols, chloroaliphatics, catechols etc. which
can affect the aquatic system by their inhibitory and mutagenic
activities.
— Certain soil-inhabiting fungi, streptomycetes, bacteria and white
rot fungi (Ganoderma lacidum, Coriolus (Trametes) versicolor, P.
1.22 Environmental Biotechnology

chrysosporium, Coprinus macrorhizus, Hericiumerinaceus, etc. have

been studied for their ability to decolourise chromophoric substrates.
White rot fungi produces a variety of lignin-degrading enzymes
(e.g. peroxidases and lacasses) that degrade phenolic substances.

1.13.8 Use of biotechnology for toxic site reclamation

Generally, incineration (drying and then burning to ashes in furnace) or
chemical treatment are being used to get rid of toxins and waste from the waste
disposal sites. Of late, biotechnological techniques involving biodegradation
as an alternative approach is being used. Companies like BioTechnica are
working on treating polluted site in situ. However, there is a lot of debate
over the issue regarding the release of genetically engineered microbes for
treatment of toxic sites and the risk involved in the whole procedure. As we
know that, the released engineered organisms that have capacity to reproduce,
spread to sites other than the initial release sites and may undergo mutations.
All this can lead to the risk of developing what are described as “super bugs”.
Some of the companies in US are experimenting and conducting their work
in the closed reactors in order to further evaluate the risk assessment and cost
effectiveness of this approach.
In order to solve the problem of soil pollution caused due to extensive
use of herbicides, pesticides and insecticides, bioremediation of soil using
microorganisms is being carried out. The most common pollutants are:
hydrocarbons, chlorinated solvents, polychlorobiphenyls and metals. The
bioremediation of soil involves:
• Biostimulation: Biostimulation involves stimulation of microorganisms
already present in the soil. This can be done by adding nutrients e.g.
nitrogen, phosphorus etc., by supplying co-substrates e.g. methane
which can degrade trichloroethylene, or by adding surfactants to
disperse the hydrophobic compounds in water.
• Bioaugmentation: Addition of specific microorganisms to the
polluted soil constitutes bioaugmentation. Some of the pollutants
like polychlorobiphenyls (PCBs), trinitrotoluene (TNT), polyaromatic
hydrocarbons (PAHs), etc. are not degraded by only native soil
microorganisms so a combination of microorganisms; referred to as
“consortium” or “cocktail” of microorganisms; is added to achieve
bioaugmentation.
• Bioventing: Bioventing involves aerobic biodegradation of pollutants
by circulating air through sub-surfaces of soil and is one of the very
cost-effective and efficient technique used for the bioremediation of
petroleum-contaminated soils. It is very effectively used for degradation
of soluble paraffins and polyaromatic hydrocarbons.
• Phytoremediation: Bioremediation by using plants is called
phytoremediation. Certain plant species which have the capability to
Introduction to Environmental Biotechnology 1.23

stimulate biodegradation of pollutants (especially near the soil adjacent

to roots- rhizosphere) are cultivated near the sites of polluted soil. This
is a cheap and environmentally friendly process but takes a long time
to finish the clean-up process.
• Land farming: Land farming is a technique for the bioremediation of
hydrocarbon contaminated soils. In this, the soil is excavated, mixed
with microorganisms and nutrients and spread out on a liner just
below the polluted soil.
• Use of slurry-phase bioreactors: In this process, the excavated polluted
soil is subjected to bioremediation under optimal controlled conditions
in specifically designed bioreactors.
Engineered bacteria used for the degradation of
xenobiotics and toxic wastes
Bacterium Substrate that can be degraded
Pseudomonas capacia 2,4,5-trichloro-phenoxyacetic acid
P. putida and other spp. (also E. coli) 2,2,5-dichloropropionate; mono and
dichloroaromatics
Alcaligenes sp. Dichlorophenoxyacetic acid, mixed
chlorophenols; 1,4-dichlorobenzene
Acinetobacter sp. 4-chlorobenzene
Pseudomonas capacia 2,4,5-trichoro-phenoxyacetic acid

1.13.9 Use of biotechnology in the removal of oil and grease

deposits
• The oil spills from oil tankers on land surface as well as in seas and
oceans are a major environmental hazard. This not only kills the
aquatic flora and fauna by destroying the habitat but also creates health
problems for the local inhabitants.
• Traditionally, chemical dispersants are being used as remediation
efforts. However, these chemical dispersants are also toxic in nature
and they persist in the environment for a long time.
• The present techniques of washing the oil off the gravel and cleaning
the area of oil spills, is very expensive and time-consuming.
• In order to overcome some of these problems, the oleophilic fertilizers
are being developed which allow rapid growth and multiplication of
microbes which further leads to the increase in the biodegradation
process for removal of oil.
• In recent years, using genetic engineering, oil utilizing microorganisms
have been produced which can grow rapidly on oil.
• The genetically engineered microbes for cleaning oil spills are mixed
with straw. At the site of oil spill, the straw mixed with microbes are
scattered over the oil spilled area.
1.24 Environmental Biotechnology

• The straw soaks the oily water and the microbes break the oil into non-
toxic and non-polluting materials thereby cleaning up the site.
• Some of the oil-utilizing microbes can also produce surface-active
compounds that can emulsify oil in water and thereby removing the
oil.
• A strain of Pseudomonas aeruginosa produces a glycolipid emulsifier
that reduces the surface tension of oil-water interface, which helps in
the removal of oil from water. This microbial emulsifier is nontoxic
and biodegradable and has shown promising results in the laboratory
experiments.
• Some of the microorganisms which are capable of degrading petroleum
include Pseudomonas, various Corynebacteria, Mycobacteria and some
yeasts.
The two methods for bioremediation of oil spills are:
(a) using a consortium of bacteria, and
(b) using genetically engineered bacteria/microbial strains.
Both bacterial and fungal cultures from the petroleum sludge have
been isolated. The fungal culture could degrade 0.4% sludge in 3 weeks.
Degradation of petroleum sludge occurred within two weeks when the
bacterial culture (Bacillus circulans CI) was used. A significant degradation of
petroleum sludge was observed in 10 days when the fungus + B. circulans and
a prepared surfactant were exogenuously added to petroleum sludge.

1.13.10  Tannery effluents and their treatment

The environment, we know, is under increasing pressure from solid and
liquid effluents from the leather industry, an offshoot of the natural resources
(animals). They are categorized as:
• Solids: Suspended solids; Settleable solids; Gross solids.
• Sulphides (S2–): They result from the use of sodium sulphide and
sodium hydrosulphide and the breakdown of hair and in the unhairing
process. In the alkaline condition, they remain largely in solution and
when the pH drops below 9.5, hydrogen sulphide, the obnoxious gas
evolves.
• Neutral salts: Sulphates (SO24–) and Chlorides (Cl–).
• Oils and grease.
• Chromium compounds: Chrome (3+) and Chrome (6+).
• Other metals: Al, Zr, etc.
• Solvents.
The tannery effluents damage the normal functioning of a river, destroy
its ecosystem and are the main causes of imminent systems collapse of the
Introduction to Environmental Biotechnology 1.25

Ganges. They are responsible for polluting the aquifers to such a significant
extent that it is becoming a serious environmental hazard (water becomes
non-potable).
Pollution remediation of the tannery effluents is very complex. A
multi-prong treatment is thus called for a combination of nanotechnology
and microbial technology with prior processing by cycloning, flotation,
microflotation or electroflotation. The suspended solids can be separated
by an initial filtration process followed by flotation, microflotation or
electroflotation or a combination depending on their surface properties
and their electrostatic behaviour whereas the semi-colloidal solids, i.e. the
protein residues can be separated by cycloning. During cycloning, the protein
material will be separated out at the outlet (at the top of the cyclone). Injection
of black iron (Fe0) nanoparticles (ZVI) or Ni/Fe bimetallic nanites15, produced
in the laboratory, into the contaminant in an effluent treatment basin (where
oxygen is available) with non-porous boundary zones will immobilize the
metals, particularly the hazardous chromium, and detoxify and dechlorinate
the other pollutants available. Biodegradation by microorganisms (discussed
earlier under biosorption) will follow the nanoremediation process if it still
contains partial degradation products that are considered hazardous.
The flow of the nanotreated material to a second basin is, therefore,
imperative. When the engineering design is carried out, the material after
treatment can be flowed out into the open system. Microorganisms like Bacillus,
Escherichia, Enterobacter, Micrococcus, Arthrobacter, Pseudomonas, etc. and fungi
like Neurospora can be utilized for biodegradation of the metals available in
these effluents, after they have been exposed to ZVI remediation technology.
The heap soil washing technology using leaching microbes is receiving much
attention these days for the remediation of large volumes of heavy metals and
radioactive elements in contaminated soil. From another perspective, such
bioremediation is gaining recognition as a metallurgical process for recovery
of metals.

1.13.11  Bioremediation of Radioactive Contaminants

Among the most toxic chemicals of global concern are the radionuclides found in
contaminated waste streams and groundwaters. For example, former nuclear-
weapons facilities are severely contaminated with plutonium, uranium, and
neptunium because of past practices that released mixtures of radionuclides
and other organic and inorganic wastes into adjacent soil and groundwater.
The greatest potential for clean-up of such sites is in situ bioremediation,
which exploits the reactions of microorganisms to directly or indirectly
alter the chemical form of the radionuclides, rendering them immobile and
less toxic. The microorganisms interact directly with the radionuclides by
catalyzing chemical redox transformations. The microorganisms act indirectly
by producing acids, bases, and complexing ligands that react with the
radionuclides.
1.26 Environmental Biotechnology

Radionuclide bioremediation is a complicated situation, since the fate of

radionuclide depends on so many different reactions, which proceed through
multiple steps and at vastly different rates. Such a complex scenario can be
understood and controlled only by using mathematical modeling. Such a
unique biogeochemical mathematical model, CCBATCH, was developed by
Dr. Rittmann and colleagues to connect all the different types of reactions that
control the fate of radionuclides and a large range of metals. Current research
is focused on applying CCBATCH towards bioremediation of plutonium, one
of the most toxic materials.
To create a clean environment for all the people around the world, it is
necessary to mitigate waste problems left behind as legacies of dangerous past
activities, such as making nuclear weapons. When understood well, such as
with CCBATCH, in situ bioremediation has great promise to deal with one
of the most difficult challenges — groundwater and soil contamination by
radionuclides, such as plutonium.

1.14 USE OF BIOSENSORS TO DETECT ENVIRONMENTAL

POLLUTANTS
Biosensors are biophysical devices which can detect the presence of specific
substances e.g. sugars, proteins, hormones, pollutants and a variety of toxins
in the environment. They are also capable of measuring the quantities of these
specific substances in the environment.
Technically, a “Biosensor” is defined as “an analytical device containing an
immobilized biological material (which could be an enzyme, or antibody, or nucleic
acid, or hormone, or an organelle/whole cell), which can specifically interact with an
analyte and produce physical, chemical or electrical signals that can be measured”.
An analyte is the compound (e.g. glucose, urea, drug, pesticide) whose
concentration has to be measured.
Biosensors basically involve the quantitative analysis of various substances
by converting their biological actions into measurable signals. Generally the
performance of the biosensors is mostly dependent on the specificity and
sensitivity of the biological reaction, besides the stability of the enzyme.
A biosensor or an enzyme or an antibody is associated with microchip
devices which is used for quantitative estimation of the substance. A biosensor
equipment has the following components
• biological component—enzyme, cell, etc.
• a physical component—a device for measuring the quantity of
this product, thus indirectly giving an estimate of the substrate e.g.
transducer, amplifier etc.
The biosensors are being used in the area of medicine, industry, etc.
however, their use in environmental monitoring is of great benefit. Special kits
Introduction to Environmental Biotechnology 1.27

have been designed to identify the specific pollutants in the environment. E.g.,
special cost-effective enzymatic tests are available which can detect pesticide
contamination in water.

1.14.1 Principle of a biosensor

The biological material in use (e.g. an enzyme) is immobilized by conventional
methods like physical or membrane entrapment, non-covalent or covalent
binding. A contact is made between the immobilized biological material and
the transducer. The analyte binds to the biological material to form a bound
analyte which in turn produces the electronic response that can be measured.
Sometimes, the analyte is converted to a product which could be associated
with the release of heat, gas (oxygen), electrons or hydrogen ions. The
transducer then converts the product linked changes into electrical signals
which can be amplified and measured.
A good example of a biosensor in frequent use is the glucose oxidase
enzyme. The enzyme is immobilized on an electrode surface which acts as
an electrocatalyst for oxidation of glucose. The biosensor gives reproducible
electrical signal for glucose concentrations as low as 0.15 mM.
Another area where biosensors are being used is “Biomonitoring” or
“biological monitoring”. Biomonitoring is defined as the measurement and
assessment of work place agents or their metabolites either in tissues, secreta,
excreta, or any combination of these systems in occupationally exposed human
subjects. The “Biological effect monitoring” refers to the biological effects
of these toxic agents in the workers exposed to these agents. A continuous
evaluation of biological monitoring methods is done in order to assess the risk
effectiveness of these tests against the various kinds of exposures to toxins. The
use of genetic engineering to create organisms specifically designed for bio
remediation also has great potential. The bacterium Deinococcus radiodurans,
which is the most radioresistant organism known, has been modified to
consume and digest toluene and ionic mercury from highly radioactive
nuclear waste.
Some of the important biosensors used in environmental pollution
monitoring are:
(a)
Gas biosensors: In order to detect gases such as sulphur dioxide
(SO2), methane, carbon dioxide, etc., microbial biosensors have been
developed. Thiobacillus-based biosensors can detect the pollutant SO2,
whereas methane (CH4) can be detected by immobilized Methalomonas.
A particular strain of Pseudomonas is used to monitor carbon dioxide
levels.
(b)
Immunoassay biosensors: Immunoelectrodes as biosensors are used
to detect low concentrations of pollutants. Pesticide specific antibodies
can detect the presence of low concentrations of triazines, malathion
and carbamates, by using immunoassay methods.
1.28 Environmental Biotechnology

(c)
BOD biosensor: Biological oxygen demand (BOD) is widely used as a
test to detect the levels of organic pollution. This requires five days of
incubation but a BOD biosensor using the yeast Trichosporon cutaneum
with oxygen probe takes only 15 minutes to detect organic pollution.
Miscellaneous biosensors: A graphite electrode with Cynobacterium
(d)
and Synechococcus has been developed to measure the degree of electron
transport inhibition during photosynthesis due to certain pollutants
e.g. herbicides. To detect phenol, phenol oxidase enzyme obtained
from potatoes and mushrooms is used as a biosensor.
Biosensors for the detection of polychlorinated biphenyls (PCBs) and
chlorinated hydrocarbons and certain other organic compounds have also
been developed. Biosensors employing acetylcholine esterase which can be
obtained from bovine RBC can be used for the detection of organophosphorus
compounds in water.

1.14.2 Use of selected and engineered microbes for removal and

recovery of strategic and precious metals from contaminated
degraded lands
The domestic and industrial effluents often contain harmful heavy metals.
These heavy metals cause soil contamination when these effluents are used for
irrigation purposes. The biotechnological methods and procedures are being
developed to prevent the contamination by these heavy metals and also restore
the contaminated soils. This involves the selective use of engineered microbes.
Plasmids have been constructed which can enhance the recovery of gold from
arsenopyrite ores, by Thiobacillus ferroxidans. Ganoderma lucidum which is a
wood rotting macrofungus, is a highly potential biosorbent material for heavy
metals and thus can be used to control contamination by heavy metals.
The metal pollution occurs through several processes. As the living
organisms including man are constantly exposed to metals, they accumulate
by a process referred to as ‘bioaccumulation.’ The continuous exposure and
accumulation of a given metal leads to increase in it’s concentration which is
referred to as ‘biomagnification’. Biomagnification occurs through food chain
and the human gets the maximum impact due to it’s being on top of the food
chain. The ‘biomethylation’ is carried out by microorganisms in the soil and
water and involves the process of transfer of methyl groups from organic
compounds to metals.
Some phytoplanktons (plants that float freely on water surface) and some
benthics (plants attached to some substratum at the bottom of aquatic bodies)
microorganisms can take up the metals from the wastewater ponds. These
natural bioscavengers not only control the water pollution by absorbing metals
but also contributes in the recovery of industrially important metals from the
effluents. The microorganisms like algae can absorb metals form the fresh
water e.g. Chlorella vulgaris takes up copper, mercury, uranium. Certain fungal
Introduction to Environmental Biotechnology 1.29

species like Rhizopus, Aspergillus, Pencillium, Neurospora are good absorbers of

heavy metals like lead, mercury, etc. Some of the bacterial species are capable
of accumulating metals on cell walls such as E. coli can take up mercury while
Bacillus circulans can accumulate copper.
The mechanism of metal scavenging by these microorganisms is
very complex and involves multiple steps. Some of the microorganisms
bioaccumulate these metals on their cell walls whereas some others have
the capacity to transport these metals to intracellular and intercellular free
space and cellular organelles. In certain cases, some of the metals occur as
immobilized metal containing crystals e.g heavy metal complexes of calcium
oxalate crystals. Some of the fungal and algal species synthesize metal binding
proteins or peptides. ‘Phytochelatin’ is an ubiquitious metal chelating
protein present in all plants and acts as a common buffering molecule for the
homeostasis of metals. It is rich in cysteine and can form salt metal complexes
through sulfhydral (SH) groups. Due to this property, phytochelatin can be
used as a biomarker for metal pollution detection.
The mechanisms involved in the removal of metals by microorganisms
are: adsorption, complexation, precipitation and volatilization.
• The process of adsorption involves the binding of metal ions to the
negatively charged cell surfaces of microorganisms.
• The process of complexation leads to production of organic acids e.g.
citric acid, oxalic acid, gluconic acid, lactic acid, malic acid, etc. which
chelate the metal ions.
• In precipitation, the metals are precipitated as hydroxides or sulfates
by some bacteria such as which produce ammonia, organic bases or
H2S. e.g. Desulfovibrio and Desulfotomaculum transform SO4 to H2S
which promotes extracellular precipitation of insoluble metal sulfides.
Klebsiella aerogenes detoxifies cadmium sulphate which precipitates on
cell surface.
• Volatilization involves bacteria that cause methylation of Hg2+ and
convert it to dimethyl mercury which is a volatile compound.
Whole cell of Bacillus subtilis have been shown to reduce gold from Au3+
to Au° through extracellular enzymatic biotransformation. Under anoxic
environment, sulphate-reducing bacteria (Desulfovibrio) oxidize organic
matter using sulphate as an electron acceptor. In yeast, Saccharomyces cerevisiae,
removal of metals is done by their precipitation as sulphides e.g. Cu2+ is
precipitated as CuS.

1.15 BIOTECHNOLOGY AND THE SAVING OF RESOURCES AND

ENERGY
Breeding of insect and pest–resistant crop strains help promote a safe
environment, save money and conserve resources. Industrial processes are
1.30 Environmental Biotechnology

very complex and chemists make use of inorganic catalysts which speed
up the rate of reaction when making new chemicals. These catalysts often
need high temperatures, and acid or alkaline conditions, in order to work
efficiently. In future, genetically engineered organisms may be able to work
effectively at lower temperatures, and require less extreme conditions. This
will save money and resources, and will also produce fewer hazardous by-
products. For example, in paper-making, the wood pulp has to be treated with
chemicals which break up the fibres and remove the lignin (the substance that
makes up the wood). The pulp is bleached so that the finished paper is white.
This process produces a large volume of chemical waste that has to be treated
before it is ready for disposal. Enzymes have been discovered in fungi which
may be suitable for use as biological alternatives to some of these chemicals.
In the near future, using the modern plant breeding techniques, it may
be possible to breed trees which have less lignin, and so require fewer
chemicals and less energy to produce the pulp. Plastic is made from oil
and its manufacture uses a lot of energy and produces a lot of polluting by-
products. There is now hope that some forms of plastic will be made by living
organisms. One biodegradable plastic, called Biopol (trade name) is made by
bacteria. One way to make larger quantities of this plastic at lower cost might
be to insert the gene into potatoes. This would save energy, and reduce both
cost and pollution. As the supply of fossil fuels (oil, gas and coal) dwindles as
a result of the global financial crisis, genetically engineered organisms may
be manufactured to produce far more materials like plastics at less energy,
reduced cost and minimal environmental pollution.

1.16 FEARS AND CONCERNS ABOUT BIOTECHNOLOGY

APPROACH IN ACHIEVING A SAFE ENVIRONMENT AND
AGRICULTURE
There are some fears and concerns about biotechnology and safe environment
and agriculture. For instance, plant breeding, an agricultural biotechnology
approach has some concerns. Genetically engineered organisms are living
things and so are much less predictable than artificial materials and chemicals.
They can reproduce, move and even mutate. Developments in genetic
engineering take place in carefully controlled laboratory conditions. However,
once a new or modified organism has been developed, it is likely to be grown
outside and once released into the environment, it cannot be recalled. The
organism could change or interbreed with others, creating new species.
Geneticists should therefore be cautious and assess the possible risks involved
so that genetically engineered plants cause no more harm than the chemicals
they are replacing. It has been found that genetic alteration of plants to resist
viruses can stimulate the virus to mutate into a more virulent form, one that
might even attack other plant species. If the genes for insect- and weed killer
resistance, introduced into crop plants, find their way into weeds, this could
Introduction to Environmental Biotechnology 1.31

result in development of super-weeds, which would be impossible to kill

using traditional weed killers.
Some scientists think that genetically engineered plants and animals will
threaten the survival of other species and reduce diversity (the number of
different plant and animal species). The genetically engineered plants, which
may be more resistant to disease and pests, when grown around the world,
may cross to the wild species. The wild population would be contaminated
which could affect local habitats and the species that grow there. For example,
oilseed, rape, cross-breeds easily with wild relatives. This may mean that
genetically engineered oilseed rape would breed with related plants, and the
new gene for resistance to weed killer could spread into the wild population.
Some scientists predict that, within just one year, a large percentage of weeds
growing near the crop would have acquired this gene. There is no way of
stopping genetically engineered crops from breeding with wild plants.

1.17 FEARS AND CONCERNS ABOUT ENVIRONMENTAL IMPACT

OF BIOREMEDIATION
Bioremediation is the use of microorganisms or microbial processes to
detoxify and degrade environmental contaminants. Microorganisms have
been used for the routine treatment and transformation of waste products for
several decades. Although bioremediation represents a promising and largely
untapped environmental biotechnology, it has some disadvantages. Additives
added to enhance the functioning of one particular group of micro-organisms
during in situ bioremediation, may be disruptive to other organisms inhabiting
that same environment. Also, stimulated microbial population or genetically-
modified micro-organisms introduced into the environment after a certain
point of time may become difficult to remove. Bioremediation is generally very
costly and labour-intensive, and can take several months for the remediation
to reach acceptable levels.

1.18 ROLE OF BIOTECHNOLOGY IN RESTORATION OF DEGRADED

LANDS
The urbanization and increased human activity has led to degradation of
habitats. The restoration of the degraded lands can be carried out by using
biotechnology which involves the manipulations of biological systems. This
restoration could be carried out by the following biotechnological methods:

1.18.1 Use of micropropagation and Mycorrhiza for reforestation

One of the approaches to tackle this problem is to develop strong and superior
species which have the capability to grow well on degraded lands. This can
be done by using mass multiplication which involves starting aseptic culture,
multiplication of shoot using shoot apical meristems or buds, rooting of in vitro
1.32 Environmental Biotechnology

formed shoots, transfer, acclimatization and adaptation of micro propagated

plantlets in the field. Using this methodology, an estimated 500 million plants
of diverse nature have been produced.
Mycorrhizae, which are symbiotic non-pathogenic associations between
plant roots and fungi, improve the seedling survival and growth by enhancing
uptake of nutrients and water. They also lengthen the root life and provide
protection against the pathogens. A list of fungi which can efficiently form
mycorrhizae has been prepared. These fungi can be used as inocula which
are applied to roots of seedlings, to allow formation of mycorrhizae. The
experimental infection of micro propagated plants during rooting increases
their survival chances in the field, which is very important in case of plantations
on degraded lands.

1.18.2 Improvement of soil infertility through the use of nitrogen

fixing bacteria, Rhizobium in association with leguminous
trees and Frankia in association with non-leguminous
species
Biotechnological methods are being developed to help the non-leguminous
plants to survive under adverse conditions such as low nutrient supply. There
are about 160 species of angiosperms, which are known to form nitrogen
fixing root nodules with the Actinomycetes bacteria belonging to the genus
Frankia which is being used for this purpose. Frankia helps in nitrogen fixation
in non-leguminous plant species; therefore, it can be used for land reclamation
through reforestation due to high biomass production without the need of
expensive nitrogen fertilizers.

1.18.3 Development of plants tolerant to abiotic stress which can

be grown on degraded lands
The techniques like tissue culture and genetic engineering are being used to
develop plants resistant to abiotic stresses e.g. salinity, acidity, aluminium
toxicity, etc. The cell lines which exhibit resistance to salt stress are selected and
then used for plantation on degraded lands. E.g., Brassica spp., Citrus aurantium,
Nicotiana tabacum etc. Research is going on to understand the molecular basis
of salt tolerance and to isolate genes responsible for this attribute so that salt
tolerant plants can be developed using genetic engineering. In vitro selection
for tolerance to abiotic stress like aluminium toxicity has been successful in
certain plant species e.g. tomato, rice, barley, rice and wheat.
“Triticale”, which is a man made synthetic crop, has been found suitable
for growing on acid soils, dry and sandy soils, on alkaline and calcareous
soils and on mineral deficient and high-boron soils especially in countries
like Kenya, Ethiopia, Ecuador, Mexico, Brazil, etc. In China, a number of new
stress resistant varieties of rice, wheat and tobacco have been developed using
anther culture.
Introduction to Environmental Biotechnology 1.33

1.19 BIOMIMICRY AND BIOTECHNOLOGY

It is difficult to achieve a four-fold improvement in environmental performance
through incremental improvements in conventional production technologies.
Improvements of this magnitude usually call for a paradigm shift. For a
growing number of companies, the inspiration for such a paradigm shift is
coming from the products and processes found in natural ecosystems and the
organisms that live in them.
Biomimicry is the name coined for this approach in which industrial
production systems imitate nature.
Industrial biotechnology is that set of technologies that come from
adapting and modifying the biological organisms, processes, products, and
systems found in nature for the purpose of producing goods and services.
The organisms, processes, products and systems found in natural
ecosystems have evolved over millions of years to become highly efficient. For
example, all energy in natural ecosystems is renewable and is initially captured
from sunlight through photosynthesis. Also, all bio-organic chemicals and
materials are renewable, biodegradable and recycled. There is no such thing
as “waste”—the by-products of one organism are the nutrients for another.
Most, if not all, metabolic processes are catalyzed by enzymes and are highly
specific and efficient. Biotechnology has evolved over the last 25-30 years
into a set of powerful tools for developing and optimizing the efficiency of
bioprocesses and the specific characteristics of bio products. This increase in
efficiency and specificity has great potential for moving industry along the
path to sustainability.
Increased efficiency allows for greater use of renewable resources
without leading to their depletion, degradation of the environment and a
negative impact on quality of life. Biotechnology can become an important
tool for decoupling economic growth from degradation of the environment
and the quality of life. Biotechnology can also enable the design of processes
and products whose performance cannot be achieved using conventional
chemistry or petroleum as feedstock. Here are some examples of some of the
industrial efficiency tools now coming from the application of biotechnology:
• Enzymes extracted from naturally occurring micro-organisms, plants
and animals can be used biologically to catalyze chemical reactions with
high efficiency and specificity. Compared to conventional chemical
processes, bio-catalytic processes usually consume less energy, produce
less waste and use less organic solvents (that then require treatment
and disposal).
• By imitating natural selection and evolution, the performance of
naturally occurring enzymes can be improved. Enzymes can rapidly
be ‘evolved’ (this technique is called “molecular evolution”) through
mutation or genetic engineering and selected using high-throughput
1.34 Environmental Biotechnology

screening to catalyze specific chemical reactions and to optimize their

performance under certain conditions such as elevated temperature.
• The metabolic pathways of micro-organisms can also be modified by
genetic engineering. The aim is to turn each cell into a highly efficient
“mini reactor” that produces in one step and at high yield what would
take an organic chemist a number of steps with much lower yield (this
technique is called “metabolic engineering”).
• A further improvement on metabolic engineering involves engineering
the enzymes in the optimal configuration onto the cell membrane, and
when the cell is ruptured, the cell membrane becomes a bio-catalytic
surface that provides the high efficiency of metabolic engineering
without the energy penalty of keeping the organism alive.
• Plant biomass can be processed and converted by fermentation and
other processes into chemicals, fuels and materials that are renewable,
and result in no net emissions of greenhouse gases. Also, these
biologically derived products (“bio products”) are generally less toxic
and less persistent than their petrochemical counterparts.
• Groups of companies can mimic the co-operative action of organisms in
natural ecosystems by clustering around the processing of a feedstock
such as biomass, so the by-product of one is the starting material for
another. Also, energy, such as waste heat, can be used efficiently. This
approach is called “industrial ecology”.
• The ability to “evolve” bioprocesses and bio production systems
allows for major improvements in both economic and environmental
performance. This permits a manufacturing facility to increase its
profitability and capacity while maintaining or even reducing its
environmental footprint.

1.20 BIOTECHNOLOGY IN THE CONSERVATION OF BIODIVERSITY

The extinction of wild species due to the destruction of habitats and ecosystems
has raised serious concerns about the biodiversity in general. Biodiversity
provides genes from wild species for biotechnology exercises and experiments
hence, biotechnology and biodiversity are interrelated. Besides taking steps to
minimize and regulate the factors responsible for causing loss of biodiversity,
efforts are on to develop the techniques of conservation of biodiversity. One
of the methods involves the establishment of “gene banks” leading to “in situ
conservation” and “ex situ conservation”. The in situ conservation involves the
conservation of plants and animals in their natural habitat and ecosystems.
The ex situ conservation includes conservation of species away from their
habitats. The ex situ conservation uses sample populations and establishes
the “gene banks” which include resource centers, zoos, botanical gardens,
national parks, culture collection centers, etc.
Introduction to Environmental Biotechnology 1.35

Biotechnology offers special methods to conserve both animal and plant

genetic resources especially in the conservation of endangered plant species.
The tissue culture method is being used to multiply an endangered plant
species. The method of embryo transfer and artificial insemination is used for
the multiplication of endangered animal species.
The way these fears and concerns about application of biotechnology
to achieving a safe environment and agriculture are addressed will have a
remarkable impact on the future of biotechnology. A detailed analysis of both
the advantages and the disadvantages would assist in directing the future
of environmental and agricultural biotechnology, since the overall goal is to
achieve a safe environment and improved agricultural productivity.

1.21 BASIC TOOLS AND METHODOLOGIES ASSOCIATED WITH

ENVIRONMENTAL BIOTECHNOLOGY
It is understood that the scope of biotechnology is widening with every
passing day. This is largely because more and more intensive research in
this field has given birth to new ideas, innovations, and need of approaching
new philosophies and new allied fields to make the maximum use of
biotechnological methodologies and tools for the benefit of mankind. As far as
the use of biotechnology in the fields of genetics, breeding, agriculture, animal
husbandry, tissue culture and cloning is concerned, this is quite known and
common now. However, another exciting and comparatively newer field of
biotechnology, where it has shown an immense progress and productivity
over a very short period, is environmental biotechnology. Environmental
biotechnology, in fact, environmental biology, deals with the application of
biotechnological methodologies to make use of living organisms and living
systems for the remediation of environmental pollution ensuring a clean
and human-friendly environment. This means that on one hand, this area
of science cleans and repairs the damaged environment, and on the other, it
reduces the hazardous implications of technological processes and renders
them environmental friendly. Environmental biotechnological techniques
make use of microorganisms and plants, and thereby preserve energy. 
A conventional example of the use of environmental biotechnology
is the release of guppy fish (Poecilia reticulata) in ponds and lakes. Guppy
fish eats mosquitoes and its larvae. This reduces mosquito population in
the respective area and helps contain malaria, dengue and other diseases
which are dependent on mosquitoes for their spread. Another technique
makes use of microorganisms to extract heavy metals from low grade ores.
Chemoautotrophic bacteria derive energy from the breakdown of inorganic
chemicals, which in this case, are ores of heavy metals. The action of such
bacteria on these ores releases the metal itself. It is also an example of symbioses,
where the bacteria derive their energy from the breakdown of ore and release
useful heavy metals. This reiterates the fact that environmental biotechnology
1.36 Environmental Biotechnology

serves to preserve energy and resources. Copper, uranium, cobalt, lead, nickel
and gold can be extracted in this way. Thiobacillus ferrioxidans is used to extract
copper and uranium.
Some species of bacteria also accumulate metals. This property enables
them to be used in both, the extraction of metals, as well as detoxification of
waste. For example, some species of Pseudomonas accumulate mercury and
uranium, while those of Thiobacillus accumulate silver. 
Another useful implication of environmental biotechnology, and a major
one at that, is bioremediation. It is defined as the use of biological agents,
such as bacteria or plants, to remove or neutralize contaminants in polluted
soil or water. This technique usually breaks down the pollutants into smaller
harmless compounds such as water and carbon dioxide. However, depending
on the various types and nature of processes used, it can also be used to
produce methane and hydrogen, which are very valuable and highly worthy
fuels. This takes place under the especially maintained anaerobic conditions,
and therefore, bacteria that thrive best under such conditions, are selectively
used for the purpose. Bioremediation is used to clean oil spills and beaches;
treat sewage water and decontaminate soil, air and water. 
Apart from this, environmental biotechnology has helped to make paper
and plastic industries environmental friendly. It has introduced biological
organisms and microbes in place of compounds that are costly, consume a
lot of energy and cause pollution if they escape into the environment. For
example, when a lignin degrading and modifying enzyme isolated from a
fungus was used in pulp processes, it reduced energy costs, increased the life
of the system and reduced the risks associated with bleach. Likewise, in plastic
industry, glucose is replacing ethylene and propylene as raw material, and
microbes are being used to convert it into alkene oxides.
However, with its vast area of applications and so many advantages,
environmental biotechnology also poses some complications. Microorganisms
used in the aforementioned techniques are more often than not genetically
engineered to make the processes efficient. Such organisms being genetically
different form their natural contemporaries pose a threat to the balance of
the ecosystem. Thus, it can be concluded in a very fair and confident way
that environmental biotechnology has some very promising and extremely
production future concerns associated with the environmental remediation of
our planet. 

1.22 DEVELOPING ENVIRONMENTALLY SOUND BIOTECHNO-

LOGIES IN INDIA
India’s National Environmental Engineering Research Institute has developed
a number of environmentally sound biotechnologies – demonstrating that not
all advances take place in developed countries. They include the following:
Introduction to Environmental Biotechnology 1.37

• A chemo-biochemical technology for desulphurizing gaseous fuels

and emissions containing hydrogen sulphide, which also recovers
elemental sulphur. The process removes 99 per cent of the hydrogen
sulphide. The recovered sulphur, with a purity of up to 99.7 per cent,
can be used commercially.
• A technology for producing biosurfactants—active compounds derived
from biological sources which, like synthetic surfactants, exhibit
characteristic physical and chemical properties. Biosurfactants can be
used in situ to enhance oil recovery, in remediation of oil spills, and as
detergents.
• The bioremediation of mine spoil dumps, which involves excavating
pits on the eroded, stony dumps, filling them with bedding material
(organic waste and spoil), and planting selected saplings pretreated
with microbial cultures. The process reclaims spoil dumps, mined land
and wastelands within three to four years without using chemicals. The
degraded ecosystems recover fast, providing carbon dioxide sinks.
• A process using a microbial treatment which removes 85 per cent of
high pyritic sulphur and 89 per cent of ash from coal before it is burned,
leaving coal which is usable in thermal power plants, coal gasification
plants and for generating cleaner liquid fuels. The institute’s cost-
benefit analysis of these and other biotechnologies shows that the
initial investments, annual operating and maintenance costs, benefits
and investment returns are attractive to small-scale enterprises in
developing countries.
 CHAPTER

2 Environmental Microbiology—Soil

Microbes are everywhere in the biosphere, and their presence invariably affects
the environment that they are growing in. The many and varied metabolic
activities of microorganisms assure that they participate in chemical reactions
in almost every environment on earth. Microorganisms require an energy
producing system (including an electron acceptor) to sustain life and nutrients,
including liquid water, in order to grow and reproduce. Since microorganisms
have been present on earth longer than other organisms, they have evolved the
ability to thrive in almost any environment that meets these minimal criteria.
Energy comes from one of two sources, light (photosynthesis), or the oxidation
of reduced molecules. Oxidizable molecules may be organic (e.g. sugar,
protein or any of the other foods we humans relish) or a variety of inorganic
molecules such as sulfur, iron, hydrogen, carbon monoxide, or ammonia or
even a combination of organic/inorganic molecules. Microorganisms exist that
prosper inside eukaryotic cells, at temperatures >100oC, in the presence of
toxic metals like copper or mercury, at pHs ~2.0 and ~11.0, down to 3.5 km
below the earth’s surface and in saturated salt solutions at 0°C.
Microorganisms have broadened the environments they can live in by
evolving enzymes that allow them to utilize sunlight for energy as well as a
diversity of electron donor/acceptor pairs; so they can perform energy-yielding
oxidative reactions on available energy sources. That this evolution is ongoing
is shown by the isolation of microorganism that can metabolize numerous
man-made chemicals (ones not found in nature). The range of electron
acceptors includes gaseous oxygen, sulfate, nitrate, nitrite, carbon dioxide,
carbon monoxide, iron and magnesium. Indeed, evolutionary principles
predict that microbes should have evolved to utilize any niche meeting the
minimal physical and chemical requirements. Recently, bacteria that live
~3.5 km below the earth’s surface in rocks at high temperatures have been
discovered. Since these conditions cover the entire earth—even that portion
under the oceans—these bacterial forms may make up the largest single mass
of life on (or in) earth.
2.2 Environmental Biotechnology

Below is an abbreviated list of the roles microbes play in our lives:

• They maintain soil fertility and soil tilth.
• They clean up all the dead organic material; without them, we would
be up to our ears in dead things, like our ancestors.
• They fix gaseous nitrogen into forms that can be used by plants to
maintain the fertility of soils.
• They can be used to extract minerals from ores.
• They are the prime food for all the marine and freshwater life; even
whales depend on them directly or indirectly for their nutrition.

2.1 SOIL AND SOIL MICROORGANISMS

Soil is the outer, loose material of earth’s surface which is distinctly
different from the underlying bedrock and the region which supports
plant life. Agriculturally, soil is the region which supports the plant life by
providing mechanical support and nutrients required for growth. From the
microbiologist viewpoint, soil is one of the most dynamic sites of biological
interactions in the nature. It is the region where most of the physical, biological
and biochemical reactions related to decomposition of organic weathering of
parent rock take place. Living organismsboth plants and animals, constitute
an important component of soil. The pioneering investigations of a number of
early microbiologists showed for the first time that the soil was not an inert
static material but a medium pulsating with life. The soil is now believed to
be a dynamic or rather a living system, containing a dynamic population of
organisms/microorganisms. Cultivated soil has relatively more population
of microorganisms than the fallow land, and the soils rich in organic matter
contain much more population than sandy and eroded soils. Microbes in the
soil are important to us in maintaining soil fertility / productivity, cycling of
nutrient elements in the biosphere and sources of industrial products, such as
enzymes, antibiotics, vitamins, hormones, organic acids etc. At the same time,
certain soil microbes are the causal agents of human and plant diseases.

2.1.1 The characteristics of soil microorganisms

Soil inhabit diverse group of living organisms, both microflora and micro-
fauna
• Microflora:
— Bacteria
— Fungi, Molds, Yeast, Mushroom
— Actinomycetes, Streptomyces
— Algae, e.g. BGA, Yellow Green Algae, Golden Brown Algae
Bacteria is again classified into: (I) Heterotrophic e.g. symbiotic & non
- symbiotic N2 fixers, Ammonifiers, Cellulose Decomposers, Denitrifiers
(II) Autrotrophic e.g. Nitrosomonas, Nitrobacter, Sulphur oxidizers, etc.
Environmental Microbiology—Soil 2.3

• Microfauna: Protozoa, Nematodes

The density of living organisms in soil is very high i.e. as much as
billions/gm of soil usually density of organisms is less in cultivated soil than
uncultivated/virgin land and population decreases with soil acidity. Top
soil, the surface layer contains greater number of microorganisms because it
is well supplied with oxygen and nutrients. Lower layer/subsoil is depleted
with oxygen and nutrients, hence, it contains fewer organisms. Soil ecosystem
comprises of organisms which are both, autotrophs (algae, BOA) and
heterotrophs (fungi, bacteria). Autotrophs use inorganic carbon from CO2 and
are “primary producers” of organic matter, whereas heterotrophs use organic
carbon and are decomposers/consumers.

2.1.1.1 Viruses
• Viruses lead a strictly parasite existence - they reproduce within
bacteria, plants, animals and human cells.
• The most important kind of viruses in the soil environment are the
viruses living in bacterial cells, called bacteriophages (phages).
• The role of phages in the soil environment depends on their ability
to eliminate some populations of bacteria and on selecting the
microorganisms both in a negative and positive way. The example
of their negative influence are the phages that attack the root nodule
bacteria (Rhizobium) which are the cause of the decline of papilionaceous
plants crops.

2.1.1.2 Bacteria
• Bacteria constitute the basic mass of all soil microorganisms. They are
characterized by high metabolic activity.
• Most soil bacteria are characterized by the ability to adhere to surfaces
of the mineral molecules and to the soil colloids.
• Especially high numbers of bacteria gather around the residue of
plants’ and animals’ tissues as well as in animal droppings that finds
its way into the soil. The environment that is especially suitable for
the development of the bacteria are the plants’ roots and their other
underground parts.
• Soil bacteria can be subdivided into two groups: those that always
occur in each one of the soils’ type (autochthonous) and the ones that
grow only after high amount of the organic matter discharge into the
soil (zymogenous).
• The largest group of soil bacteria is represented by the Actinomycetes
and rod-coccus bacteria that belong to the Arthrobacter genus.
Rod-coccus bacteria: Club-shaped bacteria that belong to the Arthrobacter
genus are dominant in numbers representative of the autochthonous soil
2.4 Environmental Biotechnology

microflora. They make up 2-60% of the whole population of soil microflora

and are characterized by the tendency to form branching and coccus forms.
The bacteria are polymorphic. In new bacterial cultures, the bacteria grow in a
form of long irregular rods whereas in old cultures, they create coccus forms.
They are characterized by a high resistance to the environmental factors
during the vegetative stage. Also, they are capable of surviving in dry soil
for a few months, whereas most of the other bacteria, that do not produce
resting spores, die out. The bacteria have the ability to utilize a wide spectrum
of organic compounds as a food substrate. They conduct biodegradation of
not easily accessible compounds and may utilize many metabolites of other
microorganisms including various polymers, growth factors and the amino
acids produced by microorganisms. The bacteria which utilize the cellulose
(Cellulomonas) also belong to the club-shaped forms.
• Bacteria play an important role in the environment. Many dead materials
are decomposed by bacteria. If there were no bacteria, the environment
would have been polluted and full of harmful microorganisms. They
degrade the dead organic matter and convert it into energy and
nutrients. For example, they decompose trees and get their food from
them in the form of nutrition.
• Organic carbon present in the environment in the form dead organism
is able to eat up all the carbon dioxide from the atmosphere if there
were no decomposers in the on earth. It can be imagined that if
carbon dioxide gets reduced from the atmosphere, there would have
been no photosynthesis in plants and as a result no food would have
been produced by plants. Decomposers or bacteria help in cycling of
minerals like carbon and sulfur. They are also helpful in making, drugs,
antibodies and hormones.
• Nitrogen is the most important component of plants. Plants totally
rely on nitrogen for their health and growth and they cannot inhale
it directly from the environment. They are dependent on soil for the
supply of nitrogen. Through the process of nitrogen fixation, nitrogen
from the atmosphere becomes available to the plants. This process
takes place through bacteria, for example, Rhizobium and Cyanobacteria.
These species of bacteria convert the atmospheric nitrogen into nitrates
and nitrites which is the part of their metabolism and make it available
to the plants. Some plants have modified themselves so well that they
are now able to store the bacteria into their tissues.
• Using soil rich with beneficent bacteria can increase the productivity,
growth and health of the plants. Just as carbon dioxide is the useful
component of plants, similarly oxygen is also necessary for them
because plants need oxygen in the process of respiration. They take
oxygen by digesting the sugars present in them and use it for their
growth. They absorb oxygen from the roots and if the roots will be able
Environmental Microbiology—Soil 2.5

to absorb nutrients and energy from the soil only then they will be able
to provide large amount of oxygen to the plants. Those soils are not
fertile which do not have proper structure and organic matter. If these
components will be present in the soil, then plants will get plenty of
oxygen for respiration.

2.1.1.3 Actinomycetes
The Actinomycetes are (chemo) organotrophic bacteria. They form elongated,
branched out mycelium-like threads that contain a large number of prokaryotic
cells. The width of the threads is 1-5 ìm. They mainly live in soil or upon
decomposing plants. Most of them lead a saprophytic type of life, and some
are pathogenic to plants and animals (for example: Streptomyces somaliensis,
Actinomyces israelii and Nocardia asteroides cause the subcutaneous infections of
feet called mycetoma). Their growth abilities in temperatures of 40-50°C give
them a wide range of decomposition potential of various substances.
• The Actinomycetes degrade steroids, lignin, chitin, hydrocarbons, fatty
and humic acids, which are not easily decomposed by other bacteria.
• During the decomposition of the above, they produce aromatic
compounds.
• The characteristic smell of freshly ploughed soil, especially in spring,
comes from the actinomycetes bacteria. The smell is caused by the
substance called geosmin (1,10- dimetylo-9-dekalol), which is produced
by Streptomyces griseus. They are the aerobic bacteria, whereas a small
group has the ability to conduct the metabolic processes in anaerobic
conditions (Eg. Actinomyces, Micromonospora).
• Many types of actinomycetes produce antibiotics such as erythromycin,
neomycin, streptomycin, tetracycline, plus others, as the by-product
of metabolism. About 90% of all actinomycetes isolated from soil are
Streptomyces.

2.1.1.4 Fungi
• Fungi belong to a group of eucaryotic organisms which are the absolute
heterotrophs. Most of them belong to the group of aerobes or fermenting
fungi. They take the carbon and energy to build their own cells from
the decomposition of the organic compounds. Fungi do not have any
chlorophyl. In contrast to bacteria, the fungal cell wall contains chitin,
glucans and other polysaccharides.
• They occur mostly in the upper layers of soil, however, they can be
found as deep as 1 m.
• They get into symbiotic relationships with algae, insects and higher
plants. Many species of fungi are pathogenic to humans, plants and
animals.
2.6 Environmental Biotechnology

• Their vegetative forms create thread-like shreds that are more or less
branched out and usually multi-cellular. Their thick weaves form
mycelium or thallus. The individual cells are the size of about 10ìm.
The most common soil fungi are the genera of Penicillium, Aspergillus,
Trichoderma, Verticillium, Fusarium, Rhizopus, Mucor, Zygorhynchus,
Chaetomium. Fungi grow strongly in acidic soils and have crucial
influence on changing of pH reaction.
Role of Fungi in environment:
• Fungi plays significant role in soils and plant nutrition.
• They plays important role in the degradation/decomposition of
cellulose, hemicellulose, starch, pectin, lignin in the organic matter
added to the soil.
• Lignin which is resistant to decomposition by bacteria is mainly
decomposed by fungi.
• They also serve as food for bacteria.
• Certain fungi belonging to sub-division Zygomycotina and
Deuteromycotina are predaceous in nature and attack on protozoa &
nematodes in soil and thus, maintain biological equilibrium in soil.
• They also plays important role in soil aggregation and in the formation
of humus.
• Some soil fungi are parasitic and cause number of plant diseases
such as wilts, root rots, damping-off and seedling blights eg. Pythium,
Phyiophlhora, Fusarium, Verticillium, etc.
• Number of soil fungi form mycorrhizal association with the roots of
higher plants (symbiotic association of a fungus with the roots of a
higher plant) and help in mobilization of soil phosphorus and nitrogen
eg. Glomus, Gigaspora, Aculospora (Endomycorrhiza) and Amanita,
Boletus, Entoloma, Lactarius (Ectomycorrhiza).

2.1.1.5 Soil phytoedaphon

Phytoedaphon consists mainly of algae, and to a lesser extent, the higher
plants. Algae are the main component of phytoedaphon. They are most
numerous upon the surface of soil reaching deeper through ploughing,
percolating water, animal activities and the ability to migrate. Two groups are
distinguishable: algae colonies that live upon the surface-epiphytoedaphon
and the ones that live in deeper layers-endophytoedaphon.
• Soil algae are obligatory photoautotrophs, however, the ones living in
deeper layers probably feed heterotrophically.
• They play a major role in soil’s ecosystem and influence its qualities
and stability. Through extracellular secretion, they fertilize the soil and
take part in nutrient discharge into the environment.
Environmental Microbiology—Soil 2.7

• Some blue-green algae (Cyanobacteria) are capable of fixing the

atmospheric nitrogen (Nostoc, Anabaena, Scytonema, Tylypothrix).
Soil inhabited by these microorganisms contains 26-400 times more
nitrogen. Due to the ability of nitrogen (N2) and carbon dioxide (CO2)
assimilation, they may be the first ones to colonize the nitrogen and
organic carbon-free ground.
• About two thousand species of algae occur in soil. They are mainly:
— Blue-green algae: Nostoc, Anabaena, Scytonema, Tolypothrix,
Microcoleus, Schizothrix
— Green algae: Ankistrodesmus, Chlorella, Chlorococcum, Chlamydomonas,
Characium, Klebsormidium
— Diatoms: Achnanthes, Cymbella, Eunotia, Fragilaria, Hantzschia,
Navicula, Nitzschia, Pinnularia
— Yellow-green algae: Botrydiopsis, Heterothrix, Heterococcus,
Pleurochloris
— Euglenoids: Euglena, Peranema
— Red algae: Porphyridium
• Among the macrobiotic plant organisms that inhabit the soil
environment, higher plants dominate making up the basic element of
biocenoses of all the land ecosystems.
Algae plays important role in the maintenance of soil fertility especially
in tropical soils.
They add organic matter to soil when die and thus increase the amount
of organic carbon in soil.
Most of soil algae (especially BGA) act as cementing agent in binding
soil particles and thereby reduce/prevent soil erosion.
Mucilage secreted by the BGA is hygroscopic in nature and thus helps
in increasing water retention capacity of soil for longer time/period.
Soil algae, through the process of photosynthesis, liberate large quantity
of oxygen in the soil environment and thus facilitate the aeration in
submerged soils or oxygenate the soil environment.
They help in checking the loss of nitrates through leaching and drainage
especially in uncropped soils.
They help in weathering of rocks and building up of soil structure.

2.1.1.6 Fauna of soil

The soil microfauna is represented by the protozoans, which mainly feed on
bacteria. Their role is to conduct selection and rejuvenate the population of
soil bacteria. Amoebae and flagellates dominate among them. Mesofauna is
represented by the nematodes (eelworms), snails, insects, myriapods, mites
2.8 Environmental Biotechnology

and others. They feed upon dead organic matter contributing to the formation
of humus.
Protozoa: These are unicellular, eukaryotic, colourless, and animal like
organisms (Animal kingdom). They are larger than bacteria and size varies
from few microns to a few centimeters. Their population in arable soil ranges
from l0,000 to 1,00,000 per gram of soil and are abundant in surface soil. They
can withstand adverse soil conditions as they are characterized by “cyst stage”
in their life cycle. Except few genera which reproduce sexually by fusion of
cells, rest of them reproduce asexually by fission/binary fission. Most of the
soil protozoa are motile by flagella or cilia or pseudopodia as locomotors
organs. Depending upon the type of appendages provided for locomotion,
protozoa are:
• Rhizopoda (Sarcondia)
• Mastigophora
• Ciliophora (Ciliata)
• Sporophora (not common Inhabitants of soil)
• Class - Rhizopoda: Consists protozoa without appendages; usually
have naked protoplasm without cell; wall, pseudopodia as temporary
locomotory organs are present some times. Important genera are
Amoeba, Biomyxa, Euglypha, etc.
• Class - Mastigophora: Comprises flagellated protozoa, which are
predominant in soil. Important genera are: Allention, Bodo, Cercobodo,
Cercomonas, Entosiphon Spiromonas, Spongomions and Testramitus. Many
members are saprophytic and some possess chlorophyll and are
autotrophic in nature. In this respect, they resemble unicellular algae,
and hence, are known as “Phytoflagellates”.
• The soil protozoa belonging to the class ciliate/ciliophora are
characterized by the presence of cilia (short hair-like appendages)
around their body, which helps in locomotion. The important soil
inhabitants of this class are Colpidium, Colpoda, Balantiophorus,
Gastrostyla, Halteria, Uroleptus, Vortiicella, Pleurotricha etc.
Protozoa are abundant in the upper layer (15 cm) of soil. Soil moisture,
aeration, temperature and pH are the important factors affecting soil protozoa.
• Most of protozoans derive their nutrition by feeding upon or ingesting
soil bacteria belonging to the genera Enterobacter, Agrobacterium,
Bacillus, Escherichia, Micrococcus, and Pseudomonas and thus, they play
important role in maintaining microbial/bacterial equilibrium in the
soil.
• Some protozoa have been recently used as biological control agents
against phytopathogens.
• Species of the bacterial genera viz. Enterobacter and Aerobacter are
commonly used as the food base for isolation and enumeration of soil
protozoans.
Environmental Microbiology—Soil 2.9

• Several soil protozoa cause diseases in human beings which are carried
through water and other vectors, eg. Amoebic dysentery is caused by
Entomobea histolytica.
Macrofauna is represented by the earthworms, moles, rodents. The
organisms break up soil material and carry it down to a significant depth. The
earthworms play the most important role among the invertebrates, by feeding
upon dead organic matter and absorbing it along with the mineral part of the
soil; non-digested residue mixed with mineral soil and the metabolites are
excreted in the form of lumps (coprolites) that contributes to the formation
of crumb texture of soil and to its loosening. During the period of one year,
earthworms, on the area of one hectare, are able to pass 7 thousand kg of soil
through their digestive tracts.

2.1.2 The Number of Soil Microorganisms

The number and composition of soil microorganisms depends on the type of
soil, its structure, humidity and on the content of the organic matter.

2.1.2.1 Viruses
The exact number of viruses in soil is not known. Their mass is estimated
at less than 0.01 tons/ha.

2.1.2.2 Bacteria
• The number of bacteria varies from a couple of million to a couple of
billion cells per each 1g of soil. The highest number of bacteria occurs
in a layer of cultivable soil at the depth of up to about 30 cm. In deeper
layers their numbers quickly lower. In the cultivable layer of soil of
about 30 cm thick, there may be anywhere from several hundred kg up
to a few tons of bacterial mass per each 1 hectare.
• In the vicinity of roots and upon their surface, the bacteria find increased
amounts of organic compounds, such as organic acids, amino acids and
vitamins that are excreted by plants. Therefore, in the layer around
roots, called rhizosphere, the number of bacteria is several times higher
than in soil far from the roots.
• In soils rich in organic compounds, there live more bacteria; usually in
1 g of cultivable soil there may be between 0.5-5.0 billion bacteria (1.5-
15 tons/ha).
• Acidic soils contain relatively low number of bacteria and a large
number of fungi.

2.1.2.3 Fungi
• Fungi, excluding yeast, occur in the forms of mycelia or spores. The
number of such units per 1 gram of soil may reach several tens of
2.10 Environmental Biotechnology

thousand; among that spores constitute anywhere from several to a

few dozen per cent depending on the soil’s humidity and the organic
substances available.
• Fungi are most widely represented in acidic peat and forest soils. There,
they may be more numerous than the bacteria.
• The main mass of fungi is found in the upper 20-30 cm layer. The
combined mass of fungi in the upper layers is almost identical to that
of bacteria, and in forest soils, it may even be greater. On an average, it
is between 0.001-1.0 billion fungi (about 1.5 tons/ha).

2.1.2.4 Algae
Algae live mainly in the upper layers of soil anywhere between 0-10 cm where
the sunlight penetrates (rarely below 50 cm). Their number may vary between
100 thousand to 3 million per 1 g of soil (0.2 tons/ha). In favourable conditions,
for instance in highly irrigated tropical soils, the numbers may be increased.

2.1.2.5 Soil fauna

Protozoa can widely develop in adequately humid soils. Their numbers reach
anywhere from a few hundred to several million per 1 g of dry soil. The mass
of protozoa in soil is between 0.1-0.5 tons/ha, whereas the mass of nematodes
is between 0-0.2 tons/ha, earthworms 0-2.5 tons/ha, and other soil animals
0-0.5 tons/ha.

2.1.3 Activity of Microorganisms in soil

Microorganisms reproduce and transform the organic matter creating
biomass of their own cells and collect substrates essential for replenishing the
supplies of humus. Additionally, they decompose and mineralize the organic
compounds, consequently recirculating the indispensable elements in plant
production based on the assimilation of CO2 from the atmosphere.
• Carbon makes up 50% of mass of the organic matter that gets into
soil in the form of plant and animal residues (falling leaves, various
remains in meadows and forests, animal corpses, roots and shoots of
dead plants).
• The fresh organic matter is composed of monosaccharides (hexose,
pentose), polysaccharides (starch, cellulose, hemicellulose, chitin),
organic acids, aromatic compounds (lignin, phenols, tannin),
hydrophobic compounds (wax, cutin, fat and others).
• Carbon is recovered during the organic compounds decomposition
and mineralization processes.
• Depending on its chemical nature, particular components of plants’
mass are decomposed and mineralized at various speeds.
Environmental Microbiology—Soil 2.11

• Soluble substances such as sugars, amino acids, organic acids are easily
washed away with water from plant and animal residues and then are
quickly metabolized by soil microorganisms; they especially regulate
the microbiological activities in the rhizosphere.
• Waxes, fats, rubbers and tannin are decomposed with great difficulty
due to their high hydrophobic properties. Lignin is the most resistant
substance to decompose among the plant materials.

2.1.3.1 Cellulose decomposition

• Cellulose occurs commonly in the walls of plant cells and is associated
with hemicellulose and lignin. In the dry mass of green plants, the
content of cellulose is at 15-30%, whereas in lignified parts and straw,
it can reach 50%.
• Cellulose is a polysaccharide that consists of a long unbranched chain of
glucose units.
• Cellulolitic bacteria belong mainly to the genera: Cytophaga, Cellfalcicula,
Cellulomonas and Cellvibro.
• The best known cellulolytic system occurs in fungi. The Trichoderma
genus releases the most active cellulase enzymes into the environment,
then the enzymatic attack occurs away from the cells. Other fungi like
Chaetomium, Fusarium and others also take active part in the above
process.
• Decomposition may also occur in oxygen-free conditions; it’s conducted
by the genera: Acetovibrio, Bacteroides, Clostridium, Ruminococcus.
Consequently, a large number of gaseous substances, such as CO2, H2,
CH4 are created.
• Decomposition of cellulose occurs faster in soils of neutral or slightly
acidic pH and is slowed down in highly acidic soils.
• Microorganisms that decompose cellulose change it into simpler
compounds and this way they create a nutrient base for all the soil
heterotrophs.

2.1.3.2 Lignin decomposition

Lignin belongs to a large group of aromatic compounds and, apart from
cellulose, is a main component of wood tissues (up to 30% of plant biomass).
• Lignin is a polymer built of phenylopropane units which contain an
aromatic ring and the methoxyl groups - OCH3.
• The most active lignin-degrading organisms are the fungi that cause, so
called, white rot of wood. They decompose wood to CO2 and H2O. They
belong to basidiomycete and ascomycyte groups and are represented
by several hundred species. Among the Basidiomycetes, the best
2.12 Environmental Biotechnology

known ones are: Trametes versicolor, Phanerochaete chrysosporium, oyster

mushroom Pleurotus ostreatus—edible mushroom and Lintunula edodes.
Among the Ascomycetes, the ones involved are: Xylaria, Libertella and
Hypoxylon. Among the mold fungi, Trichoderma lignorum demonstrates
the ability to decompose lignin.
• The enzymatic complex composition that decomposes lignin is,
among others, represented by oxidoreductases which require oxygen
or hydrogen peroxide H2O2 for oxidative tearing up of bonds that
connect phenylpropane subunits of lignin to each other. In direct
decomposition of lignin, the following takes part: laccase (may oxidize
the monophenoles), lignin peroxidase and other enzymes that haven’t
been elucidated.
• The activity of microorganisms that decompose lignin in soil stimulates
the production of humus.

2.1.3.3 Synthesis and humus decomposition

Humus is an amorphous organic substance, usually dark, that makes up the
colloidal system of a large surface area capable of adsorbing ions of water and
gases.
• It contains fractions of organic substances which have a low ratio of
C:N (from 10 to 15), whereas the ratio of these elements in dead plants’
residue is at about C:N=40:1.
• Fulvic, humic acids and humins make up the composition of humus.
These are the conglomerates of more or less carbonized compounds,
which are characterized by the presence of carboxyl, phenyl and
methoxyl groups that contain C, O, N, P and S, as well as the aromatic
skeleton with numerous side chains.
• The main humus forming system is the activity of soil microorganisms:
bacteria (including actinomycetes) and fungi.

2.1.3.3.1 The synthesis of humus (Humification)

• The process of humus formation is called humification.
• The main substrates from which humus compounds are formed are
lignin, hydrocarbons and nitrogen compounds. However, the soil type
and the climate conditions determine the kind of humus.
• Microorganisms conduct the following processes connected to the
formation of humus:
— they decompose fresh organic matter producing metabolites - the
precursors of humus compounds.
— they create biomass, which after atrophy and autolysis make up the
additional initial substrates needed for the formation of humus.
— they catalyze the processes of humus synthesis.
Environmental Microbiology—Soil 2.13

The basic way of humus compounds formation is through its synthesis

from the fragments such as polyphenols with the participation of nitrogen
components of protein origin. The source of polyphenols may be the
processes of lignin decomposition, hydrocarbon transformation and various
microbiological synthesis processes. Many polyphenols form as the metabolites
of different microorganisms. The next stage of humification is the oxidation
of polyphenols that leads to the formation of chinoid compounds. These
transformations are catalyzed by the phenol oxidases produced by different
microorganisms such as by the fungus Serpula lacrymans. The final phase of
the process is the polymerization of oxidized phenols. Transformation of the
organic matter in conditions when oxygen is available leads to the formation
of humus and in oxygen-free conditions to peat deposit formation.

2.1.3.3.2 Decomposition of humus

• Degradation of humus occurs in conditions when there is a shortage
of fresh organic matter and when there is not an adequate supply of
nitrogen in soil.
• It is believed that the decomposition is caused by the autochthonous
bacteria, which are adapted to the shortages of the available organic
substances and they are consequently utilizing components contained
in humus complexes. Particularly high degradation activity is

The role of microorganisms in nitrogen processes in soil—the nitrogen cycle

2.14 Environmental Biotechnology

demonstrated by the actinomycetes and some other bacteria such

as Micrococcus, Corynebacterium and some white rot fungi such as
Polysticus.
• It may be assumed, that both in humus formation and during its
decomposition, the entire system of soil microflora and microfauna
play a collective role.
• Due to the microbiological processes nitrogen from the atmosphere is
being incorporated into the compounds of the organic cells (so called
nitrogen fixation)
• The organic compounds contained in animal and plant residues are
mineralized by microorganisms and then are incorporated into the
nitrogen cycle. In this way, the free nitrogen level in the atmosphere
remains stable (78%).
• The Nitrogen cycle in the environment is composed of several links
such as:
— symbiotic and non-symbiotic fixation of atmospheric N2 by
microorganisms.
— microbial decomposition of the organic nitrogen compounds,
ammonification – the release of NH3 and of NH4+ions the, utilization
of NH4+ ions for the resynthesis of proteins by microorganisms the
utilization of NH4+ ions as ammonium salts by plants.
— the nitrification of NH4+ ions , nitrates are created through nitrites.
— the utilization of nitrates by higher plants as well as by some of the
microorganisms (transformation of nitrogen into protein).
— denitrification.

2.1.3.4 Atmospheric nitrogen fixation

The assimilators are capable of fixing of nitrogen only in symbiosis with plants.
• Rhizobium bacteria, living in symbiosis with papilionaceous plants,
supply soil with the most nitrogen. They get into the root system of
the plant where they multiply forming the long bacteria threads which
penetrate plant tissue. The overgrowth of the plant tissue stimulated by
bacteria causes the growth of nodules that form specific units upon the
roots. Inside the nodules, a part of plant-infecting bacteria transform
into bacteroids, which do not reproduce but are continuously active.
• It is believed, that the bacteroids take active part in the process of the
atmospheric nitrogen fixation. The bacteroids contain a red dye called
leghemoglobin. It is called that way because of the similarities to the
hemoglobin. The iron Fe3+ contained in the dye is reduced to Fe2+, thus
it is believed, that the leghemoglobin mediates the transfer of electrons
to free nitrogen, hence causing its reduction.
Environmental Microbiology—Soil 2.15

• Inside the plant roots; for instance, of black alder root, nodules contain
interacting actinomycetes (Streptomyces alnii).
• Nitrogen fixation by the free living bacteria is similar. The reduction of
N2 to NH3 is performed by pyruvate dehydrogenase and nitrogenase
enzymes.

2.1.3.5 Free-living N2 assimilators (non-symbiotic nitrogen fixation)

The following heterotrophic bacteria posses the ability to fix free nitrogen
from the air and to enrich the soil with nitrogen:
• aerobes — Azotobacter, Azotomonas, Derxia, Achromobacter, Beijerinckia.
• microaerophiles — Pseudomonas, Flavobacterium, Mycobacterium,
Arthrobacter and Aerobacter.
• anaerobes — some species of Clostridium such as: Cl. butyricum, Cl.
pectinovorum.
Within the group of autotrophs, the above capabilities are demonstrated
by the photosynthesizing bacteria: Chlorobium, Chromatium and cyanobacteria
e.g. Anabaena, Nostoc.

2.1.3.6 Ammonification
Ammonification is a process of the ammonium ion NH4+ or of the free
ammonia formation. During the first stages of this process, a break down of
protein and a liberation of the amino acids occurs. Next, deamination of the
amino acids takes place. The proteolytic break down of proteins occurs with a
participation of the exocellular enzymes. Formed amino acids are transported
to microorganisms’ cells where the process of deamination takes place.
Ammonia, being a gas, quickly spreads in dry soils, whereas in humid ones, it
dissolves in water forming NH4+. Formed ammonium ions are utilized by the
bacteria and plants for the synthesis of amino acids or undergo the process of
nitrification.

2.1.3.7 Nitrification
Nitrification is a biological process of oxidation of ammonia to nitrate
accomplished by nitrificating bacteria (chemolithotrophs). The energy
released during this process is utilized by bacteria in the synthesis of organic
compounds. The nitrification proceeds in two stages:
1. First, the ammonium is oxidized to nitrite. Bacteria oxidizing NH4
to NO2 are described as the “nitroso”: Nitrosomonas, Nitrosospira,
Nitrosocyjastis, Nitrosoglea
2. Second, the formed nitrite is oxidized to form nitrate. Nitrites are
oxidized into nitrates by the group of bacteria called “nitro”, such as
genera Nitrobacter, Nitrospira, Nitrococcus
2.16 Environmental Biotechnology

The nitrifying bacteria are sensitive to the acidification of the environment;

slowing down of their growth occurs at pH 5.0. The nitrification process
may be also conducted by the heterotrophic microorganisms. The biggest
group that conductss the heterotrophic nitrification are fungi: Aspergillus
flavus, Penicillium, Cephalosporium. The nitrification conducted by fungi is less
sensitive to acidification and more resistant to drought. Formed nitrates in soil
can be assimilated by the plants, flushed out by water, or decomposed in the
process of denitrification.

2.1.3.8 Denitrification
Denitrification is the process of nitrate reduction to form molecular nitrogen.
The above process is conducted mainly in oxygen-free conditions, and it
is when the nitrates are utilized for respiration as the terminal electron
acceptors. Several kinds of heterotrophic bacteria belonging to Pseudomonas,
Achromobacter, Bacillus, Micrococcus genera are involved in the process of
denitrification. The reduction of nitrates occurs in a few stages. During the
first stage the nitrates are reduced to nitrites (NO2-), then the nitrites are
reduced to nitric oxides (NO, N2O) and down to molecular nitrogen. The
process of denitrification is also conducted by some chemoautotrophic
bacteria such as Thiobacillus denitrificans. The above bacteria obtain the energy
from the oxidation of sulfur compounds to simultaneously reduce nitrates.
Denitrification is believed to be a disadvantageous process since it leads to
the deprivation of vital nitrogen compounds from plants. The loss of nitrogen
from soil due to the denitrification increases with excess soil moistening,
oxygen-free conditions, accumulation of nitrates and temperature increase.

2.1.4 INTERACTIONS AMONG SOIL MICROORGANISMS

Soil is the largest terrestrial ecosystem where a wide variety of relationships
exists between different types of soil organisms. The associations existing
between different soil microorganisms, whether of a symbiotic or antagonistic
nature, influence the activities of microorganisms in the soil. Microflora
composition of any habitat is governed by the biological equilibrium created
by the associations and interactions of all individuals found in the community.
In soil, many microorganisms live in close proximity and interact among
themselves in a different ways. Some of the interactions or associations are
mutually beneficial, or mutually detrimental, or neutral. The various types of
possible interactions/associations occurring among the microorganisms in soil
can be:
(a) beneficial—(i) mutualism, (ii) commensalism and (iii) proto-cooperation
or
(b) detrimental/harmful—(i) armensalism, (ii) antagonism, (iii) competition
(iv) parasitism and (v) predation
Environmental Microbiology—Soil 2.17

2.1.4.1 Beneficial Associations/Interactions

2.1.4.1.1 Mutualism (Symbiosis)
It is a relationship or a type of symbiosis in which both the interacting
organisms/partners are benefited from each other. The way/manner in which
benefit is derived depends on the type of interactions. When the benefit is
in the term of exchange of nutrients, then the relationship is termed as
“syntrophism” (Greek meaning: Syn-mutual and trophe = nourishment), for
example, Lichen (association of algae or BGA with fungus) in which algae
benefits by protection afforded to it by the fungal hyphae from environmental
stresses, while the fungus obtain and use CO2 released by the algae during
photosynthesis. Where the blue green algae are the partners in the lichen
association, the heterotrophs (Fungus), benefit from the fixed nitrogen by the
blue green algae.
Microorganisms may also form mutualistic relationships with plants,
for example, nitrogen-fixing bacteria i.e. Rhizobium growing in the roots of
legumes. In this Rhizobium-legume association, Rhizobium bacteria are
benefited by protection from the environmental stresses while, in turn, plant is
benefited by getting readily available nitrate nitrogen released by the bacterial
partner.
The Anabaena-Azolla is an association between the water fern Azolla and
the cyanobacterium Anabaena. This association is of great importance in
paddy fields, where nitrogen is frequently a limiting nutrient.
An actinorrhizal symbiosis of actinomycetes, Frankia, with the roots of
Alnus and Casurina (non-legumes) is common in temperate forest ecosystem
for soil nitrogen economy. Another type of symbiotic association which exists
between the roots of higher plants and fungus is Mycorrhiza. In this association
fungus gets essential organic nutrients and protection from roots of the plants
and allows them to multiply and, in turn, plants uptake phosphorus, nitrogen
and other inorganic nutrients made available by the fungus.

2.1.4.1.2 Commensalisms
In this association, one organism/partner in association is benefited by other
partner without affecting it. For example, many fungi can degrade cellulose
to glucose, which is utilized by many bacteria. Lignin is major constituent
of woody plants and is usually resistant to degradation by most of the
microorganisms but in forest soils, lignin is readily degraded by a group of
Basidiomycetous fungi and the degraded products are used by several other
fungi and bacteria which can not utilize lignin directly. This type of association
is also found in organic matter decomposition process.

2.1.4.1.3 Proto-cooperation
It is mutually beneficial association between two species/partners. Unlike
symbiosis, proto-cooperation is not obligatory for their existence or
performance of a particular activity. In this type of association, one organism
2.18 Environmental Biotechnology

favor’s its associate by removing toxic substances from the habitat and
simultaneously obtain carbon products made by the another associate/
partner. Nutritional proto-cooperation between bacteria and fungi has been
reported for various vitamins, amino and purines in terrestrial ecosystem and
are very useful in agriculture. Proto-cooperative associations found beneficial
in agriculture are : i) synergism between VAM fungus-legume plants and
Rhizobium in which nitrogen fixation and phosphorus availability/uptake is
much higher resulting in higher crop yields and improved soil fertility, ii)
synergism between PSM-legume plants and Rhizobium and iii) synergism
between plant roots and PGPR in rhizosphere, where rhizobacteria restrict
the growth of phytopathogens on plant roots and secretes growth promoting
substances.

2.1.4.2 Detrimental (Harmful) Associations/Interactions

2.1.4.2.1 Antagonism
It is the relationship in which one species of an organism is inhibited or
adversely affected by another species in the same environment. In such
antagonism, one organism may directly or indirectly inhibit the activities
of the other. Antagonistic relations are most common in nature and are also
important for the production of antibiotics. The phenomenon of antagonism
may be categorized into three i.e. antibiosis, competition and exploitation.
In the process of antibiosis, the antibiotics or metabolites produced by one
organism inhibits another organism. An antibiotic is a microbial inhibitor of
biological origin. Innumerable examples of antibiosis are found in soil. For
example, Bacillus species from soil produces an antifungal agent which inhibits
growth of several soil fungi. Several species of Streptomyces from soil produce
antibacterial and antifungal antibiotics. Most of the commercial antibiotics
such as streptomycin, chloramphenicol, Terramycin and cyclohexamide
have been produced from the mass culture of Streptomyces. Thus, species of
Streptomyces are the largest group of antibiotic producer’s in soil. Another
example of antibiosis is inhibition of Verticillium by Trichoderma, inhibition of
Rhizoctonia by a bacterium Bacillus subtilis, inhibition of soil fungus Aspergillus
terreus by a bacterium Staphylococcus aureus.

2.1.4.2.2 Ammensalism
In this interaction/association, one partner suppresses the growth of other
partner by producing toxins like antibiotics and harmful gases like ethylene,
HCN, Nitrite, etc.

2.1.4.2.3 Ammensalism Competition

As soil is inhabited by many different species of microorganisms, there exists an
active competition among them for available nutrients and space. The limiting
substrate may result in favoring one species over another. Thus, competition
Environmental Microbiology—Soil 2.19

can be defined as “the injurious effect of one organism on another because

of the removal of some resource of the environment”. This phenomenon can
result in major fluctuations in the composition of the microbial population in
the soil.
For example, chlamydospores of Fusarium, oospores of Aphanomyces and
conidia of Verticillium dahlae require exogenous nutrients to germinate in soil.
But other fungi and soil bacteria deplete these critical nutrients required for
spore germination and thereby hinder the spore germination resulting into
the decrease in population. Competition for free space has been reported to
suppress the fungal population by soil bacteria. Therefore, organisms with
inherent ability to grow fast are better competitors.

2.1.4.2.4 Parasitism
It is an association in which one organism lives in or on the body of another.
The parasite is dependent upon the host and lives in intimate physical contact
and forms metabolic association with the host. So, this is a host-parasite
relationship in which one (parasite) is benefited while the other (host) is
adversely affected, although not necessarily killed. Parasitism is widely
spread in soil communities, for example, bacteriophages (viruses which attack
bacteria) are stricte intracellular parasites Chytrid fungi, which parasitize
algae, as well as other fungi and plants. There are many strains of fungi
which are parasitic on algae, plants, animals parasitized by different organism
earthworms are parasitized by fungi, bacteria, viruses etc.

2.1.4.2.5 Predation
Predation is an association/exploitation in which the predator organism
directly feeds upon and kills the prey organism. It is one of the most
dramatic interrelationship among microorganisms in nature, for example, the
nematophagous fungi are the best examples of predatory soil fungi. Species
of Arthrobotrytis and Dactylella are known as nematode trapping fungi. Other
examples of microbial predators are the protozoa and slime mold fungi which
feed on bacteria and reduce their population. The bacteriophages may also be
considered as predators of bacteria.

2.1.5 Mutual interaction of plants and microorganisms

The most typical example of direct interaction of bacteria with plants are the
following symbiosis:
• bacterrhiza—the symbiosis of plants and bacteria.
• mycorrhiza—the symbiosis of plants and fungi.

2.1.5.1 The symbiosis of microbes and plants – bacterrhiza

• Symbiotic agreements are clearly exemplified in the rhizosphere,
which is the area incorporating the outer surface of plants’ roots and
the adjacent soil.
2.20 Environmental Biotechnology

• The symbiosis occurs when microorganisms settle in plants’ root

systems. Both the plants and microorganisms may greatly benefit from
such interaction.
• The best example of such interaction is the symbiosis of nodule bacteria
Rhizobium with the papilionaceous plants.
• The symbiotic Rhizobium bacteria are among the best known nitrogen-
fixing organisms. Rhizobium belongs to the heterotrophic and aerobic
bacteria. While developing inside the plant tissues, they obtain the
energy and carbon needed from its host. On the other hand, the nitrogen
assimilated from the air by the bacteria is utilized by the plant.
• The Rhizobium bacteria may live in soil for years without having any
contact with a plant, by utilizing monosaccharides and mannitol as the
source of C and energy. In such conditions, they do not exhibit any
abilities to reduce and fix nitrogen, but they obtain it from the substrate
in the form of ammonium nitrogen. Nevertheless, once in the vicinity of
a papilionaceous plant, with which they can interact, they penetrate its
root system and form nodules that participate in atmospheric nitrogen
fixation.
• The symbiotic system is formed only with particular types of
papilionaceous plants and the suitable species of Rhizobium genus.

2.1.5.1.1 Rhizosphere
The rhizosphere is the layer of soil around the roots where among others,
in great concentrations, live bacteria, fungi, protozoa, nematodes, mites,
springtails, which usually form groups of species characteristic to a given
plant.
• The rhizosphere is occupied by a large variety of forms, however
the Pseudomonas and Achromobacter as well as the denitrifiers are the
most numerous, and less numerous are the Arthrobacter and Bacillus
forms. The above organisms utilize nutrients released by the roots.
The increased number of microorganisms is accompanied by higher
activity of soil’s fauna, especially of those organisms which feed upon
roots and microorganisms.
• The number of bacteria in the rhizosphere may even be 1000 times
higher than outside the rhizosphere. The ratio of bacteria from within
the rhizosphere to the number of bacteria from outside is called the
rhizosphere effect and it is marked with the R/S symbol (R – rhizosphere,
S – soil).
• Microorganisms of the rhizosphere also have a big effect on plants. They
lead to a continuous breakdown of organic and mineral compounds,
which become available to plants. Moreover, they produce organic
and non-organic acids, influence the dissolution of mineral salts and
protect the plants against the phytopathogens.
Environmental Microbiology—Soil 2.21

• An unfavorable effect is due to the metabolic activity of microflora

that cause the depletion of the oxygen required for development of
denitrifiers. As a consequence, some phytotoxic substances may be
produced, like alcohols, antibiotics or phenolic compounds.

2.1.5.2 The symbiosis of fungi and plants

Mycorrhiza is the interaction of fungi with vascular plants. In this type
of interaction, both organisms benefit. Fungi grow into plant roots. They
penetrate its cells and stimulate their growth by producing auxin hormone.
Due to mycorrhiza, plants obtain larger absorbing surface and better access
to nutrients being broken down and absorbed by fungi. Plants supply fungi
with organic substances in the form of assimilation products transported from
leaves to roots. Mycorrhiza is a widespread phenomenon; it concerns not only
roots of trees, bushes, species of flowers but also cultivated plants such as
grains and potatoes. There are two types of mycorrhiza:
• Ectotrophic,
• Endotrophic.

2.1.5.2.1 Ectotrophic mycorrhiza

Fungus develops upon the surface of plant roots, creating a kind of muff
composed of intertwined threads of mycelium. The outer hyphae of this
mycelium penetrate the soil, while the inner ones penetrate the surface layers
of root tissues.
• As the result of the mycorrhiza, roots lose their root hairs and become
shorter since the functions of roots are taken over by the fungi. Because
mycelium’s suction force is much stronger than that of the roots the
plant is better supplied with water and mineral salts than in the cases
when there is no mycorrhiza.
• For plants interacting with mycorrhizal fungi, the absorption of
nitrogen may increase by 90%, phosphorus by 20% and potassium by
75%.
• Fungi also produce substances that stimulate roots’ growth, and some
are able to fix nitrogen. That kind of symbiotic fungi mainly belong to
the Basidiomycetes.

2.1.5.2.2 Endotrophic mycorrhiza

Endotrophic mycorrhiza occurs usually among green plants and some
deciduous trees.
• In this type of interaction, the plant roots do not differ externally from
those without mycorrhiza.
• The inside of the root cells is filled with a thick network of intertwined
hyphae which are partially digested by the plant.
• Endotrophic mycorrhiza is formed by the Fungi imperfecti.
2.22 Environmental Biotechnology

2.1.6  Soil Bioremediation

Bioremediation is a group of treatment methods or processes designed to
enhance the natural microbial degradation of organic contaminants. The
microorganisms carry out the degradation of harmful substances to a less toxic
or non-toxic state. Microorganisms utilize the environment-polluting organic
compounds as food substrates. After the degradation of polluting compounds,
the population of microorganisms reduces. Dead microorganisms or low
numbers of microorganisms once lack food substrates, no longer pose any
danger to the environment.
The goal of bioremediation is the neutralization of the organic pollution
to achieve undetectable concentrations or concentrations permissible by the
national regulations of particular countries. Bioremediation is utilized for
cleanup of grounds and groundwaters as well as sewage and sludge. During
the utilization of bioremediation for the purpose of pollution neutralization,
the following conditions must be met:
(a)
the environment undergoing bioremediation should contain
microorganisms characterized by the specific catabolic processes,
(b) microorganisms utilized within the bioremediation process should be
capable of efficiently converting chemical compounds and reducing
their concentration down to the level allowed by the regulations,
(c) metabolites produced during the biodegradation should not have
toxic, mutagenic or carcinogenic properties, and
(d) the conditions in the immediate area, where the process is being
conducted, should be favourable to the growth and activity of the
microorganisms (adequate nutrients, acceptable pH, oxygen or other
electron acceptor, acceptable redox level, favorable moisture).
The rate of biodegradation may be limited by: temperature, the toxicity of
concentrated contaminants, or mass transfer limitations.

2.1.6.1 Microorganisms used in remediation technologies

The bioremediation processes may be conducted by the autochthonous
microorganisms, which naturally inhabit the soil/water environment
undergoing purification, or by other microorganisms, that derive from
different environments. However, in both the cases, they are characterized by
a high ‘xenobiotics’ degradation activity. The choice of strains capable of being
used for the inoculation of the contaminated grounds creates many problems.
• Apart from the high efficiency in ‘xenobiotics’ decomposition, the
chosen strains should also possess many additional features that enable
their adaptation and development in a new environment.
• One of the conditions for the adaptation of the inoculants in soil is
the lack of antagonistic interaction with the natural water and soil
microflora.
Environmental Microbiology—Soil 2.23

• Moreover, they can not be pathogenic microorganisms nor ones, which,

during growth on hydrocarbons, produce substances with cytotoxic,
mutagenic or carcinogenic properties.
The selection of microorganisms specialized in degradation of particular
compounds is based on the processes of their adaptation or genetic operation.
Substances making up the environment’s contamination, as a rule, are
composed of many compounds. Therefore, a complete soil cleanup requires a
special, carefully selected, mixture of microorganisms. Utilization of mutated
microorganisms is useful only in ex situ methods where precise conditions
of the process control and the prevention of contamination by dangerous
mutants exists. In order to avoid problems related to the introduction of
foreign organisms into the environment, mixtures of microorganisms should
be created to degrade the contamination. Moreover, the techniques used
to allow microorganisms adapted to new conditions should constantly be
improved. The initial high number of microorganisms in soil may be obtained
at the beginning of the purification process by the inoculation of the ground
with microorganisms. This allows a faster and more effective bioremediation
process. Inoculation of the soil is carried out after growing the microorganisms
in a bioreactor.
The cultured natural autochthonous microorganisms or specially prepared
strains derived from a culture collection and/or the commercial preparations
are utilized in the process of inoculation. The strains are stored in the lyophilised
form, frozen or placed in a special suspension. The microorganisms may be
incorporated into both the soil and the water environment in the form of a
suspension or placed on a solid support (immobilized).
The microbial cultures usually contain a mixture of bacteria, nutrients, a
solid support and possibly enzymes. Depending on the composition of these
biopreparations used for bioremediation, the above can be subdivided into
microbiological (bacterial), enzymatic and bacterial-enzymatic. The advantage
of microbiological preparations in relation to enzymatic preparations is the
fact that the microorganisms multiply in a previously clean environment
whereas the enzymatic preparations are added, in specific dosages, without
the possibility of multiplication. One of the conditions which have to be met
in order to obtain effective biodegradation is the bacteria’s accessibility to the
contaminated layers of soil. Migration within the soil depends on the number of
microorganisms as well as on the type of soil. The effects of the biodegradation
process of xenobiotics in soil are dependent on the method used to inoculate
the soil with microorganisms. Even distribution of microorganisms within the
soil and their contact with nutrients contained in the soil has to be provided.
Surface introduction of the inoculants is not highly effective due to slow
migration of microorganisms, especially in soils, which contain clay and silt.
In order to speed up the process of migration, the following are applied:
• injections with the use of high-pressure equipment,
• immobilization of microorganisms on solid supports,
2.24 Environmental Biotechnology

• electromagnetic field to speed up bacterial migration in the soil.

Due to the fact that biopreparations are composed of living organisms,
their application requires thorough knowledge of the subject as well as a
careful supervision of the cleaning process.

2.1.6.2 Stimulation of bioremediation

Bioremediation processes are designed to optimise the conditions for microbial
growth and degradation of contaminants. Microbial growth and metabolism
of contaminants require macronutrients (phosphorus and nitrogen) and
micronutrients. The demand for nutrients is specific for each case. It has been
determined that the ratio of carbon to nitrogen and phosphorus within the
soil should range from 100:10:1(by weight). Therefore, the basic condition
for a proper biostimulation is the control of nitrogen and phosphorus
concentrations in the soil by application of mineral fertilizers. The most
suitable ones, for the above purpose, are ammonium sulphate and sodium
phosphate (sources of nitrogen and phosphorus). Moreover, magnesium
sulphate, sodium carbonate, calcium chloride, iron sulphate are also used for
the purpose. The choice of appropriate dosages of the biogenic substances
ought to be very precise and readjusted to the soil conditions, since it has been
shown that, for example, nitrogen compounds in excessive numbers may slow
down the biodegradation process. The selection of nutrients with an optimum
composition is conducted in laboratories. The type of missing nutrients is
established by growing bacteria in the presence of various sources of biogenic
elements and by careful supervision of their growth. In addition, pollution
reduction, oxygen consumption and dioxide release are measured.

2.1.6.3 Classification of bioremediation methods

The division of bioremediation methods may be done in accordance with
the level of environment oxygenation as well as the location of the cleaning
process. Depending on the level of environmental oxygenation, the types
of biological purification can be divided into aerobic, anaerobic/aerobic,
and anaerobic. Aerobic microorganisms use oxygen as an electron acceptor.
Anaerobic microorganisms use other electron acceptors such as nitrate,
sulphate, iron, manganese, or certain organic compounds. Facultative
organisms can use oxygen, if it is present or other electron acceptors, if it is not.
Most soil bioremediation processes are aerobic. In anaerobic conditions the
microbiological decomposition of pollutants is slower and the end products
may have a toxic character. The source of oxygen utilized by microorganisms
in the respiration processes may be the atmospheric air that gets into the soil
passively or by force. The passive route depends on natural ground penetration
by the atmospheric air. Active increase of the delivered oxygen can be done
by mechanical mixing of the surface layers of soil (harrowing, ploughing etc.),
the introduction of special perforated spray lances driven directly into the
Environmental Microbiology—Soil 2.25

soil or aeration with compressors and fans. In addition, the environmental

enrichment in oxygen may take place because of the utilization of hydrogen
peroxide, which breaks down in soil to water and oxygen. Depending on the
level of contamination as well as the character of the recultivated environment,
bioremediation may take place through both, in situ or ex situ methods. In
the first one, in situ, the point is that the pollution is eliminated directly at
the place where it occurs. However, in the second method, the contaminated
ground or waters are excavated before the actual regeneration procedures. In
situ techniques do not require excavation of the contaminated soils; so may
be less expensive, create less dust, and it is possible to treat a large volume of
soil and cause less release of contaminants than by ex situ methods. But in situ
techniques are slower than ex situ, may be difficult to manage, and are most
effective at sites with permeable soil. Ex situ methods are utilized where there
is danger of toxic pollutant migration into the groundwaters and when the
process of detoxification must be conducted in a short period of time.

2.1.6.3.1 In situ methods

In situ methods are based mainly on biostimulation of the organic pollutant
degradation processes by enriching waters or soils in biogenic elements, by
acidity correction as well as by their aeration. The following in situ methods
can be used:
Agricultural methods: Those are the most commonly used methods of
soil decontamination. They are effective with almost all components of fuels.
Lighter products, such as gasoline, are eliminated by vaporization or, to lesser
extent, by biodegradation. When it comes to heavy products, like diesel fuels
and kerosene, contamination is eliminated mainly by biodegradation. There
are many variations of agricultural methods depending on the technical
solutions. The simplest variation depends on spreading the contaminated soil
in a thin layer of no more than 0.5m thick followed by a period ploughing
or deep harrowing in order to aerate the soil. This activates the microflora
by delivering essential nutrients, oxygenating the soil and by deacidification
when needed. Nutrients are replenished by application of nitrogenous,
phosphate or potassium fertilizers, when necessary. Soil deacidification can
be accomplished by liming. Planting grasses or papilionaceous plants on
the contaminated grounds may also assist the cleaning processes. Detailed
programs for fertilization, mechanical and other treatments are designed for
each individual case.
Bio-extraction: The situation is significantly more complicated when the
need for removal of contaminants from within deeper layers of soils arises.
The growth stimulation of microorganisms decomposing, for example,
petroleum products, is more difficult; nevertheless, the methods are similar.
It is also essential in this case to deliver missing nutrients, supply oxygen
and, if necessary, inoculate the soil with microorganisms able to actively
decompose the contaminants. The processes of soil venting and rinsing with
2.26 Environmental Biotechnology

solution containing nutrients or active bacterial cultures are utilized, as well.

For the above, the following technical equipment is necessary: suction and
positive displacement pumps, sumps, and screens preventing the spreading
of contaminant within soils. The condition of an effective cleaning process is
also a proper geological configuration of the terrain, which allows a controlled
flow of the medium (air, water vapour, solutions). The natural decomposition
can be accelerated by utilizing the so-called bio-extraction. Optimization of
the process can be accomplished by rinsing the soil and water environments
through forced infiltration of groundwater (water stimulated bioremediation
in-situ) and aeration (soil bio-ventilation). Microorganisms and nutrients
delivered with water stimulate biodegradation of petroleum products while
the air enriches soil in oxygen thus assisting in the biodegradation.

Water stimulated bioremediation

The goal of the above process is to force a vertical and then horizontal flow of
water along with the organic contaminants in the water and soil environment.

Water stimulated soil bioremediation

This method is utilized for the cleaning of the environment from petroleum
related non-soluble substances gathered at the surface of groundwaters.
During the process, the groundwater is pumped out up to the surface, purified,
oxygenated, and returned back to the source after nutrient enrichment. In other
cases, the flow of the underground waters may be utilized. The waters are
obtained from wells, which are situated in the lowest points. These waters are
then sent to bio-reactors, in which the process of biodegradation of petroleum-
related products occurs. The same water is then returned back to its source.
Environmental Microbiology—Soil 2.27

The above method is a combination of in situ and ex situ methods which are
complementary to each other, which in turn, allows for optimisation of the
process. Water stimulated bioremediation can be assisted by surfactants. It
has been shown that synthetic surfactants and biosurfactants accelerate the
processes of hydrophobic contaminants biodegradation, particularly of heavy
fractions of petroleum products. Increased solubility and emulsion formation
result in better mobility of petroleum products in soil and in larger specific
surfaces accessible to microorganisms. Surfactants can also increase the
permeability of soils.
Bioventilation: The acceleration of the natural processes of biodegradation
may be assisted by soil ventilation. Ventilation is a physical method, which
may be utilized as an independent decontamination technique used for the
maximization of volatilization of low molecular mass hydrocarbons (for
example, products based on gasoline or solvents). However, during this
process, a very little biodegradation occurs.

Bioventilation
The ventilation process permits the removal of volatile petroleum products
from the aeration and saturation zones, while at the same time enriching the
ground with air and increasing the level of oxygenation. The aeration process
can be both, active or passive. In the case of passive ventilation, the aeration
is done by the utilization of perforated pipe networks. Active ventilation
however, depends on the creation of negative pressure (extraction of the
ground air) or positive pressure (air injection). The effectiveness of the soil
and water environment bioventilation depends on the level of oxygen in the
air contained in the soil, level of bio-genes, reduction-oxidation conditions,
presence of surface-active agents, level of saturation (moisture), pH and
temperature.
2.28 Environmental Biotechnology

The most effective way of supplying oxygen to the contaminated ground

is by introducing it with compressed air. Ozone (O3) is also utilized as an
alternative source of oxygen. However, there is some danger in using ozone as
the source of oxygen since it possesses some toxic properties.

2.1.6.3.2 Ex situ methods

Ex situ methods can be implemented when the danger of toxic pollutant
migration into the groundwaters occurs and when the process of detoxification
has to be completed in a short period of time.
Bioreactor method: The decontamination process is most effective when
run in specially designed bioreactors. They provide effective control of all
parameters, delivery of required ingredients, oxygenation and inoculation of
the soil. The cost of this method is however rather high due to the technological
requirements as well as the need for transportation of heavy masses. Therefore,
the above method is not often used and is usually employed for smaller
amounts of soil.

Schematic representation of the bioreactor method

Among the technologies based on ex situ methods, there are bio-reactors
and lagoons used for decontamination of oily sewage waters and reactors for
treatment of semi-liquid masses of contaminated soil, sludge and deposits.
Solid materials undergo mixing with liquids followed by aeration. In both
types of bioreactors, the level of dissolved oxygen may be controlled just
like the pH and the concentration of inorganic nutrients. This technique is
similar to the one that treats municipal wastes by utilizing the activated sludge
method. Moreover, the technique may be aided by surfactants or dispersants
in order to desorb the hydrocarbons from the solid particles, and to increase
the degree of dispersion of water insoluble oil contaminants. Surface-active
agents used in these methods may be synthetic or natural. Bio-surfactants have
found ever-increasing use due to the fact that they are more environmentally
friendly and are biodegradable.
Environmental Microbiology—Soil 2.29

Bioreactors may also be utilized for decontamination of groundwaters.

They are either stationary or portable, which makes the process easier and
less expensive. Portable bioreactors are used for decontamination of ground
waters after pumping out the material from the water-bearing bed or for
decontamination of waters derived from the process of contaminated ground
rinsing. There are two different types of bioreactors, fluidized and immobilized
beds. The choice of bed depends mainly on the type of contaminants and
their concentration in water that is going to be processed. The immobilized
biomass is a mixture of selected microorganisms capable of biodegradation of
particular pollutants. The microorganisms may be trapped inside the carrier’s
structure (natural polymers such as agar, alginate, collagen, or synthetic
polymers such as polyacrylamide gels, polyurethanes and others) which is
called active immobilization.
In the passive immobilization, microorganisms are bound to the surface
of a porous material (activated carbon, for instance) or may create a gelatinous
film upon the surface of stationary elements (ceramic rings, plastic tiles,
polyurethane foam). In such bioreactors, the contaminated water flows
countercurrent to the air through the solid bed containing the immobilized
microorganisms.
Biopile method: The method consists of transferring the contaminated
soil or ground into a specially prepared place.

Schematic representation of the biopile method

The excavated soil is arranged in the form of an elongated heap inside a

foil-lined ditch that is equipped with drainage and ventilation systems. The
process is also aided by aeration as well as water and nutrients addition. The
2.30 Environmental Biotechnology

air is pumped in through the system of perforated pipes equipped with a

blow-fan that creates negative pressure at the bottom of the heap. In simpler
technologies, the actual aeration is ensured by mechanical shifting of the soil.
The heap is usually covered with a foil tunnel equipped with a system of
sprinklers through which water and nutrients are delivered. In many recently
used technologies, water circulates in a closed system. Reflux from within the
ground is filtered into bioreactors and then pumped into the reservoir from
which, after enrichment in nutrients and growth of the microorganisms, it is
returned by the sprinkler system onto the heap.

2.1.7 Role of Soil Microorganisms in Biodegradation of Pesticides

and Herbicides
Pesticides are the chemical substances that kill pests and herbicides are the
chemicals that kill weeds. In the context of soil, pests are fungi, bacteria insects,
worms, and nematodes, etc. that cause damage to field crops. Thus, in broad
sense, pesticides are insecticides, fungicides, bactericides, herbicides and
nematicides that are used to control or inhibit plant diseases and insect pests.
Although wide-scale application of pesticides and herbicides is an essential
part of augmenting crop yields, excessive use of these chemicals leads to the
microbial imbalance, environmental pollution and health hazards. An ideal
pesticide should have the ability to destroy target pest quickly and should be
able to degrade non-toxic substances as quickly as possible.
The ultimate “sink” of the pesticides applied in agriculture and public
health care is soil. Soil being the storehouse of multitudes of microbes, in
quantity and quality, receives the chemicals in various forms and acts as a
scavenger of harmful substances. The efficiency and the competence to handle
the chemicals vary with the soil and its physical, chemical and biological
characteristics.

2.1.7.1 Effects of pesticides

Pesticides reaching the soil in significant quantities have direct effect on soil
microbiological aspects, which in turn influence plant growth. Some of the
most important effects caused by pesticides are :
• alterations in ecological balance of the soil microflora,
• continued application of large quantities of pesticides may cause ever
lasting changes in the soil microflora,
• adverse effect on soil fertility and crop productivity,
• inhibition of N2 fixing soil microorganisms such as Rhizobium,
Azotobacter, Azospirillum, etc. and cellulolytic and phosphate-
solubilizing microorganisms,
• suppression of nitrifying bacteria, Nitrosomonas and Nitrobacter, by
soil fumigants ethylene bromide, telone, and vapam have also been
reported,
Environmental Microbiology—Soil 2.31

• alterations in nitrogen balance of the soil,

• interference with ammonification in soil,
• adverse effect on mycorrhizal symbioses in plants and nodulation in
legumes, and
• alterations in the rhizosphere microflora, both quantitatively and
qualitatively.

2.1.7.2 Persistence of pesticides in soil

How long an insecticide, fungicide, or herbicide persists in soil is of great
importance in relation to pest management and environmental pollution.
Persistence of pesticides in soil for longer period is undesirable because of the
reasons: a) accumulation of the chemicals in soil to highly toxic levels, b) may
be assimilated by the plants and get accumulated in edible plant products, c)
accumulation in the edible portions of the root crops, d) to be get eroded with
soil particles and may enter into the water streams, and finally leading to the
soil, water and air pollutions. The effective persistence of pesticides in soil
varies from a week to several years depending upon structure and properties
of the constituents in the pesticide and availability of moisture in soil. For
instance, the highly toxic phosphates do not persist for more than three months
while chlorinated hydrocarbon insecticides (eg. DOT, aldrin, chlordane, etc.)
are known to persist at least for 4-5 years and, some times, more than 15 years.
From the agricultural point of view, longer persistence, of pesticides
leading to accumulation of residues in soil may result into increased absorption
of such toxic chemicals by plants to the level at which the consumption of
plant products may prove deleterious/hazardous to human beings as well as
livestock. There is a chronic problem of agricultural chemical having entered
in food chain at highly inadmissible levels in India, Pakistan, Bangladesh and
several other developing countries in the world. For example, intensive use
of DDT to control insect pests and mercurial fungicides to control diseases
in agriculture had been known to persist for longer period and thereby got
accumulated in the food chain leading to food contamination and health
hazards. Therefore, DDT and mercurial fungicides have been banned for use
in agriculture as well as in public health department.

2.1.7.3 Biodegradation of Pesticides in Soil

Pesticides reaching the soil are acted upon by several physical, chemical, and
biological forces. However, physical and chemical forces are acting upon/
degrading the pesticides to some extent microorganisms play major role in
the degradation of pesticides. Many soil microorganisms have the ability to
act upon pesticides and convert them into simpler non-toxic compounds. This
process of degradation of pesticides and conversion into non-toxic compounds
by microorganisms is known as “biodegradation”. Not all pesticides reaching
2.32 Environmental Biotechnology

the soil are biodegradable and such chemicals that show complete resistance
to biodegradation are called “recalcitrant”.
The chemical reactions leading to biodegradation of pesticides fall into
several broad categories:
• Detoxification: Conversion of the pesticide molecule to a non-toxic
compound. Detoxification is not synonymous with degradation. Since
a single change in the side chain of a complex molecule may render the
chemical non-toxic.
• Degradation: The breaking down/transformation of a complex substrate
into simpler products leading finally to mineralization. Degradation is
often considered to be synonymous with mineralization, e.g. Thirum
(fungicide) is degraded by a strain of Pseudomonas and the degradation
products are dimethlamine, proteins, sulpholipids, etc.
• Conjugation (complex formation or addition reaction): In which an
organism make the substrate more complex or combines the pesticide
with cell metabolites. Conjugation or the formation of addition product
is accomplished by those organisms catalyzing the reaction of addition
of an amino acid, organic acid or methyl crown to the substrate; for
e.g., in the microbial metabolism of sodium dimethly dithiocarbamate,
the organism combines the fungicide with an amino acid molecule,
normally present in the cell, and thereby, inactivate, the pesticides/
chemical.
• Activation: It is the conversion of non-toxic substrate into a toxic
molecule, for eg. Herbicide, 4-butyric acid (2, 4-D B) and the insecticide
Phorate are transformed and activated microbiologically in soil to give
metabolites that are toxic to weeds and insects.
• Changing the spectrum of toxicity: Some fungicides/pesticides are
designed to control one particular group of organisms/pests, but they
are metabolized to yield products inhibitory to entirely dissimilar
groups of organisms, for e.g. the fungicide PCNB is converted in soil to
chlorinated benzoic acids that kill plants.
Biodegradation of pesticides/herbicides is greatly influenced by the soil
factors like moisture, temperature, pH and organic matter content, in addition
to microbial population and pesticide solubility. Optimum temperature,
moisture and organic matter in soil provide congenial environment for
the break down or retention of any pesticide added in the soil. Most of the
organic pesticides degrade within a short period (3-6 months) under tropical
conditions. Metabolic activities of bacteria, fungi and actinomycetes have
significant role in the degradation of pesticides.

2.1.7.4 Criteria for Bioremediation/Biodegradation

For successful biodegradation of pesticide in soil, following aspects must
be taken into consideration: (i) Organisms must have necessary catabolic
Environmental Microbiology—Soil 2.33

activity required for degradation of contaminant at fast rate to bring down the
concentration of contaminant, ii) the target contaminant must be bioavailable,
iii) soil conditions must be congenial for microbial /plant growth and enzymatic
activity and iv) cost of bioremediation must be less than other technologies for
removal of contaminants.
According to Gales (1952) principal of microbial infallibility, for every
naturally occurring organic compound, there is a microbe/enzyme system
capable its degradation.

2.1.7.5 Strategies for Bioremediation

For the successful biodegradation/bioremediation of a given contaminant
following strategies are needed.
• Passive/intrinsic Bioremediation: It is the natural bioremediation
of contaminant by tile indigenous microorganisms and the rate of
degradation is very slow.
• Biostimulation: Practice of addition of nitrogen and phosphorus to
stimulate indigenous microorganisms in soil.
• Bioventing: Process/way of biostimulation by which gases stimulants
like oxygen and methane are added or forced into soil to stimulate
microbial activity.
• Bioaugmentation: It is the inoculation/introduction of microorganisms
in the contaminated site/soil to facilitate biodegradation.
• Composting: Piles of contaminated soils are constructed and treated
with aerobic thermophilic microorganisms to degrade contaminants.
Periodic physical mixing and moistening of piles is done to promote
microbial activity.
• Phytoremediation: Can be achieved directly by planting plants which
hyperaccumulate heavy metals or indirectly by plants stimulating
microorganisms in the rhizosphere.
• Bioremediation: Process of detoxification of toxic/unwanted
chemicals/contaminants in the soil and other environment by using
microorganisms.
• Mineralization: Complete conversion of an organic contaminant to its
inorganic constituent by a species or group of microorganisms.

2.1.8 Microbial Ecology of Petroleum Contaminant Plumes

The aerobic biodegradation of petroleum in groundwater systems is effected
by microorganisms which metabolize PHCs for organic carbon and energy.
The microorganisms involved are primarily procaryotic soil bacteria such as
Nocardia, Pseudomonads, Acinetobacter, Flavobacterium, Microcossus, Anthrobacter,
and Corynebacterium. Petroleum-degrading soil bacteria consist of two different
groups distinguished by unique respiratory capabilities.
2.34 Environmental Biotechnology

• Obligate aerobic microbes consist of those soil bacteria which metabolize

organic carbon only under oxic conditions, whereas
• facultative anaerobic microbes consist of those bacteria which
metabolize organic carbon under either, oxic or anoxic conditions.
— Intrinsic biodegradation is typically effected by a bacteria group
rather than a single bacterium. This is because ultimate bio-oxidation
to carbon dioxide and water involves a series of biotransformations
in which one bacteria converts one group of petroleum hydrocarbons
to intermediate compounds. The intermediate compounds are
themselves metabolized by a different bacteria.
— In uncontaminated groundwater systems, indigenous microbes
obtain organic carbon and energy from dissolved organic carbon
(DOC). The DOC leaches from soil organic matter in the unsaturated
zone. In petroleum-contaminated groundwater systems, certain
bacteria having the genetic capability to metabolize petroleum
constituents are stimulated by the supplemental organic carbon
supplied by PHCs.
— Bacteria metabolize DOC and PHCs by breaking carbon-carbon
and carbon-hydrogen covalent bonds. PHCs amenable to intrinsic
biodegradation include the aliphatic hydrocarbons with carbon
number ranges of C10 to C25 and the aromatic hydrocarbons
benzene, toluene, ethyl benzene, and xylenes (BTEX).
• During bio-oxidation of DOC/PHCs, microbes use O2 as a terminal
electron acceptor to collect electrons released during metabolism,
and ambient inorganic nutrients and organic carbon to maintain
cell tissue and increase biomass. Although oxgyen is consumed in
this process, nutrients are generally conserved as they are recycled
during production of waste materials and cellular tissue. Alternate
terminal electron acceptors include nitrate/nitrite, sulfate/sulfite,
and carbon dioxide. However, O2 is generally the most energetic
electron acceptor for stimulating biodegradation.

2.1.9 Molecular Microbial Ecology

The scientific core of environmental biotechnology is microbial ecology—a
scientific discipline dedicated to understanding complex communities of
microorganisms: which microorganisms are present, what is their metabolic
potential, what part of the potential are they realizing, and how they interact
with each other and their environment. Fundamental scientific research in
microbial ecology provides us with a deep understanding of how the complex
communities work.
Fortunately, we have powerful new tools to help us analyze these
intriguing organisms and their community organization. Among the tools
we can use, are molecular methods that probe the genetic information of
Environmental Microbiology—Soil 2.35

the microorganisms in microbial communities. By targeting genomic DNA,

we can identify which microorganisms are present and what reactions they
can perform; through the use of RNA-based techniques we can identify the
members in the community that are actively growing and what reactions they
are performing. Using in-situ techniques, we can also investigate the metabolic
interactions that take place among microorganisms in a mixed community.
Applying these molecular tools to understand microbial communities is called
molecular microbial ecology, and it is a critical research strength of the Center
for Environmental Biotechnology.

PCR is used to detect the presence of a specific group of microorganisms

In particular, the Center has exceptional capabilities to investigate
changes in microbial structure through the use of molecular techniques that
target the 16S rRNA gene and the 16S rRNA, such as denaturing gradient gel
electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization
(FISH). DGGE is especially powerful for doing “detective work” to identify
important, but uncharacterized strains. Real-time PCR is powerful for
quantifying the different types of microorganisms. FISH allows us to do
3-dimensinal visualization of the communities and understand the interactions
among different strains. The Center also targets specific metabolic genes and
their expression to investigate the role of critical enzymes in detoxification and
energy generating reactions occurring in natural or engineered techniques
including reverse transcriptase PCR, RNA or cDNA microarrays, and real-
time PCR to quantify over- and under-expression of specific genes and for
microarray validation. Identification of specific gene-targets will allow us to
investigate critical factors affecting the performance of natural or engineered
2.36 Environmental Biotechnology

microbial systems. The expression of such critical genes can be used to monitor
and evaluate the success of engineered processes.

2.1.10 Soil Microorganisms as Biofertilizers

Biofertilizers are microbial inoculants or carrier-based preparations containing
living or latent cells of efficient strains of nitrogen-fixing, (phosphate is
solublizing) and cellulose decomposing microorganisms intended for seed or
soil application and designed to improve soil fertility and plant growth by
increasing the number and biological activity of beneficial microorganisms in
the soil.
• The objects behind the application of biofertilizers/microbial inoculants
to seed, soil or compost pit is to increase the number and biological/
metabolic activity of useful microorganisms that accelerate certain
microbial processes to augment the extent of availability of nutrients
in the available forms which can be easily assimilated by plants. The
need for the use of Biofertilizers has arisen primarily due to two
reasons, i.e., though chemical fertilizers increase soil fertility, crop
productivity and production, but increased/intensive use of chemical
fertilizers has caused serious concern for soil texture, soil fertility and
other environmental problems and that use of Biofertilizers is both
economical as well as environment friendly. Therefore, an integrated
approach of applying both chemical fertilizers and Biofertilizers is the
best way of integrated nutrient supply in agriculture.
Organic fertilizers (manure, compost, vermicompost) are also considered
as Biofertilizers, which are rendered in available forms due to the interactions
of microorganisms or their association with plants. Biofertilizers, thus include
(i) Symbiotic nitrogen fixers viz. Rhizobium sp.,
(ii) Non-symbiotic, freeliving nitrogen fixers viz. Azotobacter, Azospirillum
etc.,
(iii) BGA-inoculants viz., Azolla-Anabaena,
(iv) Phosphate, solubilizing microorganisms (PSM) viz. Bacillus,
Pseudomonas, Penicillium, Aspergillus, etc.
(v) Mycorrhiza,
(vi) Cellulolytic microorganisms and
(vii) Organic fertilizers.
Nobbe and Hiltner (1895, USA) produced the first Rhizobium biofertilizer
under the brand name “Nitragin” for 17 different legumes.

2.1.10.1 Role of Biofertilizers in soil fertility and Agriculture

Biofertilizers are known to play a number of vital roles in-soil fertility, crop
productivity and production in agriculture as they are eco-friendly and can
not, at any cost, replace chemical fertilizers that are indispensable for getting
Environmental Microbiology—Soil 2.37

maximum crop yields. Some of the important functions or roles of Biofertilizers

in agriculture are:
• They supplement chemical fertilizers for meeting the integrated
nutrient demand of the crops.
• They can add 20-200 kg N/ha year (eg. Rhizobium sp 50-100 kg N/ha yr;
Azospirillum , Azotobacter: 20-40 kg N/ha /yr; Azolla : 40-80 kg N/ha; BGA
:20-30 kg N/ha) under optimum soil conditions and thereby increase
15-25 per cent of total crop yield.
• They can, at best, minimize the use of chemical fertilizers, not exceeding
40-50 kg N/ha under ideal agronomic and pest-free conditions.
• Application of Biofertilizers results in increased mineral and water
uptake, root development, vegetative growth and nitrogen fixation.
• Some Biofertilizers (eg, Rhizobium BGA, Azotobacter sp) stimulate
production of growth promoting substance like vitamin-B complex,
Indole acetic acid (IAA) and Gibberellic acids, etc.
• Phosphate-mobilizing or phosphorus-solubilizing Biofertilizers/
microorganisms (bacteria, fungi, mycorrhiza, etc.) converts insoluble
soil phosphate into soluble forms by secreting several organic acids
and under optimum conditions, they can solubilize/mobilize about
30-50 kg P2O5/ha due to which crop yield may increase by 10 to 20%.
• Mycorrhiza or VA-mycorrhiza (VAM fungi), when used as Biofertilizers
enhance uptake of P, Zn, S and water, leading to uniform crop growth
and increased yield and also enhance resistance to root diseases and
improve hardiness of transplant stock.
• They liberate growth-promoting substances and vitamins and help to
maintain soil fertility.
• They act as antagonists and suppress the incidence of soil borne plant
pathogens and thus, help in the bio-control of diseases.
• Nitrogen-fixing, phosphate-mobilizing and cellulolytic microorganisms
in biofertilizers enhance the availability of plant nutrients in the soil
and thus, sustain the agricultural production and farming system.
• They are cheaper, pollution-free and renewable energy sources.
• They improve physical properties of soil, soil tilth and soil health, in
general.
• They improve soil fertility and soil productivity.
• Blue green algae like Nostoc, Anabaena, and Scytonema are often employed
in the reclamation of alkaline soils.
• Bio-inoculants, containing cellulolytic and lignolytic microorganisms,
enhance the degradation/decomposition of organic matter in soil, as
well as enhance the rate of decomposition in compost pit.
2.38 Environmental Biotechnology

• BGA plays a vital role in the nitrogen economy of rice fields in tropical
regions.
• Azotobacter inoculants, when applied to many non-leguminous
crop plants, promote seed germination and initial vigor of plants by
producing growth-promoting substances.
•  Azolla-Anabaena grows profusely as a floating plant in the flooded rice
fields and can fix 100-150 kg N/ha/year in approximately 40-60 tonnes
of biomass produced.
• Plays important role in the recycling of plant nutrients.

2.1.10.2 Quality Control Measures (as per ISI Specifications)

• Since, Biofertilizers contain live cells, care should be taken during their
transportation and storage.
• They should be kept in a cold place and not exposed to sunlight.
• Biofertilizers for legumes are crop-specific; therefore, they must be
used for the crop for which they are meant.
• Biofertilizers, when used under adverse soil conditions, appropriate
remedial measures (liming and use of Gypsum) should be followed.
• Biofertilizers must be carrier-based.
• Carrier material used should be in form of powder (75-106 micron size.
• They should contain minimum of 10^8 viable cells of microorganisms
/gram of the carrier material on dry weight basis.
• They should have a minimum period of six months expiry from date of
its
• They should be free from any contaminant /contamination with other
microorganisms.
• pH should be in the range of 6.0-7.5.
• They should induce desired beneficial effects on all those crops, species/
cultivars listed on the packet before the expiry date.
• They should be packed in 50-75 micron low density polythene packets

2.1.11 Factors Affecting Distribution, Activity and Population of

Soil Microorganisms
Soil microorganisms (Flora & Fauna), just like higher plants, depend entirely
on soil for their nutrition, growth and activity. The major soil factors which
influence the microbial population, distribution and their activity in the soil
are:
• Soil fertility,
• Cultural practices,
• Soil moisture,
Environmental Microbiology—Soil 2.39

• Soil temperature,
• Soil aeration,
• Light,
• Soil pH (H-ion concentration)
• Organic matter.
• Food and energy supply,
• Nature of soil, and
• Microbial associations.
All these factors play a great role in determining not only the number
and type of organism but also their activities. Variations in any one or more
of these factors may lead to the changes in the activity of the organisms
which ultimately affect the soil fertility level. Brief account of all these factors
influencing soil microflora/organisms and their activities is activities are as
follows.
Cultural practices (Tillage): Cultural practices viz. cultivation, crop
rotation, application of manures and fertilizers, liming and gypsum
application, pesticide/fungicide and weedicide application have their effect
on soil organisms. Ploughing and tillage operations facilitate aeration in soil
and exposure of soil to sunshine, and thereby, increase the biological activity
of organisms, particularly, of bacteria. Crop rotation with legume maintains
the favorable microbial population balance, particularly of N2. Fixing bacteria
and thereby improve soil fertility.
Liming of acid soils increases activity of bacteria and actinomycetes and
lowers the fungal population. Fertilizers and manures applied to the soil for
increased crop production, supply food and nutrition not only to the crops but
also to microorganisms in soil and thereby proliferate the activity of microbes.
Foliar or soil application of different chemicals (pesticides, fungicides,
nematicides, etc.) in agriculture are either degraded by the soil organisms or
are liable to leave toxic residues in soil which are hazardous to cause profound
reduction in the  normal microbial activity in the soil.
Soil fertility: Fertility level of the soil has a great influence on the microbial
population and their activity in soil. The availability of N, P and K required
for plants as well as microbes in soil determines the fertility level of soil. On
the other hand, soil microflora has greater influence on the soil fertility level.
Soil moisture: It is one of the important factors influencing the microbial
population and their activity in soil. Water (soil moisture) is useful to the
microorganisms in two ways i.e. it serve as source of nutrients and supplies
hydrogen/oxygen to the organisms and it serve as solvent and carrier of
other food nutrients to the microorganisms. Microbial activity & population
proliferate best in the moisture range of 20% to 60%. Under excess moisture
conditions/water logged conditions due to lack of soil aeration (Oxygen),
2.40 Environmental Biotechnology

anaerobic microflora become active and the aerobes get suppressed. While
in the absence of adequate moisture in soil, some of microbes die out due to
tissue dehydration others change their forms into resting stages spores or cysts
and tide over adverse conditions. Therefore, optimum soil moisture (range 20
to 60 %) must be there for better population and activity of microbes in soil.
Soil temperature: Next to moisture, temperature is the most important
environmental factor influencing the biological, physical and chemical
processes and, of microbes, microbial activity and their population in soil.
Though microorganisms can tolerate extreme temperature (such as – 60°
or + 60 u) conditions, but the optimum temperature range at which soil
microorganisms can grow and function actively is rather narrow.
Depending upon the temperature range at which microorganisms can
grow and function, they are divided into three groups, namely:
• psychrophiles (growing at low temperature below 10°C)
• Mesophiles (growing well in the temp range of 20°C to 45°C) and
• thermopiles (can tolerate temperature above 45°C and optimum
45-60°C).
Most of the soil microorganisms are mesophilic (25 to 40°) and optimum
temperature for most mesophiles is 37°C. True psychrophiles are almost
absent in soil, and thermopiles, though present in soil, behave like mesophiles.
True thermopiles are more abundant in decaying manure and compost heaps
where high temperature prevails.
Seasonal changes in soil temperature affects microbial population and
their activity, especially in temperate regions. In winter, when temperature is
low (below 50°C ), the number and activity of microorganisms falls down, and
as the soils warms up in spring, they increases in number as well as activity.
In general, population and activities of soil microorganisms are the highest in
spring and lowest in winter season.
Soil air (Aeration): For the growth of microorganisms, better aeration
(oxygen, and sometimes, CO2) in the soil is essential. Microbes consume
oxygen from soil air and gives out carbon dioxide. Activities of soil microbes
is often measured in terms of the amount of oxygen absorbed or amount
of CO2 evolved by the organisms in the soil environment. Under high soil
moisture level/water logged conditions, gaseous exchange is hindered and the
accumulation of CO4 occurs in soil air which is toxic to microbes. Depending
upon oxygen requirements, soil microorganisms are grouped into categories,
viz aerobic (require oxygen for life processes), anaerobic (do not require
oxygen) and microaerophilic (requiring low concentration/level of oxygen).
Light: Direct sunlight is highly injurious to most of the microorganisms
except algae. Therefore, upper portion of the surface soil, a centimeter or
less—is usually sterile or devoid of microorganisms. Effect of sunlight is due
to heating and increase in temperature (More than 45°)
Environmental Microbiology—Soil 2.41

Soil Reaction/Soil pH: Soil reaction has a definite influence/effect on

quantitative and qualitative composite of soil microbes. Most of the soil
bacteria, blue-green algae, diatoms and protozoa prefer a neutral or slightly
alkaline reaction between pH 4.5 and 8.0 and fungi grow in acidic reaction
between pH 4.5 and 6.5, while actinomycetes prefer slightly alkaline soil
reactions. Soil reactions also influence the type of the bacteria present in soil.
For example nitrifying bacteria (Nitrosomonas & Nitrobacter) and diazotrophs
like Azotobacter are absent totally or inactive in acid soils, while diazotrophs
like Beijerinckia, Derxia, and sulphur oxidizing bacteria like Thiobacillus
thiooxidans are active in acidic soils.
Soil Organic Matter: The organic matter in soil being the chief source of
energy and food for most of the soil organisms, it has great influence on the
microbial population. Organic matter influence directly or indirectly on the
population and activity of soil microorganisms. It influences the structure and
texture of soil and thereby activity of the microorganisms.
Food and energy supply: Almost all microorganisms obtain their food
and energy from the plant residues or organic matter/substances added to the
soil. Energy is required for the metabolic activities of microorganisms. The
heterotrophs utilize the energy liberated during the oxidation of complex
organic compounds in soil, while autotrophs meet their energy requirement
from oxidation of simple inorganic compounds (chemoautotroph) or from
solar radiation (photoautotroph). Thus, the source of food and energy rich
material is essential for the microbial activity in soil. The organic matter,
therefore serves both as a source of food nutrients as well as energy required
by the soil organisms.
Nature of Soil: The physical, chemical and physico-chemical nature of soil
and its nutrient status influence the microbial population, both quantitatively
and qualitatively. The chemical nature of soil has considerable effect on
microbial population in soil. The soils in good physical condition have better
aeration and moisture content which is essential for optimum microbial activity.
Similarly, nutrients (macro and micro) and organic constituents of humus are
responsible for absence or presence of certain type of microorganisms and
their activity. For example, activity and presence of nitrogen-fixing bacteria is
greatly influenced by the availability of molybdenum, and absence of available
phosphate restricts the growth of Azotobacter.
Microbial associations /interactions: Microorganisms interact with each
other giving rise to antagonistic or symbiotic interactions. The association
existing between one organism and another, whether symbiotic or antagonistic,
influences the population and activity of soil microbes to a great extent. The
predatory habit of protozoa, and some mycobacteria which feed on bacteria,
may suppress or eliminate certain bacteria. On the other hand, the activities of
some of the microorganisms are beneficial to each other. For instance, organic
2.42 Environmental Biotechnology

acids liberated by fungi, increase in oxygen by the activity of algae, change in

soil reaction, etc. favors the activity of bacteria and other organisms in soil.
Root Exudates: In the soil where plants are growing, the root exudates
also affects the distribution, density and activity of soil microorganism. Root
exudates and sloughed off material of root surfaces provide an abundant
source of energy and nutrients, and thus directly or indirectly, influence the
quality as well as quantity of microorganisms in the rhizosphere region. Root
exudates contain sugars, organic acids, amino acids, sterols, vitamins and
other growth factors which have profound effect on soil microbes.

2.1.12 Rhizosphere Concept and It’s Historical Background

The root system of higher plants is associated not only with soil environment
composed of inorganic and organic matter, but also with a vast community
of metabolically active microorganisms. As living plants create a unique
habitat around the roots, the microbial population on and around the roots is
considerably higher than that of root free soil environment and the differences
may be both quantitative and qualitative.
• Rhizosphere: It is the zone/region of soil immediately surrounding the
plant roots together with root surfaces, or it is the region where soil and
plant roots make contact, or it is the soil region subjected to influence
of plant roots and characterized by increased microbial.
• Rhizoplane: Root surface along with the closely adhering soil particles
is termed as rhizoplane.
• Term “Rhizosphere” was introduced for the first time by the German
scientist Hiltner (1904) to denote that region of soil which is subjected to
the influence of plant roots. The concept of “Rhizosphere Phenomenon”
which shows the mutual interaction of roots and microorganisms came
into existence with the work of Starkey (1929), Clark (1939) and Rauath
and Katznelson (1957).
• N. V. Krassinikov (1934) found that free-living nitrogen-fixing bacteria,
Azotobacter were unable to grow in the wheat rhizosphere.
• Starkey (1938) examined the rhizosphere region of some plant species
and demonstrated the effect of root exudates on the predominance of
bacterial population, in particular, and other soil microorganisms, in
general in the rhizosphere region. Thus, he put forth the concept of
“Rhizosphere effect/phenomenon” for the first time.
• F E Clark (1949) introduced/coined the term “Rhizoplane” to denote
the root surface together with the closely adhering soil particles.
• R. I. Perotti (1925) suggested the boundaries of the rhizosphere region
and showed that it was bounded on one side by the general soil region
(called as Edaphosphere), and on the other side, by the root tissues
(called Histosphere).
Environmental Microbiology—Soil 2.43

• G. Graf and S. Poschenrieder (1930) divided the rhizosphere region

into two general areas, i.e. outer rhizosphere and inner rhizosphere, for
the purpose of describing the same site of microbial action.
• H. Katznelson (1946) suggested the R:S ratio, i.e. the ratio between the
microbial population in the rhizosphere (R) and in the soil (S), to find
out the degree or extent of plant roots effect on soil microorganisms.
R:S ratio gives a good picture of the relative stimulation of the
microorganisms in the rhizosphere of different plant species.
• R:S ratio is defined as the ratio of microbial population per unit weight
of rhizosphere soil (R), to the microbial population per unit weight of
the adjacent non-rhizosphere soil (S).
• A. G. Lochhead and H. Katznelson (1940) examined in detail the
qualitative differences between the microflora of the rhizosphere and
microflora of the non-rhizosphere region and reported that gram-
negative, rod shaped and non-spore forming bacteria are abundant in
the rhizosphere than in the non-rhizosphere soil.
• C. Thom and H. Humfeld (1932) found that corn roots in acidic soils
yielded predominantly Trichoderma, while roots from alkaline soils
mainly contained Penicillium.
• M.J. Timonin (1940) reported some differences in the fungal types
and population in the rhizosphere of cereals and legumes. R: S ratio
of fungal population was believed to be narrow in most of the plant
species, usually not exceeding 10.
• E. A. Peterson and others (1958) reported that the plant age and soil
type influence the nature of fungal flora in the rhizosphere, and the
number of fungal population gradually increases with the age of plant.
• M. Adati (1932) studied many crops and found that though
actinomycetes were relatively less stimulated than bacteria, but in
some cases, the R:S ratio of actinomycetes was as high as 62.
• R. Venkatesan and G. Rangaswami (1965) studied the rhizosphere
effect in rice plant on bacteria, actinomycetes and fungi and reported
that (i) for actinornycetes R: S was more (ranging from 0 to 25),
depending on the age of plant roots and the dominant genera reported
were Nocardia, (ii) R:S ratio reduced with the depth of soil.
• E. A. Gonsalves and V. S. Yalavigi (1960) reported the presence of
greater number of algae in the rhizosphere.
• J. W. Rouatt reported positive rhizosphere effect on protozoa, but a
negative effect on algae in wheat plants.

2.1.12.1 Microorganisms in the Rhizosphere and Rhizosphere Effect

The rhizosphere region is a highly favorable habitat for the proliferation,
activity and metabolism of numerous microorganisms. The rhizosphere
2.44 Environmental Biotechnology

microflora can be enumerated intensively by microscopic, cultural and

biochemical techniques. Microscopic techniques reveal the types of organisms
present and their physical association with the outer root tissue surface / root
hairs. The cultural technique most commonly followed is “serial dilution and
plate count method” which reveal the quantitative and qualitative population
of microflora. At the same time, the cultural method shows the selective
enhancement of certain categories of bacteria. The biochemical techniques
used are designed to measure a specific change brought about by the plant
or by the microflora. The rhizosphere effect is on most commonly found
microorganisms viz. bacteria, actinomycetes, fungi, algae and protozoa.
Bacteria: The greater rhizosphere effect is observed with bacteria (R:S
values ranging from 10-20 or more) than with actinomycetes and fungi. Gram-
negative, rod shaped, non-sporulating bacteria which respond to root exudates
are predominant in the rhizosphere (Pseudomonas, Agrobacterium), while
Gram-positive, rods, cocci and aerobic spore forming (Bacillus, Clostridium)
are comparatively rare in the rhizosphere). The most common genera of
bacteria are: Pseudomonas, Arthrobacter, Agrobacterium, Alcaligenes, Azotobacter,
Mycobacterium, Flavobacter, Cellulomonas, Micrococcus and others have been
reported to be either abundant or sparse in the rhizosphere. From the agronomic
point of view, the abundance of nitrogen-fixing and phosphate-solubilizing
bacteria in the rhizosphere assumes a great importance. The aerobic bacteria
are relatively less in the rhizosphere because of the reduced oxygen levels due
to root respiration. The bacterial population in the rhizosphere is enormous
in the ranging form 10^8 to 10^9 per gram of rhizosphere soil. They cover
about 4-10% of the total root area occurring profusely on the root hair region
and rarely in the root tips. There is predominance of amino acids and growth
factors required by bacteria, are readily provided by the root exudates in the
region of rhizosphere.
Fungi: In contrast to their effects on bacteria, plant roots do not alter/
enhance the total count of fungi in the rhizosphere. However, rhizosphere
effect is selective and significant on specific fungal genera (Fusarium,
Verticillium, Aspergillus and Penicillium), which are stimulated. The R:S ratio
of fungal population is believed to be narrow in most of the plants, usually
not exceeding to 10. The soil/serial dilution and plating technique used for
the enumeration of rhizosphere fungi may often give erratic results as most
of the spore formers produce abundant colonies in culture media giving a
wrong picture/estimate (e.g., Aspergilli and Penicillia). In fact, the mycelial
forms are more dominant in the field. The zoospore forming lower fungi such
as Phytophthora, Pythium, Aphanomyces are strongly attracted to the roots in
response to particular chemical compounds excreted by the roots and cause
diseases under favorable conditions. Several fungi, e.g. Gibberella fujikurio
produces phytohormones and influence the plant growth.
Actinomycetes, Protozoa and Algae: Stimulation of actinomycetes in
the rhizosphere has not been studied in much detail so far. It is generally
Environmental Microbiology—Soil 2.45

understood that the actinomycetes are less stimulated in the rhizosphere

than bacteria. However, when antagonistic actinomycetes increase in number
they suppress bacteria. Actinomycetes may also increase in number when
antibacterial agents are sprayed on the crop. Among the actinomycete, the
phosphate solublizers (eg. Nocardia, Streptomyces) have a dominant role to play.
As rule, actinomycetes, protozoa and algae are not significantly influenced
by their proximity to the plant roots and their R:S ratios rarely exceed 2 to
3:1 and around roots of plants, R: S ratio for these microorganisms may go
too high. Because of large bacterial community, an increase in the number or
activity of protozoa is expected in the rhizosphere. Flagellates and amoebae
are dominant and ciliates are rare in the region.

2.1.12.2 Factors affecting microbial flora of the Rhizosphere/

Rhizosphere Effect
The most important factors which affect/influence the microbial flora of the
rhizosphere or rhizosphere effect are: soil type & its moisture, soil amendments,
soil pH, proximity of root with soil, plant species, and age of plant and root
exudates.
• Soil type and its moisture: In general, microbial activity and population
is high in the rhizosphere region of the plants grown in sandy soils
and least in the high humus soils, and rhizosphere organisms are more
when the soil moisture is low. Thus, the rhizosphere effect is more in
the sandy soils with low moisture content.
• Soil amendments and fertilizers: Crop residues, animal manure and
chemical fertilizers applied to the soil cause no appreciable effect on the
quantitative or qualitative differences in the microflora of rhizosphere.
In general, the character of vegetation is more important than the
fertility level of the soil.
• Soil pH/Rhizosphere pH: Respiration by the rhizosphere microflora
may lead to the change in soil rhizosphere pH. If the activity and
population of the rhizosphere microflora is more, then the pH of
rhizosphere region is lower than that of surrounding soil or non-
rhizosphere soil. Rhizosphere effect for bacteria and protozoa is more
in slightly alkaline, soil and for that of fungi, is more in acidic soils.
• Proximity of root with Soil: Soil samples taken progressively closer to
the root system have increasingly greater population of bacteria, and
actinomycetes and decreases with the distance and depth from the root
system. Rhizosphere effect decline sharply with increasing distance
between plant root and soil.
• Plant Species: Different plant species inhabit often variable microflora
in the rhizosphere region. The qualitative and quantitative differences
are attributed to variations in the rooting habits, tissue composition
2.46 Environmental Biotechnology

and excretion products. In general, legumes show/produce a more

pronounced rhizosphere effect than grasses or cereals. Biennials, due
to their long growth period, exert more prolonged stimulation on
rhizosphere effect than annuals.
• Age of Plant: The age of plant also alters the rhizosphere microflora and
the stage of plant maturity controls the magnitude of rhizosphere effect
and degree of response to specific microorganisms. The rhizosphere
microflora increases in number with the age of the plant and reaches at
peak during flowering which is the most active period of plant growth
and metabolism. Hence, the rhizosphere effect was found to be more
at the time of flowering than in the seedling or full maturity stage of
the plants. The fungal flora (especially, Cellulolytic and Amylolytic) of
the rhizosphere usually increases even after fruiting and the onset of
senescence due to accumulation of moribund tissue and sloughed off
root parts/tissues; whereas, bacterial flora of the rhizosphere decreases
after the flowering period and fruit setting.
• Root exudates/excretion: One of the most important factors responsible
for rhizosphere effect is the availability of a great variety of organic
substances at the root region by way of root exudates/excretions.
The quantitative and qualitative differences in the microflora of the
rhizosphere from that of general soil are mainly due to influences of
root exudates. The spectrum of chemical composition of root exudates
varies widely, and hence their influence on the microflora also varies
widely.
Root exudates are composed of the chemical substances like:
S.N. Root Exudates Chemical Substances
1 Amino acids All naturally occurring amino acids.
2 Organic acids Acetic, butyric, citric, fumaric, lactic, malic,
propionic, succinic, etc.
3 Carbohydrates/ Arabinose, fructose, galactose, glucose, maltose,
sugars mannose, oligosaccharides, raffinose, ribose,
sucrose, xylose etc.
4 Nucleic acid Adenine, cystidine, guanine, undine
derivatives
5 Growth factors Biotin, choline, inositol, pyridoxine etc
(phytohormones)
6 Vitamins Thiamine, nicotinic acid, biotin etc
7 Enzymes Amylase, invertase, protease, phosphatase etc.
8 Other compounds Auxins, glutamine, glycosides, hydrocyanic
acid peptides, UV-absorbing compounds,
nematode attracting factors, spore germination
stimulators, spore inhibitors etc.
Environmental Microbiology—Soil 2.47

The nature and amount of chemical substances thus exuded are

dependent on the species of plant, plant age, inorganic nutrients, temperature,
light intensity, O2/CO2 level, root injury, etc. Another source of nutrients
for the microorganisms in the rhizosphere region is the sloughed off root
epidermis which exert selective stimulation effect on some specific groups
of microorganisms. For instance, glucose and amino acids in the exudates
readily attract Gram-negative rods which predominantly colonize the roots.
Sugars and amino acids in the root exudates stimulate the germination of
chlamydospores and other resting spores of fungi; stimulation effect of root
exudates on plant pathogenic fungi, nematodes is also well known.

2.1.12.3. Alterations in Rhizosphere Microflora

Foliar application of various chemicals leads to alterations in the rhizosphere
microflora by changing the pattern of root exudates. The pattern of the
rhizosphere microflora i.e. numbers and species composition can be changed/
altered by various factors, such as:
• Soil amendments,
• Foliar application of fertilizers/nutrients, fungicides, insecticides and
hormones, and
• Bacterization/microbial seed inoculants.
Soil amendments: Soil amendments with inorganic and organic
fertilizers can alter the rhizosphere microflora and an understanding of
the type of changes in the microflora can be useful in the indirect control
of pathogens. Dwivedi and Chaube (1985) showed that amendment of soil
with neem-cake can stimulate the activity of actinomycetes which results into
the reduction of propagules of Macrophomina phaseolina. It is also known to
control phytopathogenic nematodes in soil by stimulating nematode-trapping
fungi. Amendment of soil with castor and bean leaves stimulate the activity of
Trichoderma viride and Penicillium in the rhizosphere leading to the control of
Sclerotium rolfsii.
Foliar application of fertilizers and agrochemicals: Translocation of
photosynthate from leaves to roots takes place as a part of the normal metabolic
activity in plants. Therefore, organic substances, including plant protection
chemicals (fungicides, insecticides), growth regulators and plant nutrients
applied to foliage/leaves get absorbed into the leaf tissue and further get
translocated to roots along with photosynthates. Many workers have reported
that foliar application with various chemicals cause marked alterations in the
number and kind/qualities of microorganisms in the rhizosphere of several
cereals and leguminous crop plants. Thus, such an approach can be used as
a new tool in the biological control of root diseases, stimulation of activity of
nitrogen-fixing bacteria and other beneficial microorganisms in the soil.
Seed treatment with bio inoculants: Bio-inoculants such as Azotobacter,
Beijerinckia, Azospirillum, Rhizobium or P-solubilizing microorganisms (eg.
2.48 Environmental Biotechnology

Bacillus polymyxa, Azotobacter croococcum, Aspergillus niger, Penicillium digitatum

etc.) when applied to the seed/soil, help in the establishment of beneficial
microorganisms in the rhizosphere region which will further benefit in plant
growth, encourage inhibition of plant pathogenic organisms in the root
vicinity and enrich the soil with added microbial biomass.

2.1.12.4 Associative and Antagonistic activities in the Rhizosphere

In natural environments (eg. Soil, Air, Water, etc.) a number of relationships
exist between individual microbes, microbial species and between individual
cells. The composition of microflora of any habitat (soil/rhizosphere) is
governed by the biological equilibrium created by the associations and
interactions of all individuals found in the community. In soil and rhizosphere
region, many microorganisms live in close proximity and their interactions
with each other may be associative or antagonistic.
• Associative interactions/activities in rhizosphere: The dependence
of one microorganism upon another for extra-cellular products (eg.
amino acids & growth promoting substances) can be regarded as an
associative activity/effect in rhizosphere. There is an increase in the
exudation of amino acids, organic acids and monosaccharides by plant
roots in the presence of microorganisms. Gibberellins and gibberellin-
like substances are known to be produced by bacterial genera viz.
Azotobacter, Arthrobacter, Pseudomonas, and Agrobacterium which are
commonly found in the rhizosphere. Microorganisms also influence
root hair development, mucilage secretion and lateral root development.
Fungi inhabiting the root surface facilitate the absorption of nutrient by
the roots. Mycorrhiza is one of the best known associative/symbiotic
interactions which exist between the roots of higher plants and fungi.
This mycorrhizal association has been found to improve plant growth
through better uptake of phosphorus and zinc from soil, suppression of
root pathogenic fungi and nematodes. Another example is association
between the bacterium Rhizobium and roots of legumes and Azospirillum
with cereal crops (wheat, rye, bajara, maize, etc.).
• Antagonistic interactions/activities in rhizosphere: The biochemical
qualities of root exudates and the presence of antagonistic
microorganisms, plays important role in encouraging or inhibiting
the soil-borne plant pathogens in the rhizosphere region. Several
mutualistic, communalistic, competitive and antagonistic interactions
exist in the rhizosphere. The number and qualities of antagonistic
microorganisms in the rhizosphere could be increased through
artificial means such as fertilizer application, organic amendments,
foliar spraying of chemicals, etc.
Antagonistic microorganisms in the rhizosphere play an important role in
controlling some of the soil-borne plant pathogens. Stanier and group (1966)
Environmental Microbiology—Soil 2.49

discovered the bacterial strain Pseudomonas fluorescens and the fluorescent

pigments of this species in biological control of root pathogens. Strains of
P. fluorescence are collectively called as “Fluorescent Pseudomonads”. They
produce variety of biologically active compounds such as plant growth
substances, cyanides, antibiotics and iron chelating substances called
“Siderophores”. Rovira and Campbell (1975), showed that bacterial strains
of P. fluorescens could lyse the hyphae of Gaumannomyces graminis var. Tritici,
the causative agent of take-all disease of wheat. Fluorescent pseudomonads
(P. fluorescens, P. putida) are known to produce iron chelating substances called
Siderophores. These are low molecular weight, extra-cellular, iron-binding
agents produced by pseudomonads in response to low iron stress or when
Fe3+ is in short supply. Thus, iron stress triggers the formation of iron-binding
ligands called siderophores. Siderophores contain the pigments Pyovirdin
(Fluorescent) and Pyocyanin (non-Fluorescent) having iron chelating
properties. Another pigment “Pseudobactin” is a fluorescent chelator of iron
which is known to promote plant growth and inhibition of pathogenic bacteria
in the rhizosphere. An antibiotic called “Pyrrolnitrin” reduces damping-off
disease in cotton caused by Rhizoctonia solani. Several species of Bacillus are
known to cause mycolysis in the rhizosphere. eg. Fusarium oxysporum hyphae
are known to undergo lysis in soil due to these bacterial metabolites.
The successful antagonists among fungi are Trichoderma sp (T. viride and T.
harzianum, T. hamatum) and Gliocladium virens which parasitize, lyse or kill the
phytopathogenic fungi in the soil. Antifungal and antibacterial actinomycetes
in the rhizosphere play an important role in controlling pathogenic fungi
and bacteria, for example, Micromonospora globosa is a potent antagonist of
Fusarium udum causing wilt of pigeon pea. Amoebae are also known to play
an antagonistic role in controlling soil fungi, eg. control of take-all disease of
wheat caused by Gaumannomyces graminis through the use of Myxamoebae.
There can also occur antagonisms between two fungi-producing metabolite
and interfering the growth of the other fungus as in case of Peniophora
antagonizing Heterobasidium.

2.1.12.5 Rhizosphere in relation to Plant Pathogens

Plant root exudates influence pathogenic fungi, bacteria and nematodes in
various ways. The effect may be in the form of attraction of fungal zoospores, or
bacterial cells towards the roots; stimulation of germination of dormant spores
and hatching of cysts of nematodes. Root exudates may contain inhibitory
substances preventing the establishment of pathogens. The balance between
the rhizosphere microflora and plant pathogens and soil microflora and
plant pathogens is important in host-pathogenic relationship. In this context,
the biochemical qualities of root exudates and the presence of antagonistic
micro-organisms plays an important role in the proliferation and survival of
root infecting pathogens in soil either through soil fungi stasis, inhibition or
antibiosis of pathogens in the rhizosphere.
2.50 Environmental Biotechnology

Some of the most common interactions between plant roots and plant
pathogenic microorganisms in the rhizosphere are as follows:
• Zoospore attraction: Amino acids, organic acids and sugars in the
root exudates stimulate the movement and attraction of zoospores
towards root of the plants. For example, attraction of zoospores has
been reported in Phytophthora citrophthora (Citrus roots), P. parasitica
(tobacco roots) and Pythium aphanidermatum (pea root).
• Spore germination: The spores or conidia of many pathogenic fungi
such as Rhizoctonia, Fusarium, Sclerotium, Pythium, Phytophthora, etc.
have been stimulated to germinate by the root exudates of susceptible
cultivars of the host plants. There are some reports on the selective
stimulation of Fusarium, Pseudomonas and root infecting nematodes in
the rhizosphere region of the respective susceptible hosts. This stimulus
to germination is especially important to those plant pathogens
which are not vigorous competitors and remain in resting stage due
to shortage of nutrients or fungistasis. As a rule, germination and
subsequent hyphal development are promoted by non-host species
and also by both susceptible and resistant cultivars of the host plants.
The quantity and quality of microorganisms present in the rhizosphere
of disease resistant crop varieties are significantly different from those
of susceptible varieties.
• Changes in morphology and physiology of host plant: Changes in the
physiology and morphology of host plant influence the rhizosphere
microflora through root exudations. Hence, significant changes in
the rhizosphere microflora of diseased plants were reported which
are attributed to the nature and severity of the disease. Systemic
virus diseases cause marked changes in the plant morphology and
physiology to drastically alter the rhizosphere microflora.
• Increase in antagonists activity: Root exudates provide a food base
for the growth of antagonistic organisms which plays an important
role in controlling/suppressing some of the soil-born plant pathogens.
Generally, rhizosphere of the resistant plant varieties harbour more
number of Streptomyces and Trichoderma than that of susceptible
varieties. For example, in the rhizosphere of pigeon pea varieties
resistant to Fusarium udum, the population of Streptomyces was found
more which inhibited the growth of the pathogen. High density of
Trichoderma viride in the rhizosphere of tomato varieties resistant to
Verticillium wilt has been reported with its ability to reduce the severity
of wilt in susceptible plants.
• Inhibition of pathogen: Root exudates containing toxic substances
such as glycosides and hydrocyanic acid may inhibit the growth of
pathogens in the rhizosphere. It has been reported that root exudates
from resistant varieties of Flax (eg. Bison) excrete a glucoside which on
Environmental Microbiology—Soil 2.51

hydrolysis produces hydrocyanic acid that inhibits Fusarium oxysporum,

the flax root pathogen. Exudates of resistant pea reduce the germination
of spores of Fusarium oxysporum. In this light, the rhizosphere may be
considered as a microbiological buffer zone in which the microflora
serves to protect the plants against the attack of the pathogens.
• Attraction of bacteria and nematodes: Root exudates attracts
phytopathogenic bacteria and fungi in the rhizosphere, for example,
Agrobacterium tumefaciens have been reported to be attracted to the
roots of the host plants like pea, maize, onion, tobacco, tomato and
cucumber.
Host root exudates also influence phytopathogenic nematodes in two
ways: (i) through stimulation of egg-hatching process and (ii) attraction of
larvae towards plant roots.

2.1.13 Soil Microorganisms in Cycling of Elements or Plant Nutrient

Soil microorganisms are the most important agents in the cycling/
transformation of various elements (N, P, K, S, Fe, etc.) in the biosphere; where
the essential elements undergo cyclic alterations between the inorganic state
as free elements in nature and the combined state in living organisms. Life on
earth is dependent on the cycling of nutrient elements from their elemental
states to inorganic compounds to organic compounds and back into their
elemental states. The microbes through the process of biochemical reactions
convert/breakdown complex organic compounds into simple inorganic
compounds and finally into their constituent elements. This process is known
as “Mineralization”. Mineralization of organic carbon, nitrogen, phosphorus,
sulphur and iron by soil microorganisms makes these elements available for
reuse by plants. In the following paragraphs, the cycling/transformations of
some of the important elements are discussed.
The four most important cycles are mention below:
• Nitrogen Cycle
• Sulphur Cycle/Sulphur Transformation
• Phosphorus Cycle/Transformation
• Iron Cycle/Transformation

2.1.13.1 Nitrogen Cycle

Although molecular nitrogen (N2) is abundant (i.e 78-80 % by volume) in the
earth’s atmosphere, but it is chemically inert and therefore, can not be utilized
by most living organisms and plants. Plants, animals and most microorganisms,
depend on a source of combined or fixed nitrogen (eg. ammonia, nitrate) or
organic nitrogen compounds for their nutrition and growth. Plants require
fixed nitrogen (ammonia, nitrate) provided by microorganisms, but about
2.52 Environmental Biotechnology

95 to 98 % soil nitrogen is in organic form (unavailable) which restricts the

development of living organisms including plants and microorganisms.
Therefore, cycling/transformation of nitrogen and nitrogenous compounds
mediated by soil microorganisms is of paramount importance in supplying
required forms of nitrogen to the plants and various nutritional classes of
organisms in the biosphere. In nature, nitrogen exists in three different forms
viz. gaseous/gas (78 to 80 % in atmosphere), organic (proteins and amino
acids, chitins, nucleic acids and amino sugars) and inorganic (ammonia and
nitrates).
Biological N2 Fixation:
A. Symbiotic: Eg. Rhizobium (Eubacteria) legumes, Frankia (Actinomycete)
and Anabaena (cyanobacteria) non-legumes.
B. Non-symbiotic:
1. Free Living: e.g. Azobacter, Derxia, Bejerinkia, Rhodospirillum and BGA.
2. Associative: e.g. Azospirillum, Acetobacter, Herbaspirillim.
Nutritional categories of N2 fixing Bacteria:
A. Heterotrops
B. Photoautotrophs
The sequence of biochemical changes from free atmospheric N2 to
complex organic compounds in plant and animal tissues and further to simple
inorganic compounds (ammonia, nitrate) and eventual release of molecular
nitrogen (N2) back to the atmosphere is called “nitrogen cycle”.
In this cycle, a part of atmospheric nitrogen (N2) is converted into
ammonia and then to amino acids (by soil microorganisms and plant-microbe
associations) which are used for the biosynthesis of complex nitrogen-
containing organic compounds such as proteins, nucleic acids, amino
sugars, etc. The proteins are then degraded to simpler organic compounds
viz. peptones and peptides into amino acids, which are further degraded to
inorganic nitrogen compounds like ammonia, nitrites and nitrates. The nitrate
form of nitrogen is mostly used by plants or may be lost through leaching or
reduced to gaseous nitrogen and subsequently goes into the atmosphere, thus
completing the nitrogen cycle. Thus, the process of mineralization (conversion
of organic form of nutrients to its mineral/inorganic form) and immobilization
(process of conversion of mineral/inorganic form of nutrient elements into
organic form) are continuously and simultaneously going on in the soil.
Several biochemical steps involved in the nitrogen cycle are:
• Proteolysis
• Ammonification
• Nitrification
Environmental Microbiology—Soil 2.53

• Nitrate reduction and

• Denitrification.
Proteolysis: Plants use the ammonia produced by symbiotic and non-
symbiotic nitrogen-fixation to make their amino acids and eventually, plant
proteins. Animals eat the plants and convert plant proteins into animal proteins.
Upon death, plant and animals undergo microbial decay in the soil and the
nitrogen contained in their proteins is released. Thus, the process of enzymatic
breakdown of proteins by the microorganisms with the help of proteolysis
enzymes is known as “proteolysis”. The breakdown of proteins is completed in
two stages. In first stage, proteins are converted into peptides or polypeptides
by enzyme “proteinases” and, in the second stage, polypeptides/peptides are
further broken down into amino acids by the enzyme “peptidases”.

Proteins 
→ Peptides 
→ Amino Acids
Proteinases Peptidases
The amino acids produced may be utilized by other microorganisms for the
synthesis of cellular components, absorbed by the plants through mycorrhiza
or may be deanimated to yield ammonia. The most active microorganisms
responsible for elaborating the proteolytic enzymes (Proteinases and
Peptidases) are Pseudomonas, Bacillus, Proteus, Clostridium Histolyticum,
Micrococcus, Alternaria, Penicillium, etc.
Ammonification (Ammo acid degradation): Amino acids released during
proteolysis undergo deamination in which nitrogen containing amino (–NH2)
group is removed. Thus, process of deamination which leads to production
of ammonia is termed as “ammonification”. The process of ammonification
is mediated by several soil microorganisms. Ammonification usually occurs
under aerobic conditions (known as oxidative deamination) with the liberation
of ammonia (NH3) or ammonium ions (NH4) which are either released into the
atmosphere or utilized by plants (paddy) and microorganisms, or still under
favorable soil conditions, oxidized to form nitrites and then to nitrates.
The processes of ammonification are commonly brought about by
Clostridium sp. Micrococcus sp. Proteus sp., etc. and it is represented as follows.

CH 3CHNH 2 COOH + 1 / 2O 2 de


min ase
→ CH 3COCOOH + NH 3
Alanine Pyruvic acid Ammonia
                                                   
Nitrification: Ammonical nitrogen/ammonia released during ammoni-
fication are oxidized to nitrates and the process is called “nitrification”. Soil
conditions such as well aerated soils rich in calcium carbonate, a temperature
below 30°C, neutral pH, and less organic matter are favorable for nitrification
in soil.
Nitrification is a two stage process and each stage is performed by a
different group of bacteria as follows.
2.54 Environmental Biotechnology

Stage I: Oxidation of ammonia of nitrite is brought about by ammonia

oxidizing bacteria viz. Nitrosomnonas europaea, Nitrosococcus nitrosus,
Nitrosospira briensis, Nitrosovibrio and Nitrocystis and the process is known as
nitrosification. The reaction is presented as follows.
2 NH 3 + I / 2O 2 → NO 2 + 2H 2 + H 2 O
Ammonia Nitrite

Stage II: In the second step, nitrite is oxidized to nitrate by nitrite-oxidizing

bacteria such as Nitrobacter winogradsky. Nitrospira gracilis, Nirosococcus
mobiiis etc. and several fungi (e.g. Penicillium, Aspergillus) and actinomycetes
(e.g. Streptomyces, Nocardia).
1
NO(2− ) + O 2 → NO(3− )
2 Nitrate ions
Nitrite ions

The nitrate, thus formed, may be utilized by the microorganisms,

assimilated by plants, reduced to nitrite and ammonia or nitrogen gas or
lost through leaching depending on soil conditions. The nitrifying bacteria
(ammonia oxidizer and nitrite oxidizer) are aerobic gram-negative and
chemoautotrophic and are the common inhabitants of soil, sewage and aquatic
environment.
Nitrate Reduction: Several heterotrophic bacteria (E. coli, Azospirillum)
are capable of converting nitrates to nitrites and nitrites to ammonia. Thus,
the process of nitrification is reversed completely which is known as nitrate
reduction. Nitrate reduction normally occurs under anaerobic soil conditions
(waterlogged soils) and the overall process is as follows:
Nitrate
HNO 3 + 4H 2 Reductase
 → NH 3 + 3H 2 O
Nitrate Ammonia

Nitrate reduction leading to production of ammonia is called “dissimilatory

nitrate reduction” as some of the microorganisms assimilate ammonium for
synthesis of proteins and amino acid.
Denitrification: This is the reverse process of nitrification. During
denitrification, nitrates are reduced to nitrites and then to nitrogen gas and
ammonia. Thus, reduction of nitrates to gaseous nitrogen by microorganisms
in a series of biochemical reactions is called “denitrification”. The process is
wasteful, as available nitrogen in soil is lost to the atmosphere. The overall
process of denitrification is as follows:
NaR NIR NOR
Nitrate 
→ Nitrite 
→ Nitric Oxide →
NoR
→ Nitrogen gas
Nitrous Oxide 
This process also called dissimilatory nitrate reduction as nitrate nitrogen
is completely lost into atmospheric air. In the soils with high organic matter and
anaerobic soil conditions (waterlogged or ill-drained), rate of denitrification is
more. Thus, rice/paddy fields are more prone to denitrification.
Environmental Microbiology—Soil 2.55

The most important denitrifying bacteria are Thiobacillus denitrificans,

Micrococcus denitrificans, and species of Pseudomonas, Bacillus, Achromobacter,
Serrtatia paracoccus, etc. Denitrification leads to the loss of nitrogen (nitrate
nitrogen) from the soil which results into the depletion of an essential nutrient
for plant growth and therefore, it is an undesirable process/reaction from
the soil fertility and agricultural productivity. Although, denitrification is an
undesirable reaction from agricultural productivity, but it is of major ecological
importance since, without denitrification the supply of nitrogen including N2
of the atmosphere, would have not got depleted and NO3 (which are toxic)
would have accumulated in the soil and water.

2.1.13.2 Sulphur Cycle/Sulphur Transformations

Sulphur is the most abundant and widely distributed element in the nature
and found both in free as well as combined states. Sulphur, like nitrogen
is an essential element for all living systems. In the soil, sulphur is in
the organic form (sulphur containing amino acids—cystine, methionine,
proteins, polypeptides, biotin, thiamine, etc.) which is metabolized by soil
microorganisms to make it available in an inorganic form (sulphur, sulphates,
sulphite, thiosulphale, etc.) for plant nutrition. Of the total sulphur present is
soil, only 10-15% is in the inorganic form (sulphate) and about 75-90% is in
organic form. Cycling of sulphur is similar to that of nitrogen. Transformation/
cycling of sulphur between organic and elemental states and between oxidized
and reduced states, is brought about by various microorganisms, especially
bacteria. Thus, “the conversion of organically bound sulphur to the inorganic
state by microorganisms is termed as mineralization of sulphur”. The sulphur/
sulphate, thus released are either absorbed by the plants or escape to the
atmosphere in the form of oxides.
Various transformations of the sulphur in soil result mainly due to
microbial activity, although some chemical transformations are also possible
(eg. oxidation of iron sulphide). The major types of transformations involved
in the cycling of sulphur are:
• Mineralization
• Immobilization
• Oxidation and
• Reduction
• Mineralization: The breakdown/decomposition of large organic
sulphur compounds to smaller units and their conversion into inorganic
compounds (sulphates) by the microorganisms. The rate of sulphur
mineralization is about 1.0 to 10.0 percent/year.
• Immobilization: Microbial conversion of inorganic sulphur compounds
to organic sulphur compounds.
• Oxidation: Oxidation of elemental sulphur and inorganic sulphur
compounds (such as H2S, sulphite and thiosulphate) to sulphate (SO4)
is brought about by chemoautotrophic and photosynthetic bacteria.
2.56 Environmental Biotechnology

When plant and animal proteins are degraded, the sulphur is released
from the amino acids and accumulates in the soil which is then oxidized to
sulphates in the presence of oxygen, and under anaerobic condition (water
logged soils), organic sulphur is decomposed to produce hydrogen sulphide
(H2S). H2S can also accumulate during the reduction of sulphates under
anaerobic conditions which can be further oxidized to sulphates under aerobic
conditions.
Ionization
(a ) 2S + 3O 2 + 2H 2 O Light
→ 2H 2 SO 4 
→ 2H( + ) + SO 4 ( Aerobic)
( b) CO 2 + 2H 2 S Light
→(CH 2 O) + H 2 O + 2S

OR H 2 + S + 2CO 2 + H 2 O 
→ H 2 SO 4 + 2(CH 2 O)(anaerobic)
The members of genus Thiobacillus (obligate chemolithotrophic, non-
photosynthetic) eg, T. ferrooxidans and T. thiooxidans are the main organisms
involved in the oxidation of elemental sulphur to sulphates. These are
aerobic, non-filamentous, chemosynthetic autotrophs. Other than Thiobacillus,
heterotrophic bacteria (Bacillus, Pseudomonas, and Arthrobacter) and fungi
(Aspergillus, Penicillium), some actinomycetes are also reported to oxidize
sulphur compounds. Green and purple bacteria (Photolithotrophs) of genera
Chlorbium, Chromatium, Rhodopseudomonas are also reported to oxidize sulphur
in aquatic environment.
Sulphuric acid produced during oxidation of sulphur and H:S is of great
significance in reducing the pH of alkaline soils and in controlling potato scab
and rot diseases caused by Streptomyces bacteria. The formation of sulphate/
Sulphuric acid is beneficial in agriculture in different ways : (i) as it is the
anion of strong mineral acid (H2SO4) itcan render alkali soils fit for cultivation
by correcting soil pH. (ii) solubilize inorganic salts containing plant nutrients
and thereby increase the level of soluble phosphate, potassium, calcium,
magnesium, etc. for plant nutrition.
Reduction of Sulphate: Sulphate in the soil is assimilated by plants and
microorganisms and incorporated into proteins. This is known as “assimilatory
sulphate reduction”. Sulphate can be reduced to hydrogen sulphide (H2S) by
sulphatereducing bacteria (eg. Desulfovibrio and Desulfatomaculum) and may
diminish the availability of sulphur for plant nutrition. This is “dissimilatory
sulphate reduction” which is not at all desirable from soil fertility and
agricultural productivity viewpoint.
Dissimilatory sulphate-reduction is favored by the alkaline and anaerobic
conditions of soil and sulphates are reduced to hydrogen sulphide. For
example, calcium sulphate is attacked under anaerobic condition by the
members of the genus Desulfovibrio and Desulfatomaculum to release H2 S.

CaSO 4 + 4H 2 
→ Ca(OH)2 + H 2 S + 2H 2 O
Environmental Microbiology—Soil 2.57

Hydrogen sulphide produced by the reduction of sulphate and sulphur

containing amino acids decomposition is further oxidized by some species
of green and purple phototrophic bacteria (e.g. Chlorobium, Chromatium) to
release elemental sulphur.

Light
CO 2 + 2H 2 + H 2 S Enzyme
 → (CH 2 O) + H 2 O + 2S
Carbohydrate

The predominant sulphate-reducing bacterial, genera in soil are

Desulfovibrio, Desulfatomaculum and Desulfomonas (all obligate anaerobes).
Amongst these species, Desulfovibrio desulfuricans are most ubiquitous, non-
spore forming, obligate anaerobes that reduce sulphates at rapid rate in
waterlogged/flooded soils. While species of Desulfatomaculum are spore-
forming, thermophilic obligate anaerobes reduce sulphates in dry land soils.
All sulphate-reducing bacteria excrete an enzyme called “desulfurases” or
“bisulphate Reductase”. Rate of sulphate reduction in nature is enhanced by
increasing water levels (flooding), high organic matter content and increased
temperature.

2.1.13.3  Phosphorus Cycle or Transformation

Phosphorus is only second to nitrogen as a mineral nutrient required for
plants, animals and microorganisms. It is a major constituent of nucleic acids
in all living systems essential in the accumulation and release of energy during
cellular metabolism. This element is added to the soil in the form of chemical
fertilizers, or in the form of organic phosphates present in plant and animal
residues. In cultivated soils, it is present in abundance (i.e. 1100 kg/ha), but
most of this is not available to plants; only 15 % of total soil phosphorus is in
available form. Both inorganic and organic phosphates exist in soil and occupy
a critical position both in plant growth and in the biology of soil.
Microorganisms are known to bring a number of transformations of
phosphorus, these include:
• Altering the solubility of inorganic compounds of phosphorus,
• Mineralization of organic phosphate compounds into inorganic
phosphates,
• Conversion of inorganic, available anion into cell components i.e. an
immobilization process and
• Oxidation or reduction of inorganic phosphorus compounds. Of these,
mineralization and immobilization are the most important reactions/
processes in phosphorus cycle.
Insoluble inorganic compounds of phosphorus are unavailable to plants, but
many microorganisms can bring the phosphate into solution. Soil phosphates
are rendered available either by plant roots or by soil microorganisms
through secretion of organic acids (e.g. lactic, acetic, formic, fumaric, succinic
2.58 Environmental Biotechnology

acids, etc.). Thus, phosphate-dissolving/solubilizing soil microorganisms

(e.g. species of Pseudomonas, Bacillus, Micrococcus, Mycobacterium,
Flavobacterium, Penicillium, Aspergillus, Fusarium, etc.) play important role in
correcting phosphorus deficiency of crop plants. They may also release soluble
inorganic phosphate (H2PO4) into soil through decomposition of phosphate-
rich organic compounds.
Solubilization of phosphate by plant roots and soil microorganisms is
substantially influenced by various soil factors, such as pH, moisture and
aeration.
In neutral or alkaline soils, solubilization of phosphate is more as compared
to acidic soils. Many phosphate-solubilizing microorganisms are found in
close proximity of root surfaces and may appreciably enhance phosphate
assimilation by higher plants.
By their action, fungi, bacteria and actinomycetes make available the
organically bound phosphorus in soil and organic matter and the process
is known as mineralization. On the other hand, certain microorganisms,
especially bacteria, assimilate soluble phosphate and use it for cell synthesis
and on the death of bacteria, the phosphate is made available to plants. A
fraction of phosphate is also lost in soil due to leaching. One of the ways to
correct deficiency of phosphorus in plants is to inoculate seed or soil with
commercial preparations (e.g. Phosphobacterin) containing phosphate-
solubilizing microorganisms along with phosphatic fertilizers.
Mineralization of phosphate is generally rapid and is more in virgin
soils than cultivated land. Mineralization is favored by high temperatures
(thermophilic range) and is more in acidic to neutral soils with high organic
phosphorus content. The enzymes involved in mineralization (cleavage) of
phosphate from organic phosphorus compound are collectively called as
“Phospatases”.
The commercially used species of phosphate-solubilizing bacteria
and fungi are: Bacillus polymyxa, Bacillus megatherium, Pseudomonas strita,
Aspergillus, Penicllium avamori and Mycorrhiza.

2.1.13.4 Iron Cycle or Transformation

Iron exists in nature either as ferrous (Fe++) or ferric (Fe+++) ions. Ferrous
iron is oxidized spontaneously to ferric state, forming highly insoluble ferric
hydroxide. Plants as well as microorganisms require traces of iron, manganese
copper, zinc, molybdenum, calcium, boron, cobalt etc. Iron is always abundant
in terrestrial habitats, and it is oftenly in an unavailable form for utilization by
plants and leads to the serious deficiency in plants.
Soil microorganisms play important role in the transformations of iron in
a number of distinctly different ways such as:
A. Certain bacteria oxidize ferrous iron to ferric state which precipitate as
ferric hydroxide around cells.
Environmental Microbiology—Soil 2.59

B. Many heterotrophic species attack on insoluble organic iron salts and

convert into inorganic salts.
C. Oxidation-reduction potential decreases with microbial growth which
leads to the formation of more soluble ferrous iron from highly insoluble
ferric ions.
D. Number of bacteria and fungi produce acids such as carbonic, nitric,
sulphuric and organic acids which brings iron into solution.
E. Under anaerobic conditions, the sulfides formed from sulphate and
organic sulphur compounds, remove the iron from solution as ferrous
sulfide.
F. As microbes liberate organic acids and other carbonaceous products
of metabolism, this results in the formation of soluble organic iron
complex. Thus, iron may be precipitated in nature and immobilized
by iron oxidizing bacteria under alkaline soil reaction and on the other
hand, solubilization of iron may occur through acid formation.
Some bacteria are capable of reducing ferric iron to ferrous, which lowers
the oxidation-reduction potential of the environment (eg. Bacillus, Clostridium,
Klebsiella, etc). However, some chemoautotrophic iron and sulphur bacteria
such as ThiobacillusT. Ferroxiders and Ferrobacillus ferrooxidans can oxidize
ferrous iron to ferric hydroxide which accumulates around the cells.
Most of the aerobic microorganisms live in an environment where iron
exists in the oxidized, insoluble ferric hydroxide form. They produce iron-
binding compounds in order to take up ferric iron. The iron-binding or chelating
compounds/ligands produced by microorganisms are called “Siderophores”.
Bacterial siderophores may act as virulence factors in pathogenic bacteria
and thus, bacteria that secrete siderophores are more virulent than non-
siderophores producers. Therefore, siderophore-producing bacteria can be
used as biocontrol agents eg. Fluorescent pseudomonads are used to control
Pythium, causing damping-off diseases in seedlings. Recently Vascular–
Arbusecular–Mycorrhiza (VAM) has been reported to increase uptake of iron.

2.1.14 Organic Matter

Soil organic matter plays important role in the maintenance and improvement
of soil properties. It is a dynamic material and is one of the major sources of
nutrient elements for plants. Soil organic matter is derived to a large extent
from residues and remains of the plants together with the small quantities
of animal remains, excreta, and microbial tissues. Soil organic matter is
composed of three major components i.e. plants residues, animal remains
and dead remains of microorganisms. Various organic compounds are
made up of complex carbohydrates (cellulose, hemicellulose, starch), simple
sugars, lignins, pectins, gums, mucilages, proteins, fats, oils, waxes, resins,
alcohols, organic acids, phenols, etc. and other products. All these compounds
constituting the soil organic matter can be categorized in the following way.
2.60 Environmental Biotechnology

Organic Matter (Undecomposed)

A. Organic:
• Nitrogenous:
1. Water Soluble e.g. nitrates, ammonical compounds, amides, amino
acids, etc.
2. Insoluble e.g. proteins, nucleoproteins, peptides, alkaloids purines,
pyridines, chitin, etc.
• Non Nitrogenous:
— Carbohydrates e.g. sugars, starch, hemicellulose, gums, mucilage,
pectins, etc.
— Micellaneous: e.g. lignin, tannins, organic acids, etc.
— Ether solube: e.g. fats, oils, waxes etc.
B. Inorganic  
The organic complex/matter in the soil is, therefore made up of a large
number of substances of widely different chemical composition and the
amount of each substance varies with the type, nature and age of plants. For
example, cellulose in a young plant is only half of the mature plants; water-
soluble organic substances in young plants are nearly double to that of older
plants. Among the plant residues, leguminous plants are rich in proteins
than the non-leguminous plants. Grasses and cereal-straws contain greater
amount of cellulose, lignin, hemicelluloses than the legumes, and as the
plant gets older, the proportion of cellulose, hemicelluloses and lignin gets
increased. Plant residues contain 15-60% cellulose, 10-30 % hemicelluslose,
5-30% lignin, 2-15 % protein and 10% sugars, amino acids and organic acids.
These differences in composition of various plant and animal residues have
great significance on the rate of organic matter decomposition, in general,
and of nitrification and humification (humus formation), in particular. The
end products of decomposition are CO2, H2O, NO3, SO4, CH4, NH4, and H2S
depending on the availability of air.

Factors Influencing rate of Organic Matter Decomposition

In addition to the composition of organic matter, nature and abundance of
microorganisms in soil, the extent of C, N, P and K, moisture content of the soil
and its temperature, pH, aeration, C:N ratio of plant residues and presence/
absence of inhibitory substances (e.g. tannins), etc. are some of the major
factors which influence the rate of organic matter decomposition.
As soon as plant and animal residues are added to the soil, there is a rapid
increase in the activity of microorganisms. These are not true soil organisms,
but they continue their activity by taking part in the decomposition of organic
matter, and thereby release plant nutrients in the soil. Bacteria are the most
abundant organisms playing important role in the decomposition of organic
matter. Majority of bacteria involved in decomposition of organic matter are
Environmental Microbiology—Soil 2.61

heterotrophs and autotrophs are least in proportion which are not directly
involved in organic matter decomposition. Actinomycetes and fungi are also
found to play important role in the decomposition of organic matter. Soil algae
may contribute a small amount of organic matter through their biomass but
they do not have any active role in organic matter decomposition. The various
microorganisms involved in the decomposition of organic matter are listed in
the following table.
List of Microorganisms involved in organic matter decomposition
Constituents Microorganisms
Bacteria Fungi Actinomycetes
Cellulose Achromobacter, Aspergillus, Micromonospora,
Bacillus,  Chaetomium, Nocardia,
Cellulomonas, Fusarium, Streptomyces,
Cellvibrio, Pencillium Thermonospora
Clostridium, Rhizoctonia,
Cytophaga, Vibrio Rhizopus,
Pseudomonas, Trichoderma,
Sporocytophaga, Verticilltttm
etc.
Hemicellulose Bacillus, Aspergillus, Streptomyces,
Achromobacter, Fusarium, Actinomycetes
Cytophaga Chaetomium,
Pseudomonas, Penicillium,
Erwinia, Vibrio, Trichoderma,
Lactobacillus Humicola
Lignin Flavobacterium, Humicola, Streptomyces,
Pseudomonas, Fusarium Fames, Nocardia
Micrococcus, Pencillium,
Arthorbacter, Aspergillus,
Xanthomonas Ganoderma
Starch Achromobacter, Ftisarium, Fomes, Micromonospora,
Bacillus, Aspergillus, Nocardia,
Clostridium Rhizopus Streptomyces
Pectin Bacillus, Ftisarium,  
Clostridium, Verticillum
Pseudomonas
Chitin Bacillus, Mucor, Fusarium, Streptomyces,
Achromobacter, Aspergillus, Nocardia,
Cytophaga, Trichoderma Micromanospora
Pseudomonas
Proteins & Bacillus, Penicillium, Streptomyces
Nucleic acids Pseudomonas, Rhodotorula
Clostriddum,
Serratia,
Micrococcus
2.62 Environmental Biotechnology

Aeration: Good aeration is necessary for the proper activity of the

microorganisms involved in the decomposition of organic matter. Under
anaerobic conditions, fungi and actinomycetes are almost suppressed and
only a few bacteria (Clostridium) take part in anaerobic decomposition. The
rate of decomposition is markedly retarded. It was found that under aerobic
conditions, 65 per cent of the total organic matter decomposes during six
months, while under anaerobic conditions only 47 per cent organic matter can
be decomposed during the same period. Anaerobic decomposition of organic
matter results into production of large quantity of organic acids and evolution
of gases like methane (CH4) hydrogen (H2) and carbon dioxide (CO2).
Temperature: The rate of decomposition is more rapid in the temperature
range of 30° to 40°. At temperatures below or above this range, the rate
of decomposition is markedly retarded. Appreciable organic matter
decomposition occurs at 25°C and further fluctuation in the soil temperature
has little effect on decomposition.
Moisture: Adequate soil moisture i.e. about 60 to 80 per cent of the
water-holding capacity of the soil is must for the proper decomposition of
organic matter. Too much moisture leads to insufficient aeration which results
in the reduced activity of microorganisms and thereby, checks the rate of
decomposition.
Soil pH/soil reaction: Soil pH affects directly the kind, density and
the activity of fungi, bacteria and actinomycetes involved in the process of
decomposition and thereby rate of decomposition of organic matter. The rate
of decomposition is more in neutral soils than that of acidic soils. Therefore,
treatment of acid soils with lime can accelerate the rate of organic matter
decomposition.
C:N ratio: C:N ration of organic matter has great influence on the rate
of decomposition. Organic matter from diverse plant-tissues varies widely in
their C:N ratio (app. 8-10 %). The optimum C:N ratio in the range of 20-25
is ideal for maximum decomposition, since a favorable soil environment is
created to bring about equilibrium between mineralization and immobilization
processes. Thus, a low nitrogen content or wide C:N ratio results into slow
decomposition. Protein rich, young and succulent plant tissues are decomposed
more rapidly than the protein-poor, mature and hard plant tissues. Therefore,
C:N ratio of organic matter as well as soil should be narrow/less for better
and rapid decomposition. Thus, high aeration, mesophilic temperature range,
optimum moisture, neutral/alkaline soil reaction and narrow C:N ratio of soil
and organic matter are required for rapid and better decomposition of organic
matter.

2.1.14.1 Microbiology of decomposition of various constituents in

organic matter
When plant and animal residues are added to the soil, the various constituents
of the soil organic matter are decomposed simultaneously by the activity of
Environmental Microbiology—Soil 2.63

microorganisms and carbon is released as CO2, and nitrogen as NH4 →NO3 ,

for the use by plants. Other nutrients are also converted into plant usable
forms. This process of release of nutrients from organic matter is called
mineralization. The insoluble plant residues constitute the part of humus and
soil organic matter complex. The final product of aerobic decomposition is CO2
and that of anaerobic decomposition are hydrogen, ethyl alcohol (C2H5OH),
various organic acids and carbon dioxide (CO2). Soil organisms use organic
matter as a source of energy and food.
The process of decomposition is initially fast, but slows down considerably
as the supply of readily decomposable organic matter gets exhausted. Sugars,
water-soluble nitrogenous compounds, amino acids, lipids, starches and
some of the hemicellulases are decomposed first at rapid rate, while insoluble
compounds, such as cellulose, hemicellulose, lignin, proteins, etc., which form
the major portion of organic matter are decomposed later, slowly. Thus, the
organic matter added to the soil is converted by oxidative decomposition to
simpler substances which are made available in stages for plant growth and
the residue is transformed into humus.
The microbiology of decomposition/degradation of some of the major
constituents (viz. cellulose, hemicellulose, lignin, proteins, etc.) of soil organic
matter/plant residues are discussed in brief in the following paragraphs.
Decomposition of Cellulose:  Cellulose is the most abundant carbohydrate
present in plant residues/organic matter in nature. When cellulose is associated
with pentosans (eg. xylans & mannans), it undergoes rapid decomposition,
but when associated with lignin, the rate of decomposition is very slow. The
decomposition of cellulose occurs in two stages: (i) in the first stage, the long
chain of cellulase is broken down into cellobiose and then into glucose by the
process of hydrolysis in the presence of enzymes cellulase and cellobiase, and
(ii) in the second stage into glucose is oxidized and converted CO2 and water.
Cellulose Cellobiase
1. Cellulose 
Hydrolysis
→ Cellobiose 
Hydrolysis
→ Glucose
Oxidation Oxidation
2. Glucose → Organic Acids → CO 2 + H 2 O
The intermediate products formed/released during enzymatic hydrolysis
of cellulose (eg. cellobiose and glucose) are utilized by the cellulose-
decomposing organisms or by other organisms as source of energy for
biosynthetic processes. The cellulolytic microorganisms responsible for
degradation of cellulose through the excretion of enzymes (cellulase &
cellobiase) are fungi, bacteria and actinomycetes.
Decomposition of Hemicelluloses: Hemicelluloses are water-soluble
polysaccharides and consist of hexoses, pentoses, and uronic acids and are the
major plant constituents second only in quantity to cellulose, and sources of
energy and nutrients for soil microflora.
When subjected to microbial decomposition, hemicelluloses degrade
initially at faster rate and are first hydrolyzed to their component sugars and
2.64 Environmental Biotechnology

uronic acids. The hydrolysis is brought about by number of hemicellulolytic

enzymes known as “hemicellulases” excreted by the microorganisms. On
hydrolysis, hemicelluloses are converted into soluble monosaccharides/sugars
(e.g. xylose, arabinose, galactose and mannose) which are further converted
to organic acids, alcohols, CO2 and H2O and uronic acids are broken down
to pentoses and CO2. Various microorganisms including fungi, bacteria and
actinomycetes, both aerobic and anaerobic, are involved in the decomposition
of hemicelluloses.
Lignin Decomposition: Lignin is the third most abundant constituent of
plant tissues, and accounts about 10-30 per cent of the dry matter of mature
plant materials. Lignin content of young plants is low and gradually increases
as the plant grows old. It is one of the most resistant organic substances for
the microorganisms to degrade, however, certain Basidiomycetous fungi are
known to degrade lignin at slow rates. Complete oxidation of lignin result in
the formation of aromatic compounds such as syringaldehydes, vanillin and
ferulic acid. The final cleavages of these aromatic compounds yield organic
acids, carbon dioxide, methane and water.
Protein Decomposition: Proteins are complex organic substances
containing nitrogen, sulphur, and sometimes phosphorus in addition to
carbon, hydrogen and oxygen. During the course of decomposition of organic
matter, proteins are first hydrolyzed to a number of intermediate products,
e.g. proteases, peptides etc. collectively known as polypeptides
The intermediate products so formed are then hydrolyzed and broken
down ultimately to individual amino acids, or ammonia and amides. The
process of hydrolysis of proteins to amino acids is known as “aminization
or ammonification”, which is brought about by certain enzymes, collectively
known as “proteases” or “proteolytic” enzymes secreted by various
microorganisms. Amino acids and amines are further decomposed and
converted into ammonia. During the course of ammonification, various organic
acids, alcohols, aldehydes, etc. are produced which are further decomposed
finally to produce carbon dioxide and water.
All types of microorganisms, bacteria, fungi, and actinomycetes are able
to bring about decomposition of proteins. In acid soils, fungi are predominant,
while in neutral and alkaline soils, bacteria are dominant decomposers of
proteins.

2.1.15 Notable contributions made by several scientists in the field

of soil microbiology
There is enough evidence in the literature to believe that microorganisms
were the earliest of the living things that existed on this planet. Man depends
on crop plants for his existence and crop plants in turn depend on soil
and soil microorganisms for their nutrition. Scientists form the beginning
studied the microorganisms from water, air, soil, etc. and recognized the
Environmental Microbiology—Soil 2.65

role of microorganisms in natural processes and realized the importance

of soil microorganisms in growth and development of plants. Thus, we see
that microorganisms have been playing a significant role, long before they
were discovered by man. Today, soil is considered to be the main source of
scavenging the organic wastes through microbial action and is also a rich
storehouse for industrial microflora of great economic importance. Unlike
soil science whose origin can be traced back to Roman and Aryan times, soil
microbiology emerged as a distinct branch of soil science during the first half
of the 19th century. Some of the notable contributions made by several scientists
in the field of soil microbiology are highlighted in the following paragraphs.
• V. Leeuwenhock (1673) discovered and described microorganisms
through his self-made first simple microscope with magnification of
200 to 300 times. He observed minute, moving objects which he called
“animalcules” (small animals) which are now known as protozoa,
fungi and bacteria. He, for the first time, made the authentic drawings
of microorganisms (protozoa, bacteria, fungi).
• Robert Hook (1635-1703) developed a compound microscope with
multiple lenses and described the fascinating world of the microbes.
• J. B. Boussingault (1838) showed that leguminous plants can fix
atmospheric nitrogen and increase nitrogen content in the soil.
• J. Von Liebig (1856) showed that nitrates were formed in soil due to
addition of nitrogenous fertilizers in soil.
• S. N. Winogradsky discovered the autotrophic mode of life among
bacteria and established the microbiological transformation of
nitrogen and sulphur. He isolated for the first time nitrifying bacteria
and demonstrated role of these bacteria in nitrification (l890); further
he demonstrated that free-living Clostridium pasteuriamum could fix
atmospheric nitrogen (1893). Therefore, he is considered as “Father of
soil microbiology”.
• W. B. Leismaan (1858) and M. S. Woronin (1866) demonstrated that
root nodules in legumes were formed by a specific group of bacteria.
• Jodin (1862, France) gave the first experimental evidence of elemental
nitrogen fixation by microorganisms.
• Robert Koch (1882) developed gelatin plate/streak plate technique for
isolation of specific type of bacteria in soil, formulated Koch’s postulates
to establish causal relationship between host-pathogen and disease.
• R. Warington (1878) showed that nitrification in soil was a microbial
process.
• B. Frank (i) discovered (1880) an actinomycetes “Frankia” (Actinorhizal
symbiosis) inducing root nodules in non-legumes tress of genera Alnus
sp. and Casurina growing in temperate forests, (ii) coined (1885) the term
2.66 Environmental Biotechnology

“ Mycorrhiza” to denote association of certain fungal symbionts with

plant roots (Mycorrhiza - A symbiotic association between a fungus
and roots of higher plants. Renamed the genus Bacillus as Rhizobium
(1889).
• H. Hellriegel and H. Wilfarth (1886) showed that the growth of non-
legume plants was directly proportional to the amount of nitrogen
supplied, whereas, in legumes there was no relationship between the
quantity of nitrogen supplied and extent of plant growth. They also
suggested that bacteria in the root nodules of legumes accumulate
atmospheric nitrogen and make it available to plants. Showed that a
mutually beneficial association exists between bacteria (Rhizobia) and
legume root, and legumes could utilize atmospheric nitrogen (1988).
• M. W. Beijerinck (1888) isolated root nodule bacteria in pure culture
from nodules in legumes and named them as Bacillus radicola.
Considered as father of “Microbial ecology”. He was the first Director
of the Delft School of Microbiology (Netherland).
• Beijerinck and Winogradsky (1890) developed the enrichment culture
technique for isolation of soil organisms, proved independently that
transformation of nitrogen in nature is largely due to the activities
of various groups of soil microorganisms (1891). Therefore, they are
considered as “Pioneers in soil bacteriology”.
• S. N. Winogadsky (1891) demonstrated the role of bacteria in
nitrification, and further in fill 1983 demonstrated that free living
Clostridium pasteurianum could fix atmospheric nitrogen.
• Omeliansky (1902) found the anaerobic degradation of cellulose by soil
bacteria.
• J. G. Lipman and P. E. Brown (1903, USA) studied ammonification of
organic nitrogenous substances by soil microorganisms and developed
the Tumbler or Beaker for studying different types of transformation
in soil.
• Hiltner (Germany, 1904) coined the term “Rhizosphere” to denote
that region of soil which is subjected to the influence of plant roots.
Rhizosphere is the region where soil and plant roots make contact.
• Russel and Hutchinson (1909, England) proved the importance of
protozoa controlling/maintaining bacterial population and their
activity in soil.
• Conn (1918) developed “Direct soil examination” technique for
studying soil microorganisms.
• Rayner (192I) and Melin (1927) carried out intensive study on
Mycorrhiza.
• S. A. Waksman published the book “Principles of soil Microbiology”
and thereby encouraged research in soil microbiology (1927). Studied
Environmental Microbiology—Soil 2.67

the role of soil as the source of antagonistic organisms with special

reference to soil actinomycetes (1942) and discovered the antibiotic
“Streptomycin” produced by Streptomyces griseus, a soil actinomycets
(1944).
• Rossi (1929) and Cholondy (1930) developed “Contact Slide/Buried
Slide” technique for studying soil microflora.
• Van Niel (1931) studied chemoautotrophic bacteria and bacterial
photosynthesis.
• Bortels (1936) demonstrated the importance of molybdenum in
accelerating nitrogen fixation by nodulating legumes.
• Garrett (1936) established school in UK on “Soil fungi and ecological
classification”.
• Kubo (1939, Japan) showed/proved the role and importance of
“leghaemoglobin” (Red pigment) present in root nodules of legumes
in nitrogen fixation.
• Ruinen (1956), Dutch microbiologist, coined the term “Phyllosphere”
to denote the region of leaf influenced by microorganisms.
• Alien (1980) suggested that, VAM fungi stimulate plant growth by
physiological effects other than by enhancement of nutrient uptake.
• Jensen (1942) developed the method of studying nodulation on agar
media in test tubes.
• Barbara Mosse and J. W. Gerdemann (1944) reported occurrence of
VAM (vesicular-arbuscular Mycorrhiza) fungi (Glomus, Aculopora
genera) in the roots of agricultural crop plants which help in the
mobilization of phosphate.
• Starkey (1945) studied role of bacteria (Bacillus and Clostridium) in the
transformation of iron.
• Barker (1945) studied anaerobic fermentation by methane bacteria
(Methanococcus, Methanosarcina).
• Thornton (1947) studied root nodule bacteria forming clovers.
• Virtanen (1947) studied chemistry and mechanism of leghaemoglobin
in nitrogen fixation.
• Nutman (1948, England) studied hereditary mechanism of root
nodulation in legumes.
• Burris and Wilson (1957) developed the “Isotope technique” to
quantify the amount of nitrogen fixed and further isolated and
characterized the enzyme “Nitrogenase”.
• Bergersen (1957, Australia) elaborated the biochemistry of nitrogen
fixation in legume root nodules.
• Carnham (1960, USA) discovered nitrogen fixation by cell-free extract
of Clostridium pasteurianum.
2.68 Environmental Biotechnology

• Alexander Fleming started the “School of soil microbiology” at

Cornell University to study microbial aspects of pesticides degradation
(1961) and developed the antibiotic “Penicillin” from the fungus
Penicillium notatum (1929).
• Date, Brockwell and Roughley (1962, Australia) developed the
technique of bio-inoculants production & seed application.
• Hardy & Associates (1968, USA) developed the technique of
measurement of nitrogenase activity by acetylene-reduction test
coupled with gas chromatography and thereby, estimation of biological
nitrogen fixation.
• R J Swaby (1970, Australia) developed “Biosuper”, containing rock
phosphate sulphur, and Thiobacillus which was used to enhance the
phosphorus nutrition of plants.
• Foog and Stewart (1970, UK) intensified the work on N2 fixing blue-
green algae.
• Trinick (1973, Australia) isolated Rhizobia from root nodule of genus
Trema (Parasponia) which was an unique association of Rhizobium with
non-leguminous plants causing root nodulation.
• Dobereiner and associates (1975, Brazil) studied nitrogen fixing
potential of Azospirillum in some tropical forage grasses like Digitaria,
Panicum and some cereals like maize, sorghum, wheat, rye etc. in
their roots. He reported four species of Azospirillum viz. A. lipoferum,
A. brasilense, A. amazonense and A. serpedica. He coined the term
“Associative Symbiosis” to denote the association between nitrogen
fixing Azospirillum and cereal roots. Recently this terminology has been
changed and renamed as “Diazotrophic Biocoenocis”.
• Challham and Associates (1978) isolated an actinomycetous endophyte
Frankia sp. from root nodules of Camptonia peregrina which is again an
example of non-leguminous root nodulation.
• Dommergues & associates (France and Senegal) had discovered/
reported nodules on stem of Sesbania rostrata which could fix nitrogen,
and therefore, this legume can be used as an excellent green manure
crop in low land rice cultivation. Similarly they also discovered N2
fixing stem nodules on Casurina sp. caused by Frankia, an actinomycete.
• Louis Pasteur proved the role of soil microorganisms in biochemical
changes of elements. He also showed that decomposition of organic
residues in soil was dependent on the nature of organic matter and
environmental conditions.
• Brefeld introduced the practice of isolating soil fungi by “Single Cell”
technique and cultivating/growing them on solid media. He used
gelatin (first solidifying agent) in culture media as solidifying agent.
Environmental Microbiology—Soil 2.69

• Gerretsen & Mulder (Holland) studied “Phosphate mobilization” by

soil microorganisms and showed the importance of molybdenum in
nitrogen metabolism by microorganisms.
• Fritch, Fogg & Stewart (UK) and lyengar (India) studied fixation by
algae, in general and micro algae, in particular. They also intensified
the work on N2 fixing BGA.
• James Trappe and Don Marx worked on ectomycorrhiza, colonizing
the roots of forest trees.
• W. S. Cook, G. C. Papavizas, J. Baker and N.S. Kerr contributed to
the field of biological control of plant pathogens using antagonistic
organisms from soil. From the beginning of 20th century, emphasis
was given to the study of microorganisms in soil in relation to their
physiology, ecology, interrelationship, role in soil processes and
soil fertility. Further role of fungi and actinomycetes in cellulose-
decomposition was better understood and cellulose decomposing,
sulphur-oxidizing, iron bacteria, etc. were isolated from soil and
studied in detail.

2.1.16 Notable contributions made by Indian scientists in the field

of soil microbiology
During the last few decades, greater emphasis has been given on some of the
important aspects in soil microbiology in India, which are:
• Characterization of N2–fixing Azotobacter, Rhizobium, Beijerinckia, BGA,
etc.
• Studieson P-solubilizing bacteria and fungi, celluloytic microorganisms,
silage production role of humic acid, etc.
• Establishment (1979) of All India Coordinated Project (AICP) on BNF
at IARI and field-oriented work on BNF.
• Standardization of methods of bio-inoculants application to seed and
soil.
• Seed bacterization and response of crops to bio-inoculants.
 Some of the most important contributions made on the different aspects
in the field of soil microbiology by the scientists and research institutes in the
country are as follows:
• C. N. Acharya (1940) contributed towards the better utilization of
agricultural wastes for the production of biogas & compost.
• Sundara Rao (1962) established the “Division of Microbiology” at
IARI, New Delhi.
• Madhok (Punjab) introduced the practice of using bacterial cultures for
berseem.
• Sanyasi Raju & Rajagopalan (Coimbatore) initiated the research work
on root nodulation in legumes at Madras, Agil. College.
2.70 Environmental Biotechnology

• P. K. Dey (West Bengal) worked on free-living N2 fixing organisms

viz Azotobacter, Beijerinckia and BGA in rice fields and discovered N2
fixation by BGA in paddy.
• M.O.P. lyengar (Madras Univ.) laid foundation stone for algal research
in India.
• Sadasivan (Madras) and Saxena (Allahabad) studied ecology and
physiology of soil fungi along with rhizosphere phenomenon.
• Singh B.N. did pioneering research on soil protozoa in India.
• Bhar J. V. (Bangalore) initiated work on the role of earthworms in
the maintenance of soil fertility, biological nitrogen fixation and
microbiology of phyllosphere.
• Thirumalacher (Hindustan Antibiotics, Pune) developed antifungal
antibiotics like Haymycin and Aureofungin.
• Nandi (Bose Res. Institute, Calcutta) worked on production technology
of antibiotics and bacterial fertilizers (Biofertilizers).
• Desikachray (Madras) studied taxonomy of BGA in India.
• Thomas (BARC, Mumbai) studied physiology of algae in India.
• Raja Rammohan Rao (CRRI, Cuttak) studied on rhizosphere nitrogen
fixation phenomenon.
• Bhagyaraj (GKVK, Bangalore) studied Mycorrhiza and N2 fixation
interactions.
• Verma (JNU, Delhi) studied/worked on sulphur metabolism.
• Subramaniam & Mahadevan (Univ. Madras) studied fundamental
aspects of N2 fixation.
• Modi, Sushil Kumar, Das and Thomas carried research on “Genetics of
“Nif” gene in relation to BNF by Rhizobium, Azospirillum and Kelbsiella.
• Bharadwaj (Palampur) studied/worked on microbiology of organic
matter decomposition & role of celluloytic microorganisms.
• Gaur (IARI) and Mishra (Hissar) studied the role of celluloytic
microorganisms in accelerating the process of composting and
compost-making.
• Karla and Garcha (Ludhiana) studied the phenomenon of cellulose
degradation and legume bacteriology.
• Ranganathan & Nellakantan (NDRI, Karnal) worked on silage
microbiology and process of anaerobic decomposition in biogas
production.
• Vadher, Gupta, Sethunatathan and Raghu studied role of soil enzymes
and microbiology of pesticide degradation in soil.
• Dart & Wani (non-symbiotic N2 fixation), Thomas, Kumar Rao,
Nambiar and Rupela (symbiotic N2 fixation) and Krishna (VAM
Environmental Microbiology—Soil 2.71

fungi). These scientists at ICRISAT, Hyderabad work on symbiotic and

non-symbiotic N2 fixation in gram, groundnut, arhar, sorghum and
millets.
• N. V. Joshi (1920) reported first isolation and identification of Rhizobium
from different cultivated legumes
• Gangulee and Madhok studied physiology of Rhizobium and
production of Rhizobium inoculants.
• Sen and Pal (1957) studied solubilization of phosphate by soil
microorganisms.
• A. Sankaran (1958) standardized quality of legume inoculants for first
time in India.
• P. K. Dey and R. Bhattacharya isolated for the first time a new, non-
symbiotic N2 fixing bacterium Derixa gummosa in the world.
• V. Iswaran (1959) reported the use of Indian peat as carrier for
Biofertilizers production.
• Dube J. N. (1975) reported coal (wood coal), an alternative to peat, as
carrier material for biofertilizer production.
 CHAPTER

3 Environmental Microbiology—
Water and Air

3.1 WATER AND WATER MICROORGANISMS

Water is an essential element that makes life on earth possible. Without water,
there would be no life. Although 71% of the Earth’s surface is covered by

Schematic representation of the Water cycle

water, only a tiny fraction of this water is available to us as fresh water. About
97% of the total water available on Earth is found in the ocean and is too salty
for drinking or irrigation. The remaining 3% is fresh water. Of this, 2.997% is
locked in ice caps or glaciers. Thus only 0.003% of the Earth’s total volume
3.2 Environmental Biotechnology

of water is easily available to us as soil moisture, groundwater, water vapor

and the water in lakes, streams, rivers and wetlands. This makes water a very
precious source.
Clean water is a colourless, tasteless and odourless liquid that has a boiling
point at 100°C and freezes at 0°C (under the pressure of 760 mm Hg). Water
occurring in nature contains dissolved salts and gases, especially sea and
mineral waters. Water covers 70% of the earth’s surface, and thus, it is the most
essential habitat of life. The overall volume of inland waters is estimated at
7.5 × 105 km3, of seas and oceans at 1.4 × 109 km3, and of glaciers and continental
glaciers at 1.8 × 107 km3. Water makes up the most crucial component of living
organisms (70–90% of cell mass) and fulfils a purpose in taking part in various
biological reactions and processes.

3.1.1 Types of microorganisms in water

The biotopes of water microorganisms may be underground and/or surface
waters as well as bottom sediments.
• The underground waters (mineral and thermal springs, ground waters)
due to their oligotrophic character (nutrient-deficient) are usually
inhabited by a sparse microflora that is represented by a low number
of species with almost a complete lack of higher plants or animals.
• The surfacewaters such as streams, rivers, lakes and seawaters are
inhabited by diverse flora and fauna. Microorganisms in those waters
are a largely varied group. Next to the typical water species, other
microorganisms from soil habitats and sewage derived from living and
industrial pollution occur.
• Bottom sediments are a transient type of habitat i.e. the soil water
habitat that is almost always, typically, oxygen-free in which the
processes of anaerobic decomposition by microorganisms cause the
release of hydrogen sulphide and methane into water. In the bottom
sediment, anaerobic putrefying microflora, cellulolytic bacteria and
the anaerobic chemoautotrophs develop.

3.1.1.1 Groups of water organisms

Microorganisms occupy surfacewaters in all of the zones; they may be
suspended in water (plankton) cover stationary underwater objects, plants etc.
(periphyton), or live in bottom sediments (benthos).
Plankton: The group of organisms that passively float in water, not being
able to resist the movement and the flow of water mass, is called plankton or
bioseston. We differentiate:
• phytoplankton (plant plankton)
• zooplankton (animal plankton)
Environmental Microbiology—Water and Air 3.3

• protozoa plankton
• bacterioplankton (bacteria plankton)
• virus plankton
• Phytoplankton are mainly microscopic algae and blue-green algae. It is
a varied community in terms of the systematics and mainly composed
of forms smaller than 50 mm. Sea phytoplankton are dominated by
diatoms and dinophyta, whereas freshwater phytoplankton are
dominated by cryptophytes, diatoms, green algae, and blue-green
algae.
• Zooplankton are small water animals that occur in plankton. There
are three systematic groups that occur in fresh waters: rotifers,
branchiopods and copepods. The sea water plankton is composed of
copepods, ctenophores, urochordata, arrow-worms as well as some
species of snails. Most of them are filtrators (condensed suspended
particles) or predators.
• Protozoa plankton consist of protozoa which occupy the open water
zones like flagellates and ciliates. They are the main consumers of
bacteria. Moreover, most ciliates feed upon flagellates, algae and
smaller ciliates. The protozoa itself feeds upon the zooplankton.
• The heterotrophic bacteria plankton occupy waters which are
abundant in organic compounds. The amount of bacteria in open
waters varies between 105-107 cells in 1 ml.
• Virus plankton is composed of viruses which are the smallest element
of plankton. Their numbers may be very high (108 in 1 ml) in various
fresh and sea water habitats. Viruses are, next to the protozoa, a crucial
factor in bacteria mortality.
Distribution of plankton: The ability to hover in mid-water is possible
due to the presence of mucous membranes around the cell, gas vacuoles or
lipids contained inside the cells. The distribution of species and numbers of
water organisms differs greatly since the biotic and abiotic factors vary in
particular water basins. The distribution in lotic waters such as rivers, springs
and streams is more or less the same. Especially high numbers are found in
the mid course of the river where the bottom and the main-stream speed are
favourable for development of lakes. Where the flow of water is limited, a
closely related vertical distribution of mainly phytoplankton to stratification
has been observed. During calm, quiet weather where the air meets the water,
neuston appears upon the surface of the water. This is composed of bacteria,
algae and pleuston which is composed of larger organisms.
3.4 Environmental Biotechnology

Group of organisms living in lakes

Periphyton: Periphyton occupy the shoreline zones. They are a group of
organisms that create outgrowths upon various objects and underwater plants.
Most of the time they usually consist of small algae—diatoms, green algae
and bacteria. Moreover, various settled or semi-settled protozoa, eelworms,
oligochaetes, insect larva, and even crustaceans, make up the periphyton
biocenosis. Periphyton has a characteristic complex biocenosis and many
ecological relationships can be observed between its components.
Benthos: The bottom habitat is occupied by a group of organisms called
the benthos. The muddy bottom contains an abundance of organic compounds
that are created as a result of dead matter decomposition (fallen parts of plants
and animals). At great depths, the bottom is free from any plants which, due
to a lack of light, can not grow. However, the absence of oxygen supports the
development of, among others, an oxygen-free putrid microflora. Among the
benthos microflora, the most numerous are bacteria and fungi (decomposers)
as well as some animals (detritophages). Both of the above groups are
responsible for decomposition of the organic matter. Benthos of shallow
reservoirs may also contain some algae.

3.1.2 Factors limiting growth of microorganisms in water

The development of microorganisms in water is influenced by a large number
of chemical and physical factors which, in various ways, interact or oppose
each other. They have an influence on the size and the species composition
Environmental Microbiology—Water and Air 3.5

of the microbial biocenosis as well as on their appearance and life processes.

Within water ecosystems, two groups of factors that have a crucial influence
on the quantitative and qualitative relationships between microorganisms
may be distinguished:
• abiotic factors—light and thermal energy, water reaction, water flow,
climate and the compounds dissolved and suspended in water (dead
organic matter, non-organic compounds and gasses such as oxygen,
carbon dioxide, methan and others).
• biotic factors—all water living organisms such as plants, animals,
microorganisms and the relationship between them.

3.1.3 Characterization of water microorganisms

Bacteria: In terms of morphology, most water bacteria resemble soil bacteria–
their cells are round, cylindrical or screw like. There are also thread-like and
stem-like shapes. The threads may be or not be branched, single or in groups.
Various water bacteria may create clusters made up of various numbers of
cells in the following shapes: spherical, star-shaped, lamellar, and filiform.
Most water bacteria are active and mobile using cilia or flagella (e.g. Vibrio,
Pseudomonas). Bacteria in water may swim slowly (plankton) or occupy a fixed
substrate. Oligotrophic water bacteria in clean waters occur as microforms
with cells smaller then 1 mm, usually 0.4 mm. Water oligotrophs rarely
multiply, their generation cycle lasts from a few dozen to two hundred hours.
Polluted waters are predominantly occupied by bacteria of the gram-negative
rods group. The ratio of rods to cocci is about 90:1. Clean waters (rivers and
streams) contain a sparse microflora and the ratio of rods to cocci is 1:1.5,
which indicates the dominance of cocci.
The number of bacteria in water depends mainly on the organic matter
content. In clean waters, they occur in low numbers whereas polluted waters
contain up to several million cells per 1 ml of water.
Bacteria that occur in water habitats may be divided into the following:
• autochthonous (native), constantly occupying water habitats.
• allochthonous (foreign), finding their way from the soil or the air
as well as microorganisms that get into the water basins along with
municipal and industrial sewage.
Autochthonous bacteria: We can distinguish photoautotrophs,
chemoautotrophs and chemoorganoautotrophs.

Photosynthesizing bacteria (photoautotrophs)

Purple and green bacteria are among the photosynthesizing autotrophs. Due
to their metabolism, these bacteria can be divided into the following groups:
• Filiform green bacteria (Chloroflexaceae),
• Sulfuric green bacteria (Chlorobiaceae),
3.6 Environmental Biotechnology

• Sulfuric purple bacteria (Chromatiaceae and Ectothiorhodaceae),

• Non-sulfuric purple bacteria (Rhodospirillaceae),
• Heliobacteria (Heliobacteriaceae)
The photosynthesis of bacteria is carried out slightly differently from that
of plants. Most importantly, it is an oxygen-free process which requires the
presence of reduced mineral compounds and it is not accompanied by a release
of oxygen but by a production of oxidized non-organic or organic compounds.
The assimilating pigments of bacteria are categorized by the ability to absorb
infrared light that is not absorbed by green plants. The photosynthesis in
surface waters is conducted mainly by algae and plants and the role of the
bacterial photosynthesis is less important.

Chemosynthesizing bacteria (chemoautotrophs)

Chemoautotrophs get energy from the oxidation processes of non-organic
compounds. Depending on the nature of the oxidized substrate, the following
can be distinguished: nitrifying, ferruginous, sulfuric and hydrogen bacteria.
• The role of the nitrifying bacteria in surface waters is the oxidation
of ammonia and nitrite to nitrate. In greater concentrations, the above
compounds may be harmful to water organisms as well as to humans (in
cases when such water is utilized for water supply systems). Moreover,
the production of nitrate is a fundamental process that supplies water
plants with a source of nitrogen.
• Ferruginous bacteria grow in waters when the content of bivalent iron
ranges between 0.15-8.5 mg/dm3. Their negative influence includes
corrosion and fouling of plumbing, sewage systems and different metal
constructions. The most common ferruginous bacteria are the Leptothrix
ochracea and Crenothrix polyspora and they belong to the filamentous
bacteria which are categorized by the fact that the single cells form
thread-like forms surrounded by a gelatinous sheath of varying
thickness. Stored ferruginous substances in cells change the coloration
of cell threads into a yellow or dark-brown shade. The ferruginous
bacteria are very common in fresh bodies of water especially in waters
from wells and springs, where it is possible to observe their clusters
with naked eye. Moreover, they occur abundantly in muddy streams,
marshes and ponds.
• Sulfuric bacteria occur mainly in waters containing hydrogen sulfide
which is toxic for most microorganisms, whereas for this group, it is
one of the crucial compounds for survival. These bacteria can be found
in mineral springs that contain hydrogen sulfide of geological origin
as well as in highly polluted waters where it is produced as a result of
oxygen-free protein decomposition or desulfurication processes. The
typical representatives of the sulfuric bacteria are: bacteria that move
in sliding motions Beggiatoa alba and fixed to the bottom Thiothrix nivea.
Environmental Microbiology—Water and Air 3.7

The forms of individual sulfuric bacteria are:

Thiobacillus thioparus—stores sulfur derived from oxidation of
(i)
thiosulfate.
Thiobacillus thiooxidans—grows in acidic habitats of pH 1.0 – 4.0.
(ii)
Thiobacillus ferroxidans—besides thiosulfates and tetrationans, it
(iii)
possesses the ability to decompose ferruginous salts.
Thiobacillus denitrificans—is a relative anaerobe and it has an ability
(iv)
to utilize nitrates as the electron acceptor during the oxidation
of hydrogen sulfide. In aerobic conditions, the above function is
performed by the oxygen.
• Hydrogen bacteria posses an ability to oxidize hydrogen using
oxygen as a final acceptor of electrons. Most often, they feed
heterotrophically and switch to autotrophic feeding when hydrogen
is present in the habitat. The most widespread species belong to the
genus Hydrogenomonas. Micrococcus denitrificans belongs to a group of
the hydrogen bacteria and they conduct the oxidation of hydrogen
while simultaneously reducing nitrate down to molecular nitrogen.
Desulfovibrio desulfuricans also oxidizes hydrogen while reducing
sulphate down to hydrogen sulphide.

Heterotrophic bacteria (chemoorganotrophs)

A predominant part of autochthonous bacteria which occur in water basins
are the chemoorganotrophic bacteria which belong to a group of saprophytes
that feed upon dead plant and animal organic matter. Typical bacteria
plankton that occupy an entire water mass are the cilliated gram-negative
rods and they represent the following genera: Pseudomonas, Achromobacter,
Alcaligenes, Vibro and Aeromonas, as well as the gram-positive cocci that belong
to the Micrococcus genus, treponema and spiral bacteria of the Spirillum genus.
The underwater parts of higher plants and the underwater fixed particles
are colonized by numerous stem-like bacteria (e.g. Caulobacter), sheathed,
filiform, and gemmating bacteria (e.g. Hyphomicrobium), which are one of the
microorganisms forming the periphyton. Organisms which usually grow
in bottom sediments are oxygen-free putrefactive bacteria, then oxygen-
free cellulolytic bacteria and finally, oxygen-free chemoorganotrophs such
as Desulfovibrio genus that reduce sulfate down to the hydrogen sulphide.
In addition, there are some less numerous oxygen-free methanegenerating
bacteria which reduce organic compounds down to methane.

Allochthonous bacteria
Waters of high fertility and also highly polluted surface waters are abundant
in saprophytes and parasitic bacteria from among which, the following are
predominant: gram-negative intestinal rods of Escherichia coli as well as
3.8 Environmental Biotechnology

the Proteus genus, Klebsiella and Enterobacter, and also rods of Pseudomonas
aeruginosa and of the Arthrobacter genus. Moreover, gram-positive rods (bacilli)
of the Bacillus, Corynebacterium and Clostridium genera, which are washed out
from the soil and get into the bodies of water during heavy rainfalls, also
belong to the allochthonous bacteria. Municipal wastes are the main source of
pathogenic bacteria. Moreover, during the infiltration processes and surface
run-offs, soil bacteria find their way into the waters as well. The role of air
in water contamination is significant in densely populated areas of cities and
industrial regions.
Water fungi: In contrast to bacteria which grow best in waters of pH
between 6-8, fungi occur only in waters below pH 6.0. Usually, fungi occur in
shallow waters, right on or just below the surface, which is closely connected
to the fact that the organisms require significant amounts of oxygen. The
predominant fungi in water environments are represented by mold fungi
which belong to the Oomycota class (Leptomitus, Phytophthora) and to the class
of Zygomycota (Mucor and Rhizopus). Relatively frequently, fungi belonging to
Ascomycota as well as the Deuteromycota, are found in surfacewaters.
Almost all fungi are heterotrophs that decompose organic matter; waters
are occupied by both saprophytes and parasites which colonize water
plants and animals. They have more diverse shapes than bacteria and they
differentiate into larger cells and more complicated structures. In addition
to unicellular ones, there are also multi-cellular fungi with large mycelium.
Fungi usually, do not occur in clean waters. They grow in abundance on the
bottom of waters polluted by sewage (e.g. Leptomitus lacteus).
Blue-green algae: Blue-green algae are a group of organisms previously
considered to be algae. Currently they are classified to the Procaryota kingdom
and the sub-kingdom of Eubacteria. There are unicellular, colonial (loose
cells connected with a single mucus envelope) and filamentous forms. The
prokaryotic organisms contain a nucleoid instead of an isolated nucleus. In
contrast to other bacteria, they are capable of conducting oxygen photosynthesis.
They contain chlorophyll and sometimes disguise it in other photosynthesizing
pigments: ficocyanine and alloficocyanine. Characteristically, the blue-green
colouring of blue-green algae comes from the combination of chlorophyll and
ficocyanine. Blue-green algae reproduce mainly through proliferation by cell
fission. Their characteristic trait is that they possess gaseous vacuoles which
allow movement in water to places of better illumination. Some (Anabaena) are
capable of binding atmospheric nitrogen in structures called heterocysts. Due
to their resistance to extreme environmental conditions, they are ubiquitous.
They can be found in deserts and in hot springs. Blue-green algae can cause
blooming in lakes and other water reservoirs. Some of them produce toxic
metabolites.
Algae: Algae are the simplest autotrophic eukaryotes that incorporate over
20 thousand species. Algae occur in fresh and seawaters. They are important
Environmental Microbiology—Water and Air 3.9

producers of organic matter and oxygen. Algae live in the form of single cells
or they create multicellular body of various shapes called thallus (threads,
spheres, multilayer clusters). The composition of algae community changes
significantly with respect to quality and quantity, depending on the content
of the mineral salts in any given reservoir as well as on the characteristics
of the substances that make up the main pollutant. The following are the
characteristic algae that occur in oligotrophic waters: diatoms of the following
genera: Asterionella, Tabellaria, Melosira and some other algae (Dinobrion). In
eutrophic waters, the content of algae is completely different. Most of all, such
waters contain only a vestigial number of diatoms, and instead of them, the
algae from the Dinophyta class as well as the Spirogyra genus, appear. Algae are
subdivided into the following classes:
• Chlorophyta—green algae that contain chlorophyll a and b types, a
cellulosic cell wall and starch as reserve material. They have a diverse
constitution both unicellular and multi-cellular forms exist usually as
thread-like structures. Cells may be motile; (then they are equipped
with flagella), or non-motile. Chromatophors of various shapes have a
green coloration. They reproduce vegetatively or sexually. Vegetative
reproduction consists of the division of cells and the fragmentation of
thread-like forms.
• Chrysophyta—this group involves diatoms important for the water
environment. They are common algae and occur in fresh and sea
waters, bottom sediments and soil. Chrysophyta contain a and c types of
chlorophyll. Their cell wall is enriched in silica. They produce lipids as
reserve material.
• Euglenophyta—Euglenoids usually have an elongated shape. Their
cells are equipped with flagella that allow movement in water or they
move by crawling along the bottom. The cells are surrounded by a
soft envelope called the pellicle. Chromatophores contain chlorophyll,
carotenes and xanthophylls. Within the cell there is a clearly visible
nucleus and the eyespot called stigma, sensitive to a light stimulus.
Euglenoid cells, create cysts, which make survival in unfavorable
conditions possible. They grow in waters containing high levels of
organic compounds. There are also parasitic forms.
• Pyrrophyta—occur usually individually. Some cells are surrounded
by a cellulose wall whereas others are deprived of any cell walls.
They usually possess two flagella that allow their movement. Within
the protoplasm, there is an isolated cell nucleus and yellow-green or
yellow-brown chromatophores. They reproduce by division and some
have been observed to reproduce sexually. Pyrrophyta occur in slightly
salty or sea waters and only selected species live in fresh waters. In
lakes, there is Ceratium hirundinella, a species that sometimes appears
in large masses.
3.10 Environmental Biotechnology

• Rhodophyta—contain chlorophyll type a and b as well as other pigments

such as carotene, xanthophylls and phycobilin pigments: phycoerythrin,
phycocyanin. They store starch as a storage product. Their cell wall has
two layers: the inner one is made of cellulose whereas the outer one is
made of pectin. They reproduce asexually, through fragmentation of
the thallus, and sexually, through oogamy.
• Phaeophyta—brown algae, containing chlorophyll a and c as well as
carotenoids (fucoxanthin). Their reserve material is laminarin (â-1,
3-glycan) and chrisolaminarin, mannitol and lipids. Their cell wall is
also a bi-layer: the inner wall is made of cellulose whereas the outer
wall is made of pectin. Brown algae are multi-cellular organisms that
possess the highest level of specialization of the thallus of all algae
with high anatomic and morphological variation. They reproduce by
zoospores, and also sexually, by gametes.
Water protozoa: Protozoa live in all types of waters, from small puddles,
to inland waters, to the seas. They feed heterotrophically, absorbing the
dissolved organic compounds or feeding upon bacteria. They are most
numerous in highly polluted waters and are the element of activated sludge.
When the pollution level is not too high, ciliates become predominant, and
that concerns both the free-swimming ones (e.g. Paramecium) and the settled
ones (e.g. Vorticella).
Protozoa can be sub-divided into four classes:
• Flagellata – flagellates. These move utilizing long flagella. They feed
heterotrophically and occur in polluted waters or in inefficiently
functioning activated sludge. Besides dissolved substances, they may
also absorb bacteria or unicellular algae. Flagellates live individually
or in colonies. There are parasitic forms among them too. This is
exemplified by a human parasite Giardia lamblia and the Trypanosoma
gambiense which is transferred on to humans by the Tsetse fly causing
African sleeping-sickness and neurological disturbances.
• Rhizopoda amoebae. The cells move around utilizing the plasmatic
pseudopodia which are used for locomotion and for capture of food.
Some amoebae have a changeable shape others; however, have a
constant shape as they are equipped with a mini-skeleton or an outer
shell. Some amoebae lead a parasitic life (Entamoeba histolytica).
• Ciliata – ciliates. Most of the representatives lead a free-swimming life
style (Paramaecium, Euplotes); others crawl or are attached to the bottom.
They feed upon bacteria, algae and organic substances. Ciliates occur
in large numbers in polluted waters and in activated sludge. Some,
such as Balantidum coli which causes dysentery, are parasites of animals
and humans.
Environmental Microbiology—Water and Air 3.11

• Sporozoa. Only parasites belong to this class and representatives are

Cryptosporidium parvum, causing intestinal diseases and Plasmodium
malariae, causing the malaria. The second parasite attacks the red blood
cells. This pathogen is carried by the Anopheles mosquito.

3.1.4 Sources and types of pollutants

Waters become polluted as a result of domestic and industrial sewage disposal
into surfacewaters, which contain huge amounts of various compounds
that affect the biocenosis of water reservoirs. Besides sewage, pollution is
also caused by rain run-offs which wash away different fertilizers and crop
protection products. Moreover, the pollutants also transfer into waters from
the surrounding air. This usually results from industrial dust which falls
directly into the water or is washed away from the ground surface by rain.
Important gases are: sulphur dioxide, nitrogen oxides, carbon oxides and
dioxides which get into the waters mainly in highly industrial areas.
Some of the above compounds undergo microbiological decomposition
relatively easily, becoming food for heterotrophic microorganisms, others are
resistant to such decomposition and are harmful or toxic to microorganisms.
Examples of these are the following: cyclic compounds, engine oil, lubricants,
chlorinated hydrocarbons, pesticides, and among the mineral pollutants—
heavy metal salts.

3.1.5 Self-purification of surfacewaters

Self-purification encompasses complex co-operation between physical and
biochemical factors such as: sedimentation (settling), oxidation, an exchange
of volatile substances between the atmosphere and water, and the release of
gaseous products of metabolism into the atmosphere. However, the critical
role is played by the biological factors. A wide range of microorganisms
and higher organisms participate in self-purification processes. Bacteria and

Succession of microorganism during the self-purification process

3.12 Environmental Biotechnology

fungi are the most crucial as they are capable of mineralising various mineral
components. Proteins, simple and complex sugars, fats, cellulose, lignin, wax
and others undergo degradation during the process of self-purification. As
a result of mineralization, the following compounds are created: H2O, CO2,
NO3-, SO42-, PO43-, and other simple compounds. With the progression of self-
purification the populations of microorganisms that act in the environment,
change. The self-purification process utilizes large amounts of oxygen during
the biochemical processes. The amount of oxygen that is used up in any specified
time by water microorganisms is called the biochemical oxygen demand,
BOD. By analysing the BOD, it is possible to determine the concentration of
the organic compounds dissolved in water which are susceptible to biological
oxidation. The discharge of impurities into the water reservoir creates a sudden
change in chemical, biological and physical conditions. Simultaneously, right
below the area of the discharge, the process of self-purification begins. The
process leads to the formation of zones containing, characteristically, gradually
decreasing levels of pollution.

Saprobic zones. Self-purification of lotic waters

Zones of various levels of organic pollution are called saprobic. In particular
zones, the content of biocenoses is different and altered to fit the existing
conditions. Species, which clearly dominate other species and have adapted
to the existing conditions, are found there. Zones differ in the dynamics of
dissimilation processes, the intensity of oxygen intake, and the appearance
of the water. There are three different saprobic zones: poli-, mezo- and
oligosaprobic.
Polisaprobic zone is the zone of highest concentration of pollutants
with cloudy, dirty-grey, fetid odorous waters. The high concentration of
various organic compounds ensures development of selected heterotrophic
microflora, which, while conducting biodegradation, use up large amounts of
oxygen leading to its deficit. In anaerobic conditions, the following gases are
formed: H2S, NH3, CH4, N2 and others. There is a lack of green plants in this
zone. Among the organisms which are capable of surviving in such conditions,
the dominant ones are Zooglea ramigera and Sphaerotilus natans bacteria. There
are also sulphur bacteria (in the presence of hydrogen sulphide), especially
of the Beggiatoa and Thiothrix genera; protozoa are also numerous. Reducers
(decomposers) are the most frequently occurring organisms in this zone.
In the mezosaprobic zone there is further intensive breakdown of
the organic compounds but the amount of oxygen is sufficient to sustain a
full demand. The water becomes clear, often of green coloration, due to the
abundantly flourishing algae. The number of reducers decreases. Besides
the microorganisms mentioned above, sewage fungi Leptomitus lacteus, blue-
green algae, sparse diatoms and green algae appear. The mineralization of the
organic compounds is finished within the zone, excluding, humus compounds
Environmental Microbiology—Water and Air 3.13

which are difficult to decompose. The mezosaprobic zone is divided into a-and
b-zones. The a–mezosaprobic zone is a heterotrophic one in comparison to the
b-mezosaprobic zone which is rather autotrophic. The b–mezosaprobic zone is
cleaner and higher numbers of algae species occur here.
Oligosaprobic zone is a section where the inflow of impurities ends and
water returns to its previous state of natural water. Water is clear, odorless
and well oxidized. The zone is mainly inhabited by ferruginous and nitrifying
bacteria (the chemosynthesizing bacteria); sparse blue-green algae, many
diatoms and green algae, and few protozoa, occur in the biocenose.

Self-purification of lentic waters

A self-purification process takes a different course in lentic waters; the saprobic
zones do not evolve here even though the purifying mechanism remains the
same. The impurities introduced into water fall down to the bottom (their
density is greater than that of water) where they are decomposed.

Microbiological processes within bottom sediments

There are complicated chemical, physical, and biological processes taking
place between water and the bottom sediments, which are important for the
reservoir as a whole. The water/sediment arrangement, the microorganisms
quality-quantity ratio and the direction of bio-chemical changes have a
significant influence upon the level of biogenes (nitrogen, phosphorus, sulphur
compounds) within the reservoir (its fertilization). In the bottom sediments,
the aerobic decomposition of organic constituents takes place in the upper
layers (from a few to several mm) and are a source of soluble mineral salts.
Whereas, the anaerobic biodegradation, which takes place below, causes a
release of substances, often poisonous, to the water habitat (e.g. H2S, CH4).
Bottom sediments play an important role in lentic water self-purifying
process where the organic suspended matter falls to the bottom as a result of
lack of water movement. They have a significant influence upon the conversion
of the biogenic compounds which affect the quality of water. The most crucial
factor regulating the speed of nitrogen and phosphorus penetration (also Fe
and Mg) from within the sediments into water is the content of the dissolved
oxygen within the layer near the bottom. Active diffusion of phosphates into
water begins when the content of the dissolved oxygen falls below 1mg O2/
dm3. Moreover, the following also have an influence upon the release of
phosphates: temperature, organic compound decomposition, water pH,
redox potential. In bottom sediments of various surface water reservoirs, the
following microorganisms are the most common: aerobic cellulolytic bacteria
(of the Sporocytophaga, Cytophaga, Pseudomonas, Achromobacter genera) as
well as anaerobes such as the Clostridium genus. The latter takes part in the
decomposition process of hemicellulose. In oxygen-free conditions, numerous
putrefying bacteria (releasing H2S from proteins), SO42- reducing bacteria,
3.14 Environmental Biotechnology

denitrifying bacteria (NO3– reducing), methane generating (CH4 releasing)

and hydrogen bacteria grow. Moreover, ammonificating bacteria are abundant
in bottom sediments. Nitrifying bacteria usually occur in small numbers
in upper layers of sediments as they are obligate aerobes. The presence of
CH4-oxidizing aerobic bacteria in bottom sediments also depends upon the
concentration of oxygen and iron.

3.1.6 Water-transmitted pathogenic microorganisms

Bacteria: The group of obligate pathogenic bacteria, which occur in polluted
surface waters, contain rods causing typhoid fever (Salmonella typhi), as well
as other gram-negative bacteria of the Salmonella genus, which are the cause of
various infections of the digestive tract. Bacterial dysentery caused by Gram-
negative rods of the Shigella genus are not as common as the above. In surface
waters of tropical countries, bacteria of the Vibrio cholerae genus (cholera),
frequently occur. Moreover, Mycobacterium tuberculosis causing tuberculosis
and treponema of the Leptospira, can be also found in polluted waters. The
latter bacteria cause bacterial jaundice. Beside the obligate pathogenic bacteria,
there are also numerous gram-negative bacteria in surface waters which are
described as opportunistic microorganisms (facultatively, pathogenic). These
belong to the Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter,
Citrobacter, Serratia, Acinetobacter, Proteus and Providencia genera. All of the rods
are part of the usual flora of the intestine and are not typically pathogenic
for as long as they occur in human or animal digestive tracts. In some cases
though, these bacteria find their way into other organs becoming a potential
cause of different illnesses such as inflammation of urinary and respiratory
systems and also sepsis, which is a general infection of all internal organs.
Waterborne Bacterial Infections
Disease type Species or genera of bacteria
Typhoid Salmonella typhi
Paratyphoid Salmonella paratyphi
Animal salmonellosis Salmonella sp.
Bacterial dysentery Shigella sp.
Cholera Vibrio cholerae, Vibrio cholerae type eltor
Stomach and intestine Enteropatogenic Escherichia coli, Klebsiella
catarrhs pneumoniae,Aeromonas hydrophila, Plesiomonas
shigelloides, Pseudomonasaeruginosa, Vibrio
parahaemolyticus, Campylobacter (Vibrio)fetus subsp.
jejuni, Clostridium perfringens, Bacillus cereus
Yersiniosis Yersinia enterocolitica
Tularemia Pasteurella (Francisella) tularensis
Leptospirosis Leptospira sp.
Environmental Microbiology—Water and Air 3.15

Skin infections Pseudomonas aeruginisa, Mycobacterium (M. balnei,

M. phlei,M. marinum, M. kansasii, M. fortuitum, M.
cholonei, M.Gorgonae
Bacteremia Psudomonas aeruginosa, Pseudomonas cepacia
conjunctivitis, ear and
upper-respiratory
system infection
Fever (pyrogens) Gram-negative water rods (Pseudomonas,
Achromobacter,Xantomonas, Moraxella, Acinetobacter)
Legionnaires disease Legionella pneumophila

Viruses
Besides pathogenic bacteria of surface waters, into which municipal and
industrial sewage is disposed, the waters also contain significant amounts of
other pathogenic microorganisms such as the Polio virus. They are responsible
for causing the Heine Medina disease (polio). Enteroviruses, which cause
intestinal infections, occur even in slightly polluted rivers.
Intestinal viruses which may be transmitted by water and
diseases caused by them
Viruses Number of Diseases
types
Poliovirus 3 Palsies, meningitis, fever
ECHO 34 Meningitis, respiratory system
diseases, rash, diarrhea, fever
Coxsackie A 23 Herpangina, respiratory system
diseases, meningitis, fever
Coxsackie B 6 Cardiac muscle inflammation,
innate heart defects, rash, fever,
meningitis, respiratory system
diseases, pleurodinia
Enteroviruses 4 Meningitis, encephalitis, respira-
tory system diseases, acute
hemorrhage conjunctivitis, fever
Hepatitis virus, type A 1 Hepatitis type A
Norwalk virus 1 Epidemic diarrhea, fever
Parvovirus 3 Accompany the respiratory
system diseases
Adenoviruses 41 Respiratory system disease, eye
infections, diarrhea
Rotaviruses 4 Epidemic diarrheas (mainly
among children)
Reoviruses 3 Respiratory system diseases
3.16 Environmental Biotechnology

Protozoa
Infections of the digestive tract caused by protozoa may come from
contaminated water. Most parasitic protozoa produce cysts which are able
to survive inside their host in unfavorable conditions. When the conditions
improve, cysts transform into so called trophozoits, the vegetative form
occurring in humans. Waterborne diseases caused by protozoans:
Intestinal viruses which may be transmitted by water
and diseases caused by them
Pathogenic protozoa Disease Symptoms
Giardia lamblia giardiosis Chronic diarrhea, stomach
(flagellates) cramps, flatulence, weight
loss, fatigue
Cryptosporidium parvum cryptosporidiosis Stomach aches, loss of
(sporozoa) appetite, watery diarrhea,
weight loss
Entamoeba histolytica amoebiosis Anywhere from slight to
(amoebae) (amoebicdysentery) acute diarrhea, fever with
shivers
Acanthamoeba castellani amoebicmeningo- Symptoms from the central
(amoebae) encephalitis nervous system
Naegleria gruberi amoebicmeningo- Gets into the brain of
(amoebae) encephalitis swimmers through
the nose, causes acute
symptoms of meningitis
and encephalitis ending in
death
Balantidium coli Balantidial Hemorrhage diarrhea
(ciliates) dysentery caused by an ulceration of
the large intestine

Parasitic fungi
In polluted surface waters, parasitic fungi can also occur, for example,
Microsporum sp., Trichophyton sp. and Epidermophyton sp. They are
dermatophytes, causing ringworm and other cutaneous infections.

Parasitic worms
Human parasites are not usually included in the scope of microbiological
research, however, along with other pathogens (viruses, bacteria, protozoa)
they pose a serious threat to human health. They occur in sewage and may
find their way into waters from soils as a result of infiltration and surface run-
offs. The infectious forms of the parasitic worms are their eggs. The eggs are
Environmental Microbiology—Water and Air 3.17

excreted in great numbers outside the hosts’ body along with faeces and spread
through sewage, soil or food. The worms’ eggs are very resistant to external
factors and thus, are difficult to eliminate from sewage by chlorination.
Parasitical worms in the human body
Parasite Symptoms
Human ascarid— Ascariasis. Nematodes’ larva causes
Ascarislumbricoides inflammation reactions in various parts of the
(Nematoda) body. Sometimes, it breaks up the pulmonary
alveolus. If the intestines contain a lot of
ascarids, it may cause intestinal obstruction or
a puncture causing damage to the abdomen
lining.
Whipworm— Trichomoniasis disease is caused by nematodes
Trichiuristrichura living in human caecum and the large intestine.
(Nematoda) It creates changes in the mucosa, and at high
infestation, a serious loss of mucous membrane.
Sometimes appendicitis may occur.
Spiny—headed worms Ascanthocephaliasis disease is caused
(Acanthocephala) by invertebrates which incorporate only
parasitic forms. The parasites live in the
intestines of all vertebrate representatives. In
water environment, their hosts are usually
crustaceans. The disease manifests itself by
inflammation of the digestive system and its
physical damage.
Tapeworms— Parasites develop inside the intermediate
Taeniasaginata, T. solium host until reaching the larva stage called
(Cestodes) the cysticercus and infect humans who are
its final host. Tapeworms live in the small
intestine causing nausea, chronic dyspepsia,
stomachaches and weight loss.
Flukes— Schistosomatosis—caused by Schistosoma
Schistosomamansoni mansoni, manifests itself by the ailment
(Trematodes) of the digestive system, intestine mucosa
inflammation and cirrhosis of the liver.

3.1.7 Sanitary quality of water

The possibility of infection by water imposes a constant need to control the
hygienic-sanitary quality of not only drinking water, but also that of swimming
pools and surface waters. Water gets infected by pathogenic bacteria excreted
by ill people and carriers (people who keep excreting pathogens with faeces
3.18 Environmental Biotechnology

long after they have suffered from an illness). Pathogenic microorganisms

are present in sewage and surface waters in lower numbers than other
microorganisms. Therefore, they are more difficult to detect than the plentiful
saprophytic bacteria. Consequently, much more complex diagnostic methods
need to be used in order to detect them.

Indicator microorganisms
Current norms are based on indirect inference about the presence of pathogenic
microorganisms relying on the number of indicator microorganisms, which
permanently live in human and animal digestive tracts as saprophytes.
Their presence indicates that the water is polluted with faecal matter
and, consequently, there is danger of contamination with pathogenic
microorganisms. Bacteria, which serve as sanitary indicators, should meet the
following conditions:
1. They must be constantly present in the human digestive tract so that
they allow the detection of the water’s contamination with faecal
matter.
2. The number of indicator bacteria within the intestine and faeces should
be high.
3. Among them, there should be non-spore-forming bacteria as it enables
the detection of ‘fresh’ faecal-matter water pollution.
4. Their identification must be possible with readily available methods.
5. Their life span in the external environment should be longer than that
of pathogenic bacteria.
6. They should not be able to reproduce in a water environment under
natural conditions.

Types of indicator bacteria utilized to assess the

health quality of water
In routine laboratory work, which conducts sanitary-epidemiological
supervision, it is impossible to constantly monitor water for all pathogenic
and potentially pathogenic microorganisms, which may be found in water.
Therefore, routine monitoring concentrates mainly on detecting bacteria that
indicate faecal contamination of water. The sanitary quality of water may be
checked by utilizing the saprophytic microflora that occupy the human large
intestine. The following indicators of water contamination have been adopted:
• Coliforms
• Faecal coliforms
• Faecal streptococci
• Bacilli of Clostridium genus, sulphite-reducing bacteria and in some
instances: staphylococci–coagulase positive
• Pseudomonas aeruginosa
Environmental Microbiology—Water and Air 3.19

Coliforms: Bacteria of the coli group are mainly made up of strains of

Escherichia coli as well as the genera: Enterobacter, Citrobacter and Klebsiella.
They are detected on media containing lactose at 37°C.
Faecal coliforms (thermotolerant) are mainly strains of Escherichia coli
and only some of the strains of Enterobacter, Citrobacter and Klebsiella, which
have an ability to ferment, lactose at 44 oC. The presence of coliforms or
faecal coliforms in a water sample indicates relatively recent contamination
of water with faecal matter, sewage, soil or with decaying plants. For most
types of waters, a quantitative determination of both groups of coliforms is
recommended.

Faecal streptococci
While in a water environment, faecal streptococci are characterized by a
slightly longer period of survival and resistance to most disinfecting products
than the coliforms. Faecal streptococci include microorganisms of Enterococcus
and Streptococcus genera, which belong to the serological group of Lancefield
D. Detection of faecal streptococci in a test sample, significantly exceeding
the coli group bacteria, may suggest water contamination with animal faecal
matter or sewage from animal farms.

Bacilli of Clostridium genus

The detection of sulphite reducing bacteria (mainly strains of Clostridium
perfringens) may suggest less recent contamination with faecal matter; their
endospores are able to survive for many years in unfavourable conditions.
Sulphite–reducing clostridia are a good indicator of properly conducted water
treatment processes—coagulation, sedimentation, and filtration. Endospores
of these bacteria as well as the cysts of parasitic protozoa (Cryptosporidium
parvum, Giardia lamblia) ought to be eliminated in those stages of water
treatment, because they are especially resistant to the disinfecting agents.
Conducting analysis of a water sample, in order to detect bacteria of the
Clostridium genus, is technically less complicated than searching for parasitic
protozoa and it ensures that the treated water is free from protozoa and from
the eggs of pathogenic worms (Helminthes).

Pseudomonas aeruginosa
Currently, detection of Pseudomonas aeruginosa bacteria in drinking water,
running water, swimming pools and surface waters is recommended in
addition to the above elements of sanitary analysis. They are gram-negative
rods that do not produce spores. Their characteristic trait is the ability to
produce a blue-green pigment—pyocyanin as well as a fluorescent pigment—
fluorescein. Representatives of this species were isolated from human faeces,
and in cases of infection—from urinary tracts, inner ear, suppurating wounds,
etc. These bacteria pose a potential pathogenic danger for both humans and
3.20 Environmental Biotechnology

animals. In addition, they are widely distributed in surface waters and soil. It
is also important that the species may live in chlorinated water because it is, to
some extent, resistant to disinfection.

Staphylococci
The Staphylococcus genus is mainly used to assess sanitary quality of swimming
pools. Recreation waters are the cause of infections of respiratory tracts,
skin and eyes. For this reason, microbiological analysis based on standard
indicators (coliforms) is insufficient. Some researchers have recommended
Staphylococcus aureus to be used as an additional indicator of sanitary quality
of recreational waters, because its presence is associated with human activity
in these waters.

Total number of bacteria

In routine analysis, the total number of bacteria present in 1 ml of water is also
determined by an agar plate method. One set of plates is incubated at 37°C
for 48 h (mesophilic bacteria). Another set of plates is incubated at 22°C for
72 h (psychrophilic bacteria). After incubation, the colonies are counted and
the amount of cfu/ml (colony forming units) can be calculated.

The total number of psychrophilic bacteria

Non-pathogenic water bacteria grow mainly at lower temperatures. It is
important that gram-negative bacteria in water produce lipopolysaccharides
in their cell wall which can be toxic – like endotoxins of pathogenic bacteria.
Because of this, their numbers in water should be constantly monitored. A
large increase in their numbers is evidence of the presence of easily available
organic compounds in the water. Theoretically, the presence of 0.1 mg organic
carbon in water can result in an increase of bacteria up to 108 cfu in 1 ml.
Phosphorus is also a factor which stimulates the growth of microorganisms.
Adding even small amounts of this element (< 50 mg/l) causes 10 times the
acceleration of bacterial growth in a water treatment plant.

The total number of mesophilic bacteria

More dangerous are high numbers of bacteria growing at 37 °C, because among
this high population, pathogenic forms may be found which are dangerous for
human health. High number of bacteria in samples of water can prove that
water treatment processes proceed badly or that polluted water is siphoned.

Reasons of increasing levels of total number of bacteria in water

An increase in the total number of bacteria in water samples can also be proof
of development of microorganisms on inner surfaces, especially on pipe-joints,
seals and of the creation of the layer called a biofilm. Biofilms of microorganisms
are of concern because of the potential protection of pathogens from the action
of residual disinfectant in the water and the regrowth of indicator bacteria
Environmental Microbiology—Water and Air 3.21

such as coliforms. High number of total bacteria is an indicator of potential

pathogens and one should start looking for the source of pollution and taking
proper actions. Sometimes, additional chlorination is needed e.g. for drinking
water over 0.2 mg Cl2/l. In some cases, changes to the construction of the water
supply system and removal of the biofilm are effective protection against the
excessive level of microorganisms in water.

3.1.8 Wastewater treatment

Wastewater (sewage) is polluted water which includes all harmful liquid,
solid or gaseous substances introduced into waters or soil that may lead to a
contamination of surface or underground waters. Sewage also includes: used-
up liquids, solutions, colloids, suspensions, radio-contaminated waters, saline
waters, heated cooling waters, precipitation waters or waters which contain
various impurities from urban and rural areas.
A. Classification based on origin
• domestic sewage contains large amounts of faecal matter, plant and
animal wastes, surface-active agents, urea. The sewage comes from
households, public lavatories and industrial facilities posing a serious
hygienic and epidemiological threat,
• industrial (technological), evolve during all types of industrial
processes (manufacturing and processing),
• precipitation (rain and meltwaters) contain various atmospheric
impurities (dusts, microorganisms, gaseous substances), surface run-
offs, streets and paved surfaces run-offs (oils, liquid fuels, bacteria,
small particle suspensions), microbiological impurities (bacteria,
viruses, fungi).
B. Classification based on harmfulness
• directly harmful,
• indirectly harmful (lead to a decrease of oxygen in water below the
essential organisms’ requirement).
C. Classification based on contamination stability
• degradable—organic substances that undergo chemical transformations
to form simple compounds,
• non-degradable—substances that do not yield to any chemical
transformations and are not decomposable by microorganisms,
• stable—substances which only slightly undergo biological
decomposition and stay in the habitat in an unchanged form for a long
time.
D. Man-made
• urban and domestic—source: food serving facilities, hospitals, houses
and apartments posing a hygienic and epidemiological threat,
3.22 Environmental Biotechnology

• rural—source: farms, pig fattening houses, animal farms, intensively

fertilized fields,
• industrial—source: manufacture and processing of all branches of
industry; this type of sewage is a major source of toxins,
• radioactive—source: scientific and health facilities, nuclear reactors;
such types of wastes are especially dangerous to the habitat, therefore,
they require special storage methods.

Pollutants–Municipal sewage
Sewage is characterized by the following groups of organic and non-organic
impurities:
• soluble substances,
• settling suspensions,
• liquid-suspended suspensions.
Chemical impurities contained in sewage may be divided into:
• dissolved mineral substances (sulphates, chlorides, acids and neutral
carbonates, calcium, magnesium, sodium, bases, nitrates, phosphates
etc.),
• soluble gases (oxygen, hydrogen sulphide, carbon dioxide, nitrogen),
• soluble organic substances (proteins – about 40-60%, carbohydrates –
about 25-50%, oils and fats – about 10%).
One further classification of pollutants in sewage is as follows:
(a) physical impurities
(b) chemical impurities
(c) biological impurities
(a) physical pollutants of sewage are characterized by properties which can
be detected by the senses (sight, smell). The properties of physical pollutants
are: suspension, cloudiness, colour, smell, temperature.
(b) organic pollutants are defined by three common parameters: BOD
(biochemical oxygen demand), COD (chemical oxygen demand), TOC (total
organic carbon).
• BOD – determines the amount of oxygen required by bacteria in
order to biologically oxidize decomposable organic compounds in
aerobic conditions in a temperature of 20°C. About 50% of pollutants
are oxidized by microorganisms over a period of three days. Five
days as the representative period is assumed to determine the
characteristic of biochemical oxygen demand.
• COD – specifies the amount of oxygen required to oxidize organic
compounds chemically.
Environmental Microbiology—Water and Air 3.23

• TOC – specifies the amount of carbon contained in organic

compounds.
(c) biological pollutants include microorganisms (viruses, bacteria, fungi),
eggs of helminthes.

Biogenic pollutants: Dangers connected with them

Biogenic pollutants are made up of mineral salts of elements which are essential
for the development of living organisms. The basic ones are the compounds of
phosphorus and nitrogen. After introduction into lakes and rivers, the above
compounds increase their fertility causing eutrophication.
Eutrophication is a term that describes a complex of unfavorable
symptoms connected with over-fertilization. Urban sewage contains
phosphates from human excrements, washing detergents and liquids, food
wastes, food additives and other products. Another significant source of
phosphate pollution of water is sewage from the agricultural industry. The
presence of phosphorus in sewage introduced into water along with nitrates
and nitric dioxides causes increased development of algae in both lotic and
lentic waters. Increased eutrophication has been considered to be hazardous to
water reservoirs as a consequence of uncontrollable growth of plant biomass.

Refraction pollutants
Refraction pollutants are those which do not, or only to a minimal extent,
undergo biological decomposition by microorganisms. Some of them
demonstrate characteristics of dangerous poisons e.g. heavy metals, PAH
(polycyclic aromatic hydrocarbons), PCB (polychlorinated biphenyls), dioxins,
pesticides, nitrosamines. Elimination of contamination from industrial and
municipal wastes, prior to their reintroduction to a receiving body of water,
results from a need for rational management of water supply, environment
protection and adequate sanitary conditions. Introduction of pollutants,
depending on the watercourse, may decrease the water’s physical, chemical
and sanitary conditions or even cause the disturbance of biological balance.

Main objectives of the wastewater treatment process

The objectives of the wastewater treatment process are:
• lowering the content of organic carbon including compounds which are
difficult to biodegrade as well as the toxic, mutagenic and carcinogenic
ones,
• reduction of biogenic substances: mineral salts of nitrogen and
phosphorus,
• elimination or inactivation of pathogenic microorganisms and
parasites.
3.24 Environmental Biotechnology

Methods of the wastewater treatment

Depending on the type of pollutants, there are different methods of purification
used prior to reintroduction into a receiving body of water. The methods are
classified as follows:
• mechanical – in this method only non-soluble pollutants are removed
by utilizing the following processes: gravitational and centrifugal
sedimentation, flotation, source filtration, separation in hydrocyclones,
which allow the removal of organic and mineral suspensions as well as
floating bodies;
• physical-chemical – utilizes the following operations and processes:
coagulation, coprecipitation, sorption, ion exchange, electrolysis, reverse
osmosis, ultrafiltration;
• chemical – utilizes neutralization, oxidation, reduction;
• biological – consists of sewage purification (elimination of organic
pollutants as well as biogenic and some refraction compounds) during
biochemical processes of mineralization conducted naturally by
microorganisms in a water habitat (e.g. sprinkling of wastewater onto
agricultural lands), or in special devices (on trickling filters or activated
sludge).

Stages of sewage treatment

A typical process of sewage treatment consists of four stages of purification:
• mechanical (stage I of purification),
• biological (stage II of purification),
• elimination of biogenic compounds (stage III of purification),
• water renovation (stage IV of purification).
Stage I of purification, primary treatment includes the so-called initial
or mechanical purification. The goal of this stage is the removal of solid
impurities. This stage is considered to be the preparation of sewage for
further purification. By utilizing simple mechanical operations, the following
impurities are removed during, the first stage:
• floating solid impurities,
• settling suspensions,
• oils and fats.
Stage II of purification, secondary treatment includes biological
purification, which leads to the biodegradation of soluble organic impurities,
colloidal systems and suspensions not removed during the first stage. The
intensification of purification processes is obtained by utilizing trickling filters
and activated sludge.
Environmental Microbiology—Water and Air 3.25

Stage III of purification, tertiary treatment includes processes used to

thoroughly clean sewage. The largest impurities removed during this process
are the biogenic compounds (compounds of phosphorus and nitrogen).
The nitrogenous compounds are removed during the process of biological
nitrification and denitrification, whereas the compounds of phosphorus
are eliminated by a process of chemical precipitation. The role of thorough
cleaning of sewage in this stage is the prevention of water eutrophication.
Stage IV of purification (water renovation) includes the processes
of residual sewage removal, which are left over from the previous stages
of purification. Water regeneration involves a set of methods which confer
the properties of natural water onto the sewage so that it can be utilized
in industrial facilities. Water regeneration allows the recycling of sewage,
which is a significant element in water resource management, especially in
regions low in water. There are several systems of water regeneration, from
very simple ones, that use rapid filters or straining through microsieves, to
very complex physical chemical processes: coagulation, membrane processes
and disinfection, sedimentation, expelling of ammonium, recarbonization,
absorption, ion exchange, and water demineralization.

3.1.9 Biological methods of wastewater treatment

In biological methods of sewage treatment, bacteria which form zoogleal
clusters in sewage play a crucial role. Methods of biological purification
of sewage consist of inducing the enzymatic processes of saprophytic
microorganisms that include partial oxidation of organic substances (sources
of carbon) contained in sewage as well as their partial assimilation by
microorganisms. As a result of these processes, an increase in cell mass of
the active microorganisms occurs. Microorganisms flourish when the ratio of
three basic elements C:N:P = 100:10:1.
The biological processes of purification can be divided into natural and
artificial, depending on where the processes take place—whether they occur in
natural conditions or are intentionally triggered in specially designed artificial
equipment. Biological purification can be conducted in oxygen-rich, oxygen-
poor or oxygen-free conditions. It is a process of oxidation and mineralization
of organic compounds from sewage using micro- and macroorganisms. During
the process of biological purification the following phenomena take place:
• breakdown of organic substances down to CO2, H2O, NH3 (dependent
on pH)
• nitrification (oxidation of NH3 by Nitrosomonas bacteria down to
nitrites, and then by Nitrobacter bacteria down to nitrates),
• denitrification (transformation of nitrates to gaseous nitrogen N)
3.26 Environmental Biotechnology

Natural methods
Natural methods of wastewater treatment include: purification in soil, field
and forest irrigation (the method of irrigation and filtration fields) and soil
filters.

Purification in soil
Biological purification in a field soil consists in irrigation of a field with
sewage. Biogenic substances contained in sewage lead to an average of 20%
yield increase. A field used for agricultural purposes can receive an annual
dose of 600 mm effluent per annum. After spreading, the sewage seeps into
the soil and the contents of impurities are absorbed by the soil particles.
Prior to introducing sewage onto the fields, sewage undergoes mechanical
purification (screens, sand traps, primary settling tank) and is disinfected.
Moreover the irrigated soil is checked for the content of metals. Field
irrigation can be conducted only during the period of plant vegetation and
the amount of the applied sewage has to be altered at different times. In the
winter, sewage is purified on filtration fields. After some time, the absorbed
organic compounds and microorganisms create a microscopic film around the
particles of soil and the surface soil layer works like a biological filter. The
final products of the mineralization process taking place in this layer, act as
fertilizer for the soil. Only a limited amount of sewage can be purified by this
method, otherwise the field becomes excessively loaded with sewage. In such
situations, oxygen-free processes are triggered, that are accompanied by the
formation of toxic substances and, odour release, causing the plant growth to
stop. Due to sanitary reasons, prior to irrigation, sewage must be cleared of
any helminth eggs. During the infiltration in soil, sewage gets purified and
then carried over to a receiving body of water by a drainage system.

Soil filters
Purification by soil filters consists of spreading waste upon the surface of soil
that leads to its biological purification. Most often non-cultivable fields are
utilized for such forms of purification. The lack of agricultural use allows
utilization of greater amounts of sewage (annual dosage of sewage may
go as high as 3000 mm/a). Loose and sandy soils with a grain diameter of
0.2-0.5 mm, with strata thickness between 1.5-2.0 m and with a low level of
underground water are best suited for this purpose. The field is divided into
drying beds (about 0.5 hectare area). Not cultivating the soil can result in
greater amounts of sewage being purified. Prior to pouring out the sewage,
it has to be mechanically cleaned, in order to eliminate the oily suspension
that clogs up the source. The drying beds are flooded with wastes (thickness
5-10 cm) every 0.5-4 days.
Purified waste is drained by a drainage system installed in the ground.
After some time, the soil filters lose the ability to purify and have to be
periodically excluded from operation in order to regenerate. Sewage may
Environmental Microbiology—Water and Air 3.27

undergo purification in the winter when it is upon the filtrating fields. The
number of active plots is then lowered in order to minimize losses, and
instead, the depth of the flooding increases to 20-30 cm. The surface of the
sewage covers with ice, under which the sewage is supplied and undergoes
the process of purification while decreasing the quality of the outlet.

Sewage ponds
Sewage ponds are earth reservoirs, in which the process of biological
purification occurs naturally (utilizing microbes) thus; they are used in
smaller towns, where the number of inhabitants does not exceed 20,000.
Sewage ponds are either natural or artificial ground reservoirs, in which solar
radiation reaches the bottom. Prior to introduction of sewage into the pond,
sewage has to be preliminarily cleared of suspended matter. Sewage ponds,
usually consist of a series of ponds: bacterial, algal and crustaceal. Oxidation
of organic compounds by bacteria takes place in the bacterial pond, which
leads to their mineralization, i.e. transformation into non-organic compounds
known as biogenic salts.
Sewage purified in that way is then directed to algal ponds, where algae
flourish on it, assimilating the mineral compounds that evolved during the
process of biodegradation. The final stage of such purification is conducted in
the crustacean pond, where algae eating crustaceans flourish. Such systems
of purification allow the elimination not only of organic substances but also
of the excess biogenes, whose presence in the receiving body of water could
cause eutrophication and consequently water blooming which lowers the
oxygen content in water. This pond can be used for fish and duck breeding
without a need for artificial feeding.

Hydrobotanic purification
Hydrobotanic purification consists of utilization of self-purifying processes
that take place in waterlogged ecosystems, thus it belongs to so-called wetland
systems. Purification is a result of the co-operation of soil microorganisms and
boggy plants. Microorganisms decompose the organic compounds contained
in sewage, turning them into non-organic compounds, whereas plants absorb
the produced mineral compounds creating a plant biomass. The adsorption
of impurities by the particles of soil is improved, due to very small mineral
particles (silt) present in the substrate. This type of treatment unit utilizes
wetland vegetation (common-reed, reed-mace, basket willow, etc.) that has a
high requirement for food, thus it absorbs large quantities of mineral salts. As
a result, the vegetation desalts the sewage and does not lead to eutrophication
of water reservoirs.
There are three types of hydrobotanic purification plants:
soil-plant filters—are the types of filters with horizontal (most common
and the longest in use), vertical or combined flow; mainly sandy with rooted
boggy vegetation (common-reed, reed-mace, schrubby willows).
3.28 Environmental Biotechnology

shallow reservoirs with rooted vegetation—these include water

reservoirs or ducts of depths between 10-50 cm, they are occupied by boggy
vegetation (common-reed, reed-mace, sedge).
sealed reservoirs with floating vegetation—ponds of depth between
1-2 m, with sealed bottoms and side walls, with floating vegetation – in our
climatic conditions it is duckweed – Lemna minor. Exploitation problems are
connected with an even spread of duckweed throughout the surface of the
pond and removal of the rapidly growing plants. In the winter, due to lack of
vegetation, the purification plant plays the role of a normal pond.

3.1.10 Artificial methods of wastewater treatment

Trickling filters: The treatment of sewage by trickling filters is conducted in
reservoirs filled with loose, grainy and porous material. Sewage is sprayed
upon the upper layer of the bed with sprinklers and then left to seep through
its content. A mucous biological film forms upon the content of the bed. The
film is composed of microorganisms such as: bacteria, protozoa and fungi.
The role of the filter involves a constant supply of sewage and its flow
through the trickling filter while maintaining contact with the biological film.
During the flow, the sewage undergoes mineralization as a result of aerobic
decomposition by microorganisms. The biological film is initially composed of
zoogleal bacteria which produce mucous sheets. With time, the composition
of species of the mucous membrane changes due to their succession. Besides

Trickling filter

The trickling filter process

Environmental Microbiology—Water and Air 3.29

bacteria, the following appear: fungi, protozoa, annelida and fly larvae.
Depending on the amount of treated sewage, the trickling filters may be
subdivided into percolating and flushing filters.
Depending on the amount of the organic load, the following types of
biofilters are distinguished:
• Low–loaded – may be filled with natural or artificial material. The
supplied organic material is less than 0.4kg BZT5/m3·d. In percolating
filters, the film is more developed and the biological process of
decomposition is almost complete. In the final phase of purification,
intensive processes of nitrification occur, which lead to an increase of
nitrates in a run-off to the secondary settling tank.
• Mid-loaded – are filled with natural-synthetic material and work with
a load between 0.4-0.65 kg BZT5/m3·d. In order to ensure an adequate
concentration of the supplied sewage, the recirculation of part of the
purified sewage is utilized with this type of filters. The reduction of
organic compounds upon these filters is adequate, and the processes
of nitrification partially occur. The introduction of additional processes
of purification is not necessary.
• High loaded (flushed) — are filled with natural-synthetic material,
the filter is loaded with: 0.65-1.6kg BZT5/m3·d. In flushing filters, the
intensity of sewage flow is greater, however, the biofilm is composed
almost entirely of bacteria and does not develop as much as in the
above stated case. Flowing sewage washes out used and dead biological
material from the filter. The washed out material is transported in
the form of flocy sediment. Only a partial mineralization of organic
compounds occures on that type of filter and the nitrification process
is inhibited. A low content of nitrates in effluent from filters testifies
to partial mineralization of organic compounds. In complex systems,
after these types of filters, re-purification is utilized, as the quality of
the purified sewage does not usually meet the required standards.

Activated sludge
The process of activated sludge relies on sewage purification by freely suspended
matter. It consists of producing 50-100 mm flocs with highly developed surface
areas. The floc is made up of brown or beige mineral nucleus, while on its
surface, it contains heterotrophic bacteria within the mucous envelopes. The
method of activated sludge requires delivery of oxygen into the substrate for
bio-oxidation of organic pollutants, which should be > 0.5 mg/dm3 in order to
ensure proper oxygen conditions for the bacteria.

Activated sludge characteristics

Activated sludge is a type of flocculent suspended matter created during
the aeration of sewage. Treating sewage with activated sludge consists of
3.30 Environmental Biotechnology

mineralization of organic compounds, conducted mainly by bacteria and

following the same biochemical processes as observed in self-purification.
However, the speed of the process is much greater. This results from the fact
that the conditions of intensive aeration, triggered during sewage flow through
aeration tanks, are conducive to the development of impurity-decomposing
bacteria. Agglomeraters (flocs), which consist of heterotrophic bacteria
coagulated with mucous, form during the process of aeration in aeration tanks
(flocculation). The floccules absorb impurities contained in sewage, whereas
microorganisms in floc decompose the absorbed substances.
Activated sludge has a spongy, loose structure, made of small openings
of various shapes. Undisturbed floccules easily settle and thus, allow the
separation of the activated sludge from sewage.
Biocenosis of activated sludge is, for the most part, composed of
heterotrophic bacteria. In small percentages—and only under particular
conditions and in some arrangements—it’s made up of chemolithotrophic
bacteria, especially nitrifying bacteria. The most common species of activated
sludge are: Zooglea ramigera , Pseudomonas fluorescens, Pseudomonas putida
as well as bacteria of Achromobacter, Bacillus, Flavobacterium and Alcaligenes
genera. The process of selection occurs naturally. The conditions in an aeration
tank, especially the chemical composition, pH value and air conditions, are the
determining factors for the diversity of the bacterial complex. In unfavourable
conditions (overloading of aeration tanks with easily available substrates, high
oxygen deficit), excess development of flocs occurs causing the so-called active-
sludge swelling. There are two distinguishable types of swelling: fibrous and
non-fibrous swelling. Fibrous swelling is caused by excess filiform bacteria
(Sphaerotilus natans, Beggiatoa alba or Thiothrix nivea) or fungi development.
Non-fibrous swelling is caused by bacterial development, which produce
excess amounts of mucous.
Active sludge biocenosis is made up of not only bacteria but also
protozoa, nematodes and rotifers. Even though these microorganisms do not
play a major role, their presence is equally important. Protozoa feed upon
bacterial cells forcing them to reproduce quickly, which essentially make them
an important renewal and reactivating factor of the activated sludge. The
most common protozoa are: Vorticella, Carchesium and Opercularia as well as
Anthophysa, Oxytricha, Stylonychia and Lionotus.
There is an inverse relationship between flagellates and ciliates within
activated sludge. While a large number of flagellates indicate an overload of
sludge, the presence of ciliates goes to show it is functioning properly. During
the course of sewage purification with activated sludge, a characteristic
succession of biocenosis is observed.
Activated sludge process: Sewage is directed to aeration tanks filled with
activated sludge (thick suspension of microorganisms) after its mechanical
purification. The content of the aeration tank is constantly aerated in order
Environmental Microbiology—Water and Air 3.31

to provide an adequate amount of oxygen, to keep the activated sludge in

a suspended state and to ensure its constant mixing. The aeration tank
is a device, in which the development of the activated sludge results from
continuous cultivation. There is a state of equilibrium between the rate of
sewage inflow, concentration of nutrients, bacterial reproductive rate, and the
rate of the sewage outflow containing some activated sludge in it.
During the time of contact of sewage with the activated sludge, the
decomposition processes occurring simultaneously enable the development of
activated sludge biomass. Separation of purified sewage is done in a secondary
settlement tank. Both sedimentation and clarification of the purified sewage,
which is then carried off to a receiving body of water, occurs in the device.
Activated sludge may be used again for purification; it is then recycled into the
aeration chamber. However, quite often, before reuse, the sludge is directed to
a regenerative chamber, where it is aerated in order to bring back its particular
physiological properties. When the sludge collected in a secondary settling
tank is not recycled, then, as an excess sludge, it is removed and subjected to
additional processing.

The activated sludge process

3.1.11 Methods of chemical wastewater treatment

Purification of industrial sewage that contains mineral and organic compounds,
and heavy metals, utilizes physical-chemical and chemical methods. They
include the following processes: neutralization, coagulation, oxidation,
reduction, sorption, flotation, membrane processes, extraction, electrolysis,
distillation.
Neutralization: It is a process of chemical neutralization of sewage in
relation to the pH. Depending on the make-up of sewage and the type of the
reacting substance used, neutralization may be accompanied by a chemical
process of precipitation and coprecipitation. Neutralization may be conducted
by mixing acidic sewage with bases. Hydroxides are substances most often
3.32 Environmental Biotechnology

used in the process of neutralization: NaOH in the form of 20-30% solution,

Ca(OH)2 in the form of 5-15% milk of lime, Na2CO3 in solution form CaCO3,
MgCO3, MgO, dolomite in the form of a grainy filter. Mineral acids are used
for the neutralization of basic sewage: H2SO4, HCl, H3PO4 in the form of
solutions as well as CO2 in the form of a clear gas.
Coagulation is a process of binding colloidal particles and the suspension
into clusters of particles called the agglomerates, which results in precipitation
of the sediment in the form of coagulate. The factors which most often cause
coagulation are: addition of an electrolyte solution to lower the electrolytic
potential, addition of colloids of an opposite charge into the colloidal particles,
creation of metal hydroxides that absorb ions, colloids and suspensions.
Oxidation: An oxidation process is conducted in order to remove organic
compounds, non-organic compounds and microorganisms from sewage.
The reacting substances used in oxidation are: chlorine, chlorine-oxidizing
compounds (NaOCl, Ca(OCl)2, chlorinated lime, chlorine dioxide, ozone.
Reduction: The process of reduction used in sewage purification mainly
concerns chromium. Chromium salts (VI) are toxic, carcinogenic, bacteriocidal
and are irritants to skin. Its bacteriocidal properties slow down the process of
water self-purification. Reduction of chrome from oxidation state of 6+ down
to 3+ is conducted through reduction and precipitation of hydroxide, which
belongs to a group of barely soluble compounds. Reduction is conducted
either chemically or electrochemically.
Sorption: Sorption consists of binding liquid soluble substances to the
surface of solids. Depending on the characteristics of the process, it may be
irreversible (chemiosorption), or reversible—adsorption. The characteristic of
the process of sorption is determined by one of the components of force:
• physical sorption — the result of van der Waals forces,
• chemical sorption — the result of valence forces,
• ion sorption — between groups of cations and anions in the structure
of the substrate,
• sieve sorption — at the molecular level according to the mechanism of
a molecular sieve.
Flotation: A process of structural separation consisting of raising the
hydrophobic impurities into the foam along with the rising gas bubbles. As
a result, the foam formed has a much higher concentration of pollutants than
the rest of the sewage.
Membrane processes: These processes consist of separation of particles
by flowing through a porous layer (membrane). The following are the types
of membrane processes: reversed osmosis, nanofiltration, ultrafiltration,
electrodialysis.
Extraction: This consists of transfer of components from one phase of the
solution into the second liquid phase (dissolvent). Consequently, a solution of
Environmental Microbiology—Water and Air 3.33

the component in the dissolvent is obtained. The required condition for the
process is the presence of two liquid phases.
Electrolysis: The process in which electrical energy invokes chemical
changes of the electrolyte. As a result of the electrical field, the movement of
ions toward the electrodes (upon which the process occurs) occurs:
cathode Me+ + e– → Me (reduction)
anode X– → X + e– → (oxidation)
Distillation: Process that utilizes the difference between the composition
of a liquid and vapour in the state of equilibrium.

3.1.12 Nucleic Acid - Based Techniques for Analyzing the Diversity,

Structure, and Dynamics of Microbial Communities in
Wastewater Treatment
Biological wastewater treatment systems, like activated sludge basins,
trickling filters, or anaerobic digesters, essentially can be interpreted as
specialized aquatic ecosystems, where microorganisms are the main players.
In order to fully characterize, understand, and, in the long run, control those
microbial communities, knowledge of both their structure and function is
necessary. Attention should, therefore, be given to identification, enumeration,
and spatial distribution (structural parameters) as well as to in situ activities
(functional parameter) of the community members. Furthermore, from
an ecological and from an engineering point of view stability or dynamics
of these microbial communities are important both for theory and practice.
Until recently, identification of microorganisms required the isolation of
pure cultures and the investigation of physiological and biochemical traits.
Enumeration had to be done by plate counts or most probable number (MPN)
techniques. However, since all cultivation-dependent techniques are not only
time-consuming and laborintensive but select for certain organisms, they are
inadequate for determining reliable cell numbers, and in many cases even for
the identification of the main catalysts of a system. Questions like micro-scale
distribution and in situ activity of microorganisms are almost impossible to
address by classical methods.
To circumvent these limitations, identification techniques based on nucleic
acids have been developed and successfully applied to wastewater treatment
systems during the last five years. This chapter intends to summarize
the potential, current applications, and limitations of nucleic acid-based
techniques for analyzing the microbial communities present in wastewater
treatment systems.

Ribosomal Ribonucleic Acid (rRNA)-Based Methods

Macromolecules like rRNA or proteins can be used as “molecular clocks” for
evolutionary history. By comparative sequence analysis, reconstruction of
3.34 Environmental Biotechnology

evolution and classification of organisms based on their phylogeny is possible.

For several reasons 16S, rRNA sequence comparison is currently considered
the most powerful tool for the classification of microorganisms.
• Ribosomes and consequently rRNA molecules are present in all
organisms. As an essential component of the protein synthesis
apparatus, they have a homologous origin and show functional
constancy. No lateral gene transfer has been shown for rRNA genes so
far. Therefore, it is a valid assumption to reconstruct the phylogeny of
the organisms based on these molecules.
• Some positions of the rRNA molecules are evolutionary more conserved
than others. Consequently, sequence regions can be found that allow
differentiation at any taxonomic level from species and genera up to
kingdoms or domains.
• 16S rRNA sequences have been determined for many of the described
bacterial species and deposited in public databases.
• The natural amplification of rRNA within microbial cells (usually
more than 1,000, frequently several 10,000 copies) makes it easier and
more sensitive to assay this molecule and gives, e.g., the opportunity
for identification of single bacteria by fluorescent oligonucleotide
hybridization.
• The presence and abundance of ribosomes and, consequently, rRNA
in individual cells is connected to their viability and general metabolic
activity, or at least their metabolic potential. Cells in a rapidly growing
E. coli culture need and have more ribosomes than those in a slowly
growing culture.
The applications of rRNA-based nucleic acid techniques to the analysis
of wastewater treatment systems today range from a simple identification
of isolates over the detection of bacterial diversity and population dynamics
to attempts at fully and quantitatively describing the complex microbial
communities.

Molecular Characterization of Isolates

Despite the success of molecular methods in analyzing microbial communities,
classical isolation of bacteria is still absolutely necessary for, e.g., investigations
on the metabolic potential of the community members. However, nucleic acid-
based techniques can support this classical approach by providing tools for
screening, characterization, and identification of isolates. Compared with
microbiological or biochemical methods, molecular methods are more rapid
and more reliable since they are not affected by growth conditions and culture
media.
Environmental Microbiology—Water and Air 3.35

Restriction Fragment Length Polymorphism (RFLP) and

Amplified Ribosomal DNA Restriction Analysis (ARDRA)
A common methodology for a rapid molecular characterization of isolates
is based on the generation of so-called “genetic fingerprints”. DNA of an
organism is digested by rare cutting restriction enzymes, and the resulting
fragments are separated by length using pulsed-field gel electrophoresis.
Different species will have differing fragment lengths due to mutations of the
restriction sites, insertions, or deletions, and these restriction fragment length
polymorphisms (RFLPs) are used for comparison of the isolates. Since cutting
the whole genome requires high quality DNA extraction prior to RFLP analysis
and time-consuming, complicated separation of the rather large fragments by
pulsed-field gel electrophoresis, most recent applications of RFLP are based
only on part of the genome. For example, amplified ribosomal DNA restriction
analysis (ARDRA) starts with the amplification of the genes encoding 16S
rRNA by polymerase chain reaction (PCR), followed by restriction digestion
and analysis of the fragment lengths by standard gel electrophoresis. The
resulting patterns or fingerprints are compared to reference strains and can
be used for cluster analysis of different isolates. In principle, a database of
restriction patterns of reference organisms can be established to facilitate
rapid identification of isolates by ARDRA.
ARDRA allows the processing of numerous isolates in a very short time,
and can yield valuable information on the similarity of isolates. Consequently,
appropriate isolates can be selected for further physiologic and phylogenetic
investigations. On the other hand, reliable identification using ARDRA
is hampered by the enormous amount of reference patterns required.
Furthermore, the information content in an ARDRA pattern is limited
(frequently, only few bands) and this could cause false-positive identification.

Other Methods
Besides ARDRA, the methods described in the following sections can also be
applied. Denaturing gradient gel electrophoresis is ideal to screen a large set
of isolates for redundancy. Hybridization with sets of rRNA–targeted probes
also allows rapid screening of isolates for their phylogenetic affiliation. By
this technique, e.g., the majority of isolates from a municipal activated sludge
was characterized as members of the gamma subclass of Proteobacteria. In a
second example, genus or species-specific probes were used for confirming the
identification of Paracoccus sp., initially based on physiological tests. Finally, the
full description of a new species, identified to be important in a given system,
should always include the determination of its 16S rRNA sequence for valid
phylogenetic classification and, if applicable, the DNA–DNA hybridization
with related species or strains.
3.36 Environmental Biotechnology

Diversity and Dynamics of Microbial Communities

Microbial diversity and population dynamics can be rapidly monitored using
rRNA-based pattern or fingerprinting methods. These encompass extraction
of total nucleic acids (DNA and/or RNA) from an environmental sample (like
activated sludge or biofilms), amplification of part of the genes encoding 16S
rRNA by PCR, and subsequent separation of the resulting gene fragments on
a gel to form a pattern or fingerprint of the community.

Denaturing Gradient Gel Electrophoresis (DGGE)

Method: By DGGE, DNA fragments of the same length but with different
sequences can be separated. 16S rRNA gene fragments of a length of typically
200–500 bp are amplified by PCR with an additional 40 bp GC-rich sequence
at the 5b end of one of the primers. When analyzed on a polyacrylamide gel
containing an increasing gradient of DNA denaturants (a mixture of urea and
formamide), for each of these DNA molecules (ds DNA), a transition from
a double-stranded, helical to a partially single-stranded secondary structure
will occur at a certain position in the gel. This will stop, or at least strongly
slow down, the migration of the respective gene fragment. The GC-rich region

Flow chart of a community analysis by denaturing gradient

gel electrophoresis (DGGE)
Environmental Microbiology—Water and Air 3.37

acts as a “clamp” to prevent formation of single-stranded DNA (ssDNA). As

sequence variations cause a difference in the melting behavior of ds DNA,
sequence variants of particular fragments will stop at different positions in
the denaturing gradient gel and hence can be separated effectively (Fig.1).
The result of DGGE analysis of PCR products obtained from a microbial
community is a band pattern, and the number of bands is a rough estimate for
the microbial diversity of a given system.
Applications: The patterns are frequently used for comparisons of
different systems, e.g., aerobic and anaerobic biofilms or different activated
sludge plants. DGGE is particularly useful to detect population changes
addressing the question of stability and dynamics of microbial communities.
The method can be modified by using rRNA instead of DNA as a template
for 16S rDNA amplification. For that purpose, extracted rRNA is transcribed
into ribosomal copy DNA (rcDNA) by the enzyme reverse transcriptase prior
to the PCR amplification. While the rDNA-based DGGE pattern is solely
determined by the presence of DNA and, therefore, in first approximation by
the abundance of populations, the rRNA-based DGGE pattern should more
strongly represent the metabolically active and, therefore, rRNA-rich parts of
the community.
A band at a given site of a DGGE gel has per se no biological meaning.
Therefore, to learn more about the population represented by a certain band,
this band needs to be further characterized, which can be achieved, in principle,
by two techniques. The DGGE gel can be blotted to a nylon membrane and
the pattern can be examined by hybridization analysis with taxon-specific
probes. Alternatively, bands can be retrieved from the gel and subsequently
sequenced; comparative sequence analysis then allows identification or at
least affiliation with the closest relative.
Limitations: Whereas DGGE analysis of rDNA PCR products is a
powerful tool to analyze diversity and dynamics of microbial communities, it
has severe limitations in the analysis of community structures, and is like any
other method prone to specific biases:
(1) The method involves extraction of nucleic acids and subsequent
tPCR, which may both cause some bias: Not all cells lyse under the
same conditions, and preferential amplification of certain templates
can occur. Therefore, different intensities of DGGE bands must not
be interpreted as quantitative measures of the abundance of species
relative to each other. For qualitative statements on the development
of a certain band (population) over time, i.e., to monitor its appearance
or disappearance, identical treatment of all samples has to be ensured.
(2) Separation of DNA fragments with high resolution is restricted to
a maximum size of about 500 bp. Consequently, the phylogenetic
information that can be retrieved by sequencing is relatively little. In
case of full identity with an rRNA sequence in a database, it might
3.38 Environmental Biotechnology

be sufficient for an identification, but in cases in which only distantly

related sequences are available, classification becomes difficult if not
impossible. Also characterization of bands by hybridization analysis
with rRNA-targeted oligonucleotide probes is only possible if the
probe target region is within the amplified fragment, which is only the
case for a small fraction of the full set of available probes.
(3) The main difficulty, however, is the “one band – one species” hypothesis.
Especially in complex communities, bands might originate from two
or more fragments that co-migrate on the denaturing gradient gel.
Furthermore, single species might result in two or more DGGE bands
due to inter-operon microheterogeneity.

Terminal Restriction Fragment Length Polymorphisms (T-RFLP)

Recently, RFLP analysis has been modified for application to microbial
communities: After extraction of total DNA from an environmental sample,
16S rDNA is amplified by PCR with one of the two primers being fluorescently
labeled. The fluorescent PCR products are then digested with frequently
cutting restriction enzymes, and analyzed by a standard high resolution gel
electrophoresis in which the restriction fragments are separated solely by
size. The abundance and length of only the fluorescent terminal fragments
is determined in automated sequencing devices or by fluorimetry. This
again yields a pattern or “community fingerprint”, like in the case of DGGE.
The method may be suitable for analyzing diversity and dynamics of
microbial communities as demonstrated for activated sludge. Besides similar
advantages, it suffers even more from similar limitations as DGGE: The length
of the terminal rDNA fragments ranges from 60–500 bp, too little information
for valid phylogenetic affiliation or application of specific oligonucleotide
probes. Different species may have in many cases the same terminal restriction
fragment length, hence leading to an underestimation of the actual bacterial
diversity. T-RFLP seems to be an easier, but also a less sensitive alternative to
DGGE.

Community Structure
The structure of a microbial community is mainly defined by two parameters:
Identity and abundance of its members. A broader definition of structure
would also include the spatial arrangement of species relative to each other,
an information which might be especially important in stratified habitats like
biofilms. Since both cultivation and fingerprint methods are not sufficient to
address these questions, hybridization techniques applying rRNA-targeted
oligonucleotide probes have been developed.
Oligodeoxynucleotides are single-stranded pieces of DNA with a length
of 15–25 nucleotides. When such oligonucleotides are labeled, e.g., with
radioisotopes or fluorescent dye molecules, they become so-called probes
that allow detection of complementary target sites by specific base pairing.
Environmental Microbiology—Water and Air 3.39

For the reasons outlined before, target molecules are rRNAs, extracted and
immobilized on a membrane (dot blot hybridization), or maintained in fixed
cells (whole cell hybridization), respectively. In both cases, quantification is
possible, either relative to total extracted rRNA, or relative to total cell counts.

3.2 AIR AS AN ENVIRONMENT OF MICROORGANISMS

• Air is an unfavorable environment for microorganisms, in which they
cannot grow or divide. It is merely a place which they temporarily
occupy and use for movement.
• Therefore, there are no metabolic connections occurring between
different microorganisms in air (such as in soil or water). As a
result, they form only a random collection of microorganisms, not a
microbiocenosis.
• Microorganisms get into air as a consequence of wind movement,
which sweeps them away from various habitats and surroundings (soil,
water, waste, plant surfaces, animals, and others), or are introduced
during the processes of sneezing, coughing, or sewage aeration.
Why are the air conditions unfavourable for the microorganisms?
There are 3 elementary limiting factors in the air:
• a lack of adequate nutrients,
• frequent deficit of water, threat of desiccation,
• solar radiation.
It is obvious that the first factor limits cell growth. As a matter of fact, air,
and especially polluted air, contains some organic substances, but they are
usually poorly decomposed and there is not enough to be utilized as food.
Besides, there are other unfavorable factors contributing.
Microorganisms contained in air are constantly subjected to drying, which
definitely stops all processes. Some bacteria are especially sensitive to water
deficits which cause bactericidal effects (e.g., gonococci or spirochete which die
as soon as they enter the air). Many organisms, however, can successfully cope
with water deficits and, although they cannot function properly, their dried
up forms survive months and even years (endospores, fungi spores). Solar
radiation is also damaging to microorganisms suspended in air as it causes
mutation and desiccation (in water and soil, the solar radiation is usually very
weak or simply does not exist)

3.2.1 Adaptation of microorganisms to the air environment

What types of microorganisms occur in air?
There are 3 main groups of microorganisms that occur in air:
• viruses
• bacteria
• fungi
3.40 Environmental Biotechnology

Bacteria may exist as vegetative or resting forms, however, fungi occur

in the form of spores or fragments of mycelium. Especially in the vegetative
season, pollen of anemophilous plants (e.g., grasses and some trees) is
abundant in the air. Besides the above, the following can be found in air as
well: algae and protozoa cysts and small invertebrates such as worms in forms
of eggs or cysts and mites.
Besides living microorganisms, their fragments and products, which often
exhibit toxic or allergic activities, may also occur in air.
Which microorganisms are best adapted to a prolonged existence in air?
The atmosphere can be occupied for the longest time by those forms which,
due to their chemical composition or structure, are resistant to desiccation and
solar radiation. They can be subdivided into the following groups:
• bacterial resting forms,
• bacterial vegetative forms which produce carotenoidal dyes or special
protective layers (capsules, special structure of cell wall),
• spores of fungi,
• viruses with envelopes.

Resting forms of bacteria

Endospores are the best known resting forms. These structures evolve within
cells and are covered by a thick multi-layer casing. Consequently, endospores
are unusually resistant to most unfavorable environmental conditions and
are able to survive, virtually endlessly, in the conditions provided by the
atmospheric air. They are only produced by some bacteria, mainly by Bacillus
and Clostridium genera. Because each cell produces only one endospore, these
spore forms cannot be used for reproduction.
Another type of resting form is produced by very common soil bacteria,
the actinomycetes. Their special vertical, filiform cells, of the so-called
air mycelium, undergo fragmentation producing numerous ball-shaped
formations. Due to the fact that their production is similar to the formation
of fungal, they are also called conidia. Contrary to endospores, the conidia
are used for reproduction. There are also other bacterial resting forms,
among others, the cysts produced by azotobacters — soil bacteria, capable of
molecular nitrogen assimilation. The production of carotenoidal dyes ensures
cells with solar radiation protection.
Carotenoids, due to the presence of numerous double bonds within a
molecule (–C=C), serve a purpose as antioxidants, because, as strong reducing
agents, they are oxidized by free radicals. Consequently, important biological
macromolecules are being protected against oxidation (DNA, proteins, etc.).
Bacteria devoid of these dyes quickly perish due to the photodynamic effect
of photooxidation. That explains why the colonies of bacteria, which settle
upon open agar plates, are often coloured (Fig. 2.2). The ability to produce
Environmental Microbiology—Water and Air 3.41

carotenoids is possessed especially by cocci and rod-shaped actinomycetes.

Rod-shaped actinomycetes, e.g. Mycobacterium tuberculosis, besides being
resistant to light, also demonstrate significant resistance to drying due to a
high content of lipids within their cell wall. High survival rates in air are also
a characteristic for the bacteria which possess a capsule, e.g. Klebsiella genus,
that cause respiratory system illnesses.
Fungal spores: Spores are special reproductive cells used for asexual
reproduction. Fungi produce spores in astronomical quantities, for example
the giant puffball (Calvatia gigantea) produces 20,000,000,000 (20 billion!)
spores, which get into the air and are dispersed over vast areas. A very common
type of spores found in air is that of conidia.
Conidia (gr. konia - dust) are a type of spore formed by asexual reproduction.
They form in the end-sections of vertical hyphae called conidiophores and are
dispersed by wind. The spores of common mould fungi such as Penicillium and
Aspergillus are examples of the above. Spore plants such as ferns, horsetails
and lycopods also produce spores. Plant pollen is also a kind of spores.
Resistant viruses: Besides cells, the air is also occupied by viruses. Among
those that demonstrate the highest resistance are those with enveloped
nucleocapsids, such as influenza viruses. Among viruses without enveloped
nucleocapsids, enteroviruses demonstrate a relatively high resistance.
Of course, besides the previously mentioned resistant forms, the air is
also occupied by more sensitive cells and viruses, but their survival is much
shorter. It is believed, that among vegetative forms, gram-positive bacteria
demonstrate greater resistance than gram-negative bacteria (especially for
desiccation), mainly due to the thickness of their cell wall. Viruses are usually
more resistant than bacteria.

Biological aerosols
Microorganisms suspended in air as a colloidal system: Microorganisms
in air occur in a form of colloidal system or the so-called bioaerosol. Every
colloid is a system where, inside its dispersion medium, particles of dispersed
phase occur whose size is halfway between molecules and particles visible
with the naked eye. In the case of biological aerosols, it’s the air (or other gases)
that has the function of the dispersion medium, whereas microorganisms are
its dispersed phase. However, it is quite rare to have microbes independently
occurring in air. Usually, they are bound with dust particles or liquid
droplets (water, saliva, etc.), thus the particles of the bioaerosol often exceed
microorganisms in size and may occur in two phases:
• dust phase (e.g. bacterial dust) or
• droplet phase (e.g. formed as the result of water-vapour condensation
or during sneezing).
The dust particles are usually larger than the droplets and they settle faster.
The difference in their ability to penetrate the respiratory tract is dependent on
3.42 Environmental Biotechnology

the size of the particles; particles of the droplet phase can reach the alveoli, but
dust particles are usually retained in the upper respiratory tract. The number
of microorganisms associated with one dust particle is greater than in the
droplet phase.

The size of bioaerosols

The average size of bioaerosols ranges from about 0.02 ìm to 100 ìm. The sizes
of certain particles may change under the influence of external factors (mainly
humidity and temperature) or as a result of larger aggregates formed. By using
size criterion, the biological aerosol can be subdivided into the following:
• fine particles (less than 1 µm), and
• coarse particles (more than 1 µm)
Fine particles are mainly viruses, endospores and cell fragments. They
possess hygroscopic properties and make-up the so-called nucleus of
condensation of water vapour. At high humidity, water collects around these
particles creating a droplet phase. Then, the diameter of the particles increases.
Coarse particles consist mainly of bacteria and fungi, usually associated with
dust particles or with water droplets.
 CHAPTER

4 Environmental Microbiology—
Methods and Applications

4.1 MICROORGANISMS-METAL TRANSFORMATIONS

Microorganisms can mobilize metals and radionuclides through autotrophic
and heterotrophic leaching, chelation by microbial metabolites and
siderophores, and methylation, which can result in volatilization. Conversely,
immobilization can result from sorption to cell components or exopolymers,
transport into cells and intracellular sequestration or precipitation as insoluble
organic and inorganic compounds, e.g. oxalates, sulphides or phosphates.
In bioremediation, solubilization provides a route for removal from solid
matrices such as soils, sediments, dumps and industrial waste. Alternatively,
immobilization processes may enable metals to be transformed in situ into
insoluble and chemically inert forms and are also particularly applicable to
removing metals from mobile aqueous phases.

Simple model of microbial roles in the environmental mobility of metals.

Metal entry into the environment [1] is shown as well as the importance
of abiotic environmental components in affecting metal speciation and
microbial populations [2] The major influence of microbes in effecting
transformations between soluble and insoluble phases is emphasised [3,4]

Metal Mobilization
Autotrophic (chemolithotrophic) leaching: Metals can be leached from solid
matrices as a result of autotrophic metabolism. Most autotrophic leaching
4.2 Environmental Biotechnology

is carried out by chemolithotrophic, acidophilic bacteria which fix carbon

dioxide and obtain energy from the oxidation of ferrous iron or reduced
sulphur compounds. These metabolic processes yield Fe(III) or H2SO4 as the
respective end products. The microorganisms involved in autotrotrophic
leaching include sulphur-oxidizing bacteria, e.g. Thiobacillus thiooxidans,
iron- and sulphur-oxidizing bacteria, e.g. Thiobacillus ferrooxidans and iron-
oxidizing bacteria, e.g. Leptospirillum ferrooxidans. As a result of sulphur and
iron-oxidation by these bacteria, metal sulphides are solubilized and the pH of
their immediate environment is decreased, which enhances the solubilization
of other metal compounds. The autotrophic leaching of metal sulphides by
Thiobacillus species and other acidophilic bacteria is well established for use in
industrial scale biomining processes. In a bioremediation context, production
of sulphuric acid by Thiobacillus species has been used to solubilize metals
from sewage sludge, thus enabling separation from the sludge which can then
be used as a fertiliser. Autotrophic leaching has been used to remediate other
metal-contaminated solid materials including soil and red mud, the main
waste product of Al extraction from bauxite.
Heterotrophic (chemoorganotrophic) leaching: Heterotrophic meta-
bolism can also lead to leaching as a result of the efflux of protons, organic
acids and siderophores. Organic acids provide both protons and a metal-
chelating anion to complex the metal cation. Citrate and oxalate anions can
form stable complexes with a large number of metals. Uranium forms very
stable 1:1 and 1:2 uranium-citrate complexes with stability constants that are
much higher than those of uranyl acetate, uranyl lactate, UEDTA and uranyl
ascorbate complexes. Many metal citrates are highly mobile and not readily
degraded and the presence of citric acid in soil may enhance contaminant
metal solubility for a significant time. Oxalic acid can also act as a leaching
agent for those metals that form soluble oxalate complexes, including Al and
Fe. Many fungi are able to leach metals from industrial waste and byproducts,
low-grade ores and metal-bearing minerals. Heterotrophic solubilization
can have consequences for other remedial treatments for contaminated
soils. Pyromorphite (Pb5(PO4) 3Cl) is a stable lead mineral and can form
in urban and industrially-contaminated soils. Such insolubility reduces
lead bioavailability and the formation of pyromorphite has been suggested
as a remediation technique for lead-contaminated land, if necessary by
means of phosphate addition. However, pyromorphite can be solubilized
by phosphate-solubilizing fungi, e.g. Aspergillus niger, and plants grown
with pyromorphite as a sole phosphorus source accumulate both P and Pb.
During fungal transformation of pyromorphite, biogenic production of lead
oxalate dihydrate was observed for the first time. This study emphasises the
importance of considering microbial processes in developing remediation
techniques for metal-contaminated soils. Another method for treatment
of metal-contaminated sandy soil relied on siderophore-mediated metal
solubilization by Alcaligenes eutrophus. Solubilized metals were adsorbed to
Environmental Microbiology—Methods and Applications 4.3

the biomass and/or precipitated, with biomass separated from a soil slurry by
a flocculation process. This resulted in a complete decrease in Cd, Zn and Pb
bioavailability. Related to heterotrophic solubilization is fungal translocation
of, e.g. Cs, Zn and Cd, which can lead to concentration in specific regions
of the mycelium and/or in fruiting bodies. Whether the concentration factors
observed in vitro can be reproduced in the field and, whether such amounts
can contribute to soil bioremediation, remains uncertain.
Reductive mobilization: Fe(III) and Mn(IV) oxides absorb metals strongly
and this may hinder metal extraction from contaminated soils. Microbial
reduction of Fe(III) and Mn(IV) may be one way for releasing such metals and
this process may be enhanced with the addition of humic materials, or related
compounds. Such compounds may also act as electron shuttles, for e.g., U(VI)
and Cr(VI), converting them to less soluble forms, especially if located in tight
pore spaces where microorganisms cannot enter. The solubility of certain
radionuclides can also be increased by reduction and this may favour their
removal from matrices such as soils. For example, iron-reducing bacterial
strains solubilized 40% of the Pu present in contaminated soils within 6-7
days through reduction of Pu(IV) to the more soluble Pu(III) and both iron-
and sulphate-reducing bacteria were able to solubilize Ra from uranium mine
tailings, although solubilization occurred largely by disruption of reducible
host minerals. The mechanism of bacterial Hg2+ resistance is enzymic reduction
of Hg2+ to non-toxic volatile Hg0 by mercuric reductase. Hg2+ may also arise
from the action of organomercurial lyase on organomercurials. Since Hg0 is
volatile, this could provide one means of mercury removal.
Methylation of metalloids: Microbial methylation of metalloids to yield
volatile derivatives, e.g. dimethylselenide or trimethylarsine, can be effected
by a variety of bacteria, algae and fungi. Microbial methylation of selenium,
resulting in volatilization, has also been used for in situ bioremediation of
selenium-containing land and water at Kesterson Reservoir, California.

Metal Immobilization
Biosorption: Biosorption is the uptake of organic and inorganic metal
species, both soluble and insoluble, by physico-chemical mechanisms such as
adsorption. In living cells, metabolic activity may also influence this process.
Almost all biological macromolecules have some affinity for metal species with
cell walls and associated materials being of the greatest significance. Biosorption
can also provide nucleation sites for the formation of stable minerals as well
as sorption to cellular surfaces cationic species can be accumulated within
cells via transport systems of varying affinity and specificity. Once inside
cells, metal species may be bound, precipitated, localised within intracellular
structures or organelles, or translocated to specific structures depending on
the element concerned and the organism. Freely-suspended and immobilized
microbial biomass has received attention with immobilized systems possessing
4.4 Environmental Biotechnology

advantages which include higher mechanical strength and easier biomass/

liquid separation. Immobilized living biomass has mainly taken the form
of bacterial biofilms on inert supports and is used in a variety of bioreactor
configurations including rotating biological contactors, fixed bed reactors,
trickle filters, fluidized beds and air-lift bioreactors. A range of specific and
non-specific metal-binding compounds are also produced by microorganisms.
Non-specific metal-binding compounds range from simple organic acids and
alcohols to macromolecules such as polysaccharides, humic and fulvic acids.
The metal-binding abilities of siderophores, metallothioneins, phytochelatins
and other biomolecules also have potential for bioremediation. However, the
earlier commercial promise and development of biosorption appears to have
largely ceased and there is no adoption of biosorption as a commercially viable
treatment method to date. The lack of commercial development is somewhat
perplexing although the lack of specificity and lower robustness of biomass-
based systems compared to ion exchange resins is often cited as a reason.
Metal precipitation by metal-reducing bacteria: Where reduction of a
metal to a lower redox state occurs, mobility and toxicity may be reduced,
offering potential bioremediation applications. Such processes may also
accompany other indirect reductive metal precipitation mechanisms, e.g. in
sulphate-reducing bacterial systems where reduction of Cr(VI) can be a result
of indirect reduction by Fe2+ and sulphide. A diverse range of metal reducing
bacteria can use oxidized species of metallic elements, e.g. Fe(III), Cr(VI) or
Mn(IV) as terminal electron acceptors. For example, a strain of Shewanella
(Alteromonas) putrefaciens which reduces Fe(III) and Mn(IV) also reduces U(VI)
to U(IV), forming a black precipitate of U(IV) carbonate. Bacterial uranium
reduction has also been combined with chemical extraction to produce a
potential process for soil bioremediation. Desulfovibrio desulphuricans can
reduce Pd(II) to cell-bound Pd(0) with hydrogen-dependent reduction being
O2-insensitive, providing a means of aerobic Pd recovery.
Reduction of metalloid oxyanions: Se(VI) reduction to elemental insoluble
Se(0) has been employed to remediate contaminated waters and soils. Some
bacteria can use such reduction to support growth, making this a natural
process for in situ applications. Though reduction of oxyanions of As and
Se can occur by different mechanisms, the most environmentally significant
process is dissimilatory reduction. Oxyanions of arsenic and selenium can
be used in microbial anaerobic respiration as terminal electron acceptors,
providing energy for growth and metabolism. Their reduction can be coupled
to organic substrates, e.g. lactate, acetate and aromatics, with the bacteria
found in a range of habitats and not confined to any specific genus. These
organisms, and perhaps even the enzymes themselves, may have applications
for bioremediation of selenium- and arsenic-contaminated environments.
Exposed reservoir sediments were flooded to create anoxic conditions,
in which the natural bacterial population reduced and immobilized large
Environmental Microbiology—Methods and Applications 4.5

quantities of the selenium that was present in the sediments. The incidental
ability of a variety of microorganisms from all major groups to reduce Se(VI)
and Te(VI) by additional, often uncharacterised, mechanisms offers additional
scope for bioreactor-based approaches.

Metal precipitation by sulphate-reducing and other bacteria

Sulphate-reducing bacteria (SRB) oxidize organic compounds or hydrogen
coupled with the reduction of sulfate, producing sulphide. The solubility
products of most heavy metal sulphides are very low, in the range of
4.65 × 10-14 (Mn) to 6.44 × 10-53 (Hg) so that even a moderate output of sulphide
can remove metals to levels permitted in the environment with metal removal
being directly related to sulphide production. Sulphate-reducing bacteria can
also create extremely reducing conditions which can chemically reduce metals
such as uranium(VI). In addition, sulphate reduction partially eliminates
acidity from the system as a result of the shift in equilibrium when sulphate
(dissociated) is converted to sulphide (largely protonated). This can result in
the further precipitation of metals such as copper or aluminium as hydroxides
as well as increasing the efficiency of sulphide precipitation. The sulphide
produced from sulphate reduction plays a major role in metal sulphide
immobilization in sediments but has also been applied to bioremediation of
metals in water and leachates. Large-scale bioreactors have been developed
using bacterial sulphate-reduction for treating metal-contaminated water. A
process integrating bacterial sulphate-reduction with bioleaching by sulphur-
oxidizing bacteria has also been developed to remove contaminating toxic
metals from soils. Sulphur- and iron-oxidizing bacteria liberated metals from
soils in the form of an acid sulphate solution that enabled almost all the metals
to be removed by bacterial sulphate reduction. SRB biofilm reactors may offer
a means of process intensification and entrap or precipitate metals, e.g. Cu
and Cd, at the biofilm surface. Regarding other organisms, the thiosulphate
reductase gene from Salmonella typhimurium has been expressed in Escherichia
coli. This resulted in sulphide production from inorganic thiosulphate which
precipitated metals as metal-sulphide complexes (1). A Cd-resistant Klebsiella
planticola also precipitated significant amounts of cadmium sulphide when
grown in thiosulphate-containing medium. As an alternative to anaerobic
sulphate reduction, a novel aerobic sulphate reduction pathway has been
engineered for sulphide production. The assimilatory SO42--reduction pathway
was redirected to cysteine production which was converted to S2- by cysteine
desulphydrase, leading to CdS precipitation on bacterial cell surfaces. Another
form of bacterial bioprecipitation is mediated by a phosphatase enzyme,
which liberates inorganic phosphate from a supplied organic phosphate donor
molecule, e.g. glycerol 2-phosphate. Metals/radionuclides are precipitated as
phosphates on the biomass.
Therefore, microorganisms play an important roles in the environmental
fate of toxic metals and metalloids with physico-chemical and biological
4.6 Environmental Biotechnology

mechanisms effecting transformations between soluble and insoluble phases.

Such mechanisms are important components of natural biogeochemical
cycles with some processes being of potential application to the treatment
of contaminated materials. Although the biotechnological potential of most
of these processes has only been explored at the laboratory scale, some
mechanisms, notably bioleaching, biosorption and precipitation, have
been employed at a commercial scale. Of these, autotrophic leaching is an
established major process in mineral extraction but has also been applied
to the treatment of contaminated land. There have been several attempts to
commercialise biosorption using microbial biomass but success has been
limited, primarily due to competition with commercially-produced ion
exchange media. As a process for immobilizing metals, precipitation of metals
as sulphides has achieved large-scale application, and this holds out promise
of further commercial development. Exploitation of other biological processes
will undoubtedly depend on a number of scientific, economic and political
factors, including the availability of a market.

4.2 MICROORGANISMS IN ENVIRONMENTAL MONITORING

In the past few decades, environmental pollution has become one of the
world’s major concerns. A great number of toxic compounds, originating
mostly from industrial and agricultural activities, are being released in to our
environment continuously. In some cases, harmful chemicals induce strong
acute toxic effects to exposed organisms when released to the environment, but
frequently, the consequences are delayed due to the effects of bioaccumulation
and biomagnification. Early detection of toxic chemical compounds in the
environment, particularly in water, and their biological effects on organisms
has therefore become increasingly important.
The traditional approach to environmental pollution assessment is based
on chemical analytical methods which only provide information about the
absolute concentrations of known chemicals in the environmental sample
without an adequate interpretation of its toxicity to biota in the context of
bioavailability, which means it only provides information about their potential,
not actual toxicity. Moreover, compounds that are toxic below the detection
limit of chemical analytical method or new compounds that are not yet
deposited in the databases, can not be detected this way. Another disadvantage
of chemical methods is the lack of information about the combined toxicity of
different compounds such as additive, synergistic or antagonistic effects. In
order to get more relevant information about environmental pollution risk,
it is therefore inevitable to supplement the chemical analytical data with the
results of methods providing information on biological impacts.
The negative biological effects of pollutants present in all kinds of
environmental samples can be assessed using different living organisms or
cells as ‘analytical devices’. The biological response following the exposure of
Environmental Microbiology—Methods and Applications 4.7

living organisms or cells to environmental sample usually gives an information

on toxicity, genotoxicity, estrogenicity, etc. of the whole mixture of chemical
compounds present in that particular sample. Besides being sensitive only
to the bioavailable fraction of pollutants, biotests also have the power to
assess the integrated effect of interacting chemical compounds and to detect
the compounds, which are toxic only due to bioactivation. According to the
technical principle, methods of biological monitoring can be classified to
• bioassays,
• biosensors,
• immunoassays,
• estrogenicity tests and
• ecological methods,
but there also exist other types of classifications, for example, division to
biomarkers, whole cell biotests and early warning biological systems.
The first biotests for environmental monitoring were based on
multicellular eucaryotic organisms, in particular, fish and mammals. As
they were relatively expensive, time-consuming, difficult to standardize
and ethically questionable, the need for alternative biological methods for
environmental monitoring based on 3R strategy (Reduction, Replacement,
Refinement) soon became evident. The development and standardisation of
toxicity tests based on procarotic (bacteria) or eucarotic (protozoa, unicellular
algae, yeasts) microorganisms, instead of higher organisms, has enabled fast
and inexpensive screening of environmental samples for toxic and genotoxic
effects. The first generation of biotests has been based on different naturally
sensitive microbes, while the second generation includes genetically modified
microorganisms to attain better sensitivity and/or specifity. The next step
forward was combinig microbial cells or parts of the cells to physicochemical
detection elements, forming new integrated devices, called biosensors.

Microbial toxicity and genotoxicity tests

Bioassay or ecotoxicity assay is an experiment in which living test-species
are exposed directly to an environmental sample (soil, sediment, surface
water, ground water, waste water, etc.) or extract of an environmental sample
to measure a potential biological effect due to the presence of potential
contaminants. Microbial bioassays can roughly be divided to (general) toxicity
assays and genotoxicity assays. The purpose of ecotoxicity bioassays is to
assess the integral effect of an environmental sample on general physiological
state of the test-species, while genotoxicity tests specifically show the effects
resulting in changes of genetic material. Regarding the exposure time of the
test-species to the investigated sample, bioassays can be subdivided into
acute and chronic. The first group is formed of bioassays, where exposure
time does not exceed 96 hours, while the chronic assay subgroup includes
4.8 Environmental Biotechnology

tests with longer exposure times. Common parameters calculated from acute
bioassay data are EC50 (estimated toxicant concentration in a sample at which
50 per cent of the test organisms show an effect following a given exposure
time) and LC50 (estimated toxicant concentration in a sample, at which
50 per cent of the test organisms die following a given exposure time), while
the chronic test reference parameter is NOEC value (represents the highest
toxicant concentration at which no significant effect can be detected when
compared to the control sample).

Toxicity bioassays
Several toxicity bioassays applying bacteria, microalgae, protozoa and yeast
have already been developed. Many of them are also standardized and
commercially available. Most common parameters measured by microbial
toxicity assays are population growth, substrate consumption, respiration, ATP
luminescence and bioluminescence inhibition. Vibrio fischeri bioluminescence
inhibition assay has been most frequently used and is claimed to be the
most sensitive across a wide range of chemicals compared to other bacterial
assays (nitrification inhibition, respirometry, enzyme inhibition and ATP
luminescence). Vibrio fischeri is a gram-negative marine bacterium possessing
natural bioluminiscence properties. Light production in a culture of test species
is directly proportional to the metabolic activity of the bacterial population,
therefore, any inhibition of enzymatic activity causes a corresponding decrease
in bioluminescence. The biochemical principle behind the bioluminescence
reaction is the oxidation of reduced flavin mononucleotide (FMNH2) in
to FMN and H2O upon reaction with molecular oxygen in the presence of
aldehyde and luciferase enzyme. The surplus energy formed in this reaction
is emitted as blue-green light of wavelength 490 nm, that can be measured by
a luminometer.
Vibrio fischeri bioluminescence inhibition assay is applicable for almost all
kinds of environmental samples such as surface and groundwater, sediments,
municipal and industrial waste effluents, etc. Other commonly used
bacterial bioassays are based on assessing the inhibitory effect of a sample
on b-galactosidase activity in E. coli. Commercially available variations of this
assay use different chromogenic b-galactosidase substrates for colorimetric
determination of enzymatic activity products. A similar principle is applied
with Bacillus sp. dehydrogenase activity assay, where redox-potential
induced changes in tetrazolium salt colour indicate inhibition of microbial
respiration. Bioassays with unicellular algae have found wide application in
environmental biomonitoring, too. A generally recognized method for testing
the effects of pollutant chemicals, especially pesticides, is based on measuring
the growth inhibition of green microalga Scenedesmus supspicatus following
72 hours exposure. Besides various modifications of this standardized test,
several new algal bioassays based on different approaches have also been
developed. An improvement on the standard microalgal growth inhibition
Environmental Microbiology—Methods and Applications 4.9

test has been made by using flow cytometry. This is a rapid and sensitive
method for quantitative measurement of light scattering and fluorescence of
flowing cells, which enables lower cell density algal cultures to be used for the
assay. Algae contain chlorophyll a, a pigment molecule which autofluoresces
when excited by blue light, therefore, additional fluorophores for cell count
experiments are not needed. By using biochemically specific fluorescent dyes,
it is possible to get additional information about the physiological status of the
cells and mechanisms of toxicant action.
Staining of algal cells with fluorescein diacetate (FDA) enables measurement
of algal esterase activity as an indicator of their physiological status. Healthy
cells take up and hydrolise FDA-producing fluorescent fluorescein, therefore,
decreased fluorescence at 530-560 nm indicates damage of algal cells, such as
impaired enzyme activity or loss of cell membrane integrity.
Short period incubation algal tests for toxicity estimation are based on ‘in
vivo’ chlorophyll prompt (PF) and delayed fluorescence (DF) measurements,
which indicate photosynthesis inhibition due to toxic chemicals. Many
herbicides and some other chemical compounds like mercury and
3,5-dichlorophenol have already been detected in environmental water
samples this way. An important difference between the 72-h algal growth
inhibition test and shorter period tests is, that the first group of tests involve
multiple cell generations, whereas the second group of tests only determine
the effects of tested chemicals on one cell generation, which might result in
lower sensitivity of short period tests to chemicals affecting multiple cell
generations.
Protozoa are eucaryotic microorganisms, known to be very sensitive to
environmental changes, therefore, being an ideal early-warning indicators
of aquatic ecosystem deterioration, as well as an important test-species
for toxicological assays. Non-pathogenic, free-living ciliates Tetrahymena
pyriformis and Tetrahymena termophila, being the first protozoa cultured
axenically, are the organisms of choice for most toxicological studies. Common
end points assessed in ecotoxicity protozoan assays are growth, viability/
mortality, grazing ability, ATP (adenosine-5’-triphosphate) content, ACP (acid
phosphatase) activity and MMT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) reduction capacity.
Growth impairment bioassays are traditionally based on microscopic
observations of morphological changes (cell shape and motility), what makes
them simple and inexpensive techniques. Nevertheless, problems such as
underestimating the true number of viable cells because of the assumption
that all non-motile or shape-altered cells are dead, forced the development of
an alternative method to standard direct counting. A novel method is based
on using two fluorescent dyes: calcein/AM – non-fluorescent substance, which
diffuses passively into cells where it is converted to green fluorescent calcein
by intracellular esterases of viable cells and EthD-1, that enters only damaged
4.10 Environmental Biotechnology

(dead) cells and represent a source of red fluorescence when bound to DNA.
Biotests based on detection of changes in metabolic state of protozoa often
include colorimetric determination of acid phosphatase or dehydrogenase
activity (MMT/tetrazolium reduction capacity) and determination of ATP
content.

Genotoxicity assays
Genotoxicity assays are used to measure the potential of environmental sample
to induce changes in genetic material of test organisms. Types of DNA damage
that may be assessed using these tests include mutagenicity, clastogenicity
and aneuploidy. Mutagenicity results in changes of one or a small number of
DNA base-pairs (point mutations), comprising substitutions, additions and
deletions of base pairs. Clastogenicity involves structural changes in larger
areas of chromosome, while changes in number of whole chromosomes lead
to aneuploidy.
Some widely used microbial genotoxicity assays are based on bacterium
Salmonella typhimurium. The most widespread is the Ames test, which has also
been established as a routine method of environmental water monitoring. It is
based on a hystidine-dependent strain of S. typhimurium (TA98). Mutagenicity
of the sample is determined by frequency of back mutations, which enable
the growth of revertants on the medium without hystidine. Genotoxicity
is detected by measuring the transcription of SOS-response genes, which
code for enzymes involved in DNA repair. Fusion of SOS-response genes
with â-galactosidase encoding reporter gene enables colorimetric detection
of genotoxic compounds. The same principle is applied in SOS-chromotest,
which applies Escherichia coli as test-species and is frequently used, too,
because of its high sensitivity to certain groups of pollutants, such as chloride
pesticides and chlorophenols.
The genotoxicity bioassay developed by scientist Zimmermann and his
group is based on recombinant Saccharomyces cerevisiae strains and enables not
only detection of mutations, but also recombinations and loss of chromosomes.
A novel commercially available yeast genotoxicity reporter assay has been
developed recently. Green Screen assay (GSA) is sensitive to broad spectrum
of mutagens and clastogenes. In this assay, the reporter system in yeast cells
employs the DNA damage inducible promoter of the RAD54 gene, fused to
green fluorescent protein. Genotoxicity indicator assay, which has recently
attracted much attention, is Comet assay (also called the Single-Cell Gel
Electrophoresis Assay), which primarily measures single and double-DNA
strand-breaks in single cells. Adapted protocols enable also the detection of
oxidized bases and abasic sites. The protocol has originally been developed
for detection of DNA damage in blood cells, but it has later been adapted for
eukaryotic microorganisms, too. T. termophila have already been used for the
purposes of environmental and drinking water genotoxicity monitoring by
Environmental Microbiology—Methods and Applications 4.11

comet assay. Yeast cells have been applied equally successfully in monitoring
of wastewater genotoxicity reduction by biological wastewater treatment
plants.

Microbial biosensors
A biosensor is defined as a self-contained, integrated device, consisting of a
biological recognition element interfaced to a physical signal transducer, that
together reversibly respond to a chemical species in a concentration-dependant
manner. A wider definition also includes some other forms of biological
sensors, including genetically engineered microorganisms, which respond
in observable ways to target analyte or group of related analytes. A wide
range of biological recognition elements have already been used in biosensors
constructed for potential environmental applications. Whole microbial cells,
cellular organelles and molecules such as enzymes, antibodies, different
kinds of receptors or DNA are the most common bio-recognition elements of
microbial origin. Regarding the type of transducer, biosensors could roughly
be classified to electrochemical, optical, thermometric and piezoelectric.
Microbial biosensors for environmental applications range in their
development stages from proof of concept to full commercial availability.
Regarding the target detection specificity, they may fall in one of two groups.
• Biosensors, which measure general biological effects/parameters or
• Biosensors for specific detection of target compounds.
The first group of biosensors is aimed to measure an integral toxicity,
genotoxicity, estrogenicity or other general parameters of the sample, which
affect living organisms. They essentially include whole microorganisms as
biorecognition elements. The most often reported cell-based biosensors include
genetically modified bacteria with artificially constructed fusions of particular
regulatory system (native promoter) with reporter genes. The presence of
an effector (non-specific stressor such as DNA damaging agents, heat shock,
oxidative stress, toxic metals, organic environmental pollutants) results in
transcription and translation of fused target genes, generating recombinant
proteins which produce some measurable response. Frequently used reporter
genes are lux (coding for luciferase) and gfp (coding for green fluorescence
protein), expression of which correlates with luminescence– or fluorescence–
based light emission. Colorimetric determination of target gene expression is
possible by fusing it to reporter genes coding for â-galalactosidase (lacZ) or
alkaline phosphatase (phoA).
Recently, E.coli biosensor, capable of detecting both genotoxic and oxidative
damage has been developed. This was achieved by introducing two plasmids:
first one, with fusion of katG (gene encoding for an important antioxidative
enzyme) promoter to the lux reporter genes, and another, with recA (gene
encoding crucial enzyme for DNA repair) promoter with the gfp reporter gene.
Besides genetically modified microorganisms (also named bioreporters), some
4.12 Environmental Biotechnology

other types of cellular biosensors have also been constructed. An example

is an algal biosensor, based on amperometric monitoring of photosynthetic
O2 evolution — the process affected by toxic compounds, which has been
developed by coupling Clark electrode to cyanobacterium Spirulina subsalsa.
Biosensors for specific determination of chemical compounds frequently
contain molecules like enzymes, receptors and metal-binding proteins as
recognition elements. A number of enzymes have been shown to be inhibited
by toxic metals, pesticides and some other important contaminants, like
endocrine-disrupting compounds. Limitations for the potential applications
of many enzyme biosensors include limited sensitivity and selectivity, as
well as interferences by environmental matrices. One recently introduced
strategy to overcome the first two of these limitations uses inhibition ratio of
two enzymes for the detection of specific compounds. Acetylcholinesterase
and urease, co-entrapped in the sol–gel matrix with the sensing probe, FITC-
dextran, have succesfully been used for Cu, Cd and Hg detection, for example.
Besides molecular biosensors, bioreporter cells may also be used for detection
of specific target compounds. Recently, for example, a biosensor for nitrate
monitoring has been constructed by transformation of E. coli with plasmid
containing nitrate reductase operon fused to gfp reporter gene.

Immunoassays
Immunochemical methods are based on specific and reversible binding
of immunoglobulin molecules (antibodies) to their target antigenes. The
most popular immunochemical technique in environmental analyses
today is immunoassay, which has been shown to detect and quantify many
compounds of environmental interest such as pesticides, industrial chemicals,
and products of xenobiotic metabolism. Basic immunoassays are performed
by detection of a specific marker molecule immobilized either to antibody
(Ab) or the antigene (Ag). Marker molecules may be in the form of fluorescent
or chemiluminescent compounds, radioisotopes or enzymes. Enzyme-based
immunoassay offer many advantages over other immunotechniques, because
of the great amount of product molecules, which results in signal amplification.
The main enzymes used are horseradish peroxidase, alkaline phosphatase and
â-galactosidase. A widely used immunoassay for environmental purposes
is enzyme-linked immunosorbent assay (ELISA), which can be carried out
according to different formats – direct competitive, indirect competitive or
sandwich-type. Competitive assays are most common and can be performed
in different ways. Analyte and the tracer (direct competitive ELISA) or analyte
and the immobilised ligand (indirect ELISA) may compete for a limited
number of binding sites. Sandwich-type ELISA is non-competitive assay, in
which the analyte is recognised by two different antibodies-immobilized Ab
and marker Ab.
Flow-injection immunoassay (FIIA) is a technique, based on the
introduction of the sample into carrier stream, which enters the reaction
Environmental Microbiology—Methods and Applications 4.13

chamber where the immunoreaction takes place. FIIA has been successfully
used for detection of different pollutants, e.g., triazines. At present, this method
is integrated into different immunosensors. The role of microorganisms,
related to immunoassays is mostly indirect, but still significant. They are
used as artificial factories (expression systems) for recombinant antibody
production. Since Escherichia coli provides the most popular expression
system, much research has been done to maximise the expression levels of
recombinant antibodies (rAbs) in this system. Main problems associated with
prkaryotic expression systems are reducing environment inside microbial
cell, that does not favour disulfide bond formation and leads to production
of insoluble recombinant proteins in the form of inclusion bodies. The current
approach to overcome this problem is to export the rAbs to the periplasm of
E.coli. However, this strategy is still limited by the amount of proteins that
can be exported. Eukryotic expression systems are also in use. They enable
higher levels of Ab expression, whereas the functionality of Ab produced is
highly dependent on individual single-chain antibody fragments. Different
microscopic fungi have been used for recombinant antibody production,
including yeast species Saccharomyces cerevisiae and Pichia pastoris.
Besides being used as antibody-production systems, microorganisms
may also represent a source of marker enzymes (alkaline phosphatase,
â-galactosidase) used in certain type of immunoassays.

Endocrine disruptor (EDC) assays

Endocrine disrupting compounds are a newly defined category of
environmental contaminants, which interfere with the endocrine system
function, which results in alternating the reproductive systems in wildlife
and humans. Compounds, acting as agonists or antagonists of hormone
(estrogene, androgene) receptors include a wide range of molecules, such
as organochlorine pesticides, pthalates, alkylphenols, phyto-and myco-
estrogenes, pharmaceutical estrogenes and many others. Estrogens are
hormones, that play crucial functions in growth, differentiation and
homeostasis of male and female reproductive organs. Besides, they also
influence non-reproductive tissues, such as bone, liver and the cardiovascular
system. When estrogenes enter the cells, they bind to specific receptors, forming
homodimeric complexes. Ligand-receptor complexes induce the transcription
of target genes by binding to specific regions on DNA, called ERES. Other
mechanisms of action, which do not include hormone receptors, also exist.
Faster responses to compounds with estrogenic activity, which take place in
the cytoplasm or on membranes and involve different effector molecules are
also of importance.
Several bioassays have been developed to assess substances with
estrogenic activity. Most of the ‘in vivo’ assays are based on a variety of end
points and are therefore, time-consuming, expensive and require sacrification
4.14 Environmental Biotechnology

of numerous animals. Consequently, ‘in vitro’ assays applying microorganisms

have been developed for the purpose of large-scale screening. Most of them
are based on simple cell models, that express hormone receptor coding genes
coupled to reporter genes, such as â-galactosidase or luciferase, when induced
by estrogene-like compounds. The estrogene-induced signaling pathways
are highly conserved in yeast and mammalian cells, which makes yeast cells
a suitable system for modeling cellular response of mammalian cells when
exposed to endocrine disrupters. Besides being less expensive and easier to
culture, one important advantage of using yeasts instead of mammalian cells
is their resistance to different contaminants, usually present in environmental
samples. Numerous tests, using genetically modified yeasts for the detection
of estrogenic and androgenic compounds, have been developed. They monitor
either the transcriptional activation of the steroid receptor itself or its ligand-
induced interaction with a transcriptional co-activator. Commercially available
assay for estrogene screening (YES/Yeast Estrogen Screen by Glaxo) is based
on genetically engineered Saccharomyces cerevisiae with human estrogene
receptor (hER) fused to lacZ reporter gene.

Ecotoxicogenomic approaches in environmental monitoring

Rapid progress in the fields of genomics is lately beginning to provide tools
that may assist our understanding of how chemicals can impact human and
ecosystem health. A new scientific discipline, which integrates genomics
(transcriptomics, proteomics and metabolomics) into ecotoxicology is named
ecotoxicogenomics. It is defined as the study of the response of the genome
to environmental toxicant exposure. Although the application of gene and
protein expression analysis to ecotoxicology is still at an early stage, this holistic
approach seems to have several potentials in different fields of ecological risk
assessment.
The most important advantage in using a gene expression profiling
approach compared to most standardized methods used to asses the potential
impact of chemicals on organisms, is the power of an insight into the precise
mode of action (MOA) of toxicants. This may potentially be useful for
prioritization of substances for extensive testing regarding their MOA and
may therefore, allow optimization of resources and limit the use of animals for
testing purposes. Moreover, the knowledge of precise toxicity pathways may
reveal novel molecular biomarkers for early detection of environmental stress.
Comparison of gene expression profiles of different test microorganisms
and higher organisms may provide useful information about the possibility
of extrapolation of the effects of toxic chemicals across species. Determining
similarities and dissimilarities in toxicity mechanisms across species would
give the answer where the extrapolation of chemical hazards from one species
to another is technically valid. The knowledge about conservation of toxicity
mechanisms in organisms will therefore enable to choose appropriate model
Environmental Microbiology—Methods and Applications 4.15

organisms at lower levels of biological organization (e.g. microorganisms) for

relevant monitoring of specific environmental toxicants. The use of microarray,
proteomic and metabolomic techniques may also provide the possibility to
predict toxic potential of unknown chemicals by comparing specific patterns
of gene expression (fingerprints), reflecting mode of action of unknown
chemical, with expression profiles of known Toxicants.
To conclude, by providing both, mechanism of action and predictive tools,
ecotoxicogenomic approach seems especially promising for studying the effect
of pollutants at low, environmentally relevant concentrations, improvement of
toxic mixture analysis and long-term exposure assessment of organisms. The
use of biological methods in environmental monitoring is essential in order
to complement chemical analysis with information about actual toxicity or
genotoxicity of environmental samples. Microorganisms are widely applied
test-species in different bioassays because of the ease and low costs of their
culturing as well as the lack of ethical issues often accompanying the use of
higher organisms. Combining biology to engineering skills has enabled the
development of biosensors—new generation of analytical devices coupling
biological recognition elements to physical signal transducers. Besides
the direct application of whole microorganisms or their isolated parts for
general toxicity assessment or detection of specific compounds, genetically
modified microbes also represent an important source of recombinant
antibody production, which makes them important also when talking about
immunoassays. With the development of toxicogenomic approaches, the
use of microorganisms for environmental monitoring purposes is expected
to become even more extensive because of better knowledge about potential
analogies in toxicity mechanisms between higher organisms and microbes.

4.3 APPLICATIONS OF THE POLYMERASE CHAIN REACTION IN

ENVIRONMENTAL MICROBIOLOGY
The application of the polymerase chain reaction (PCR) to explore various
areas of environmental microbiology has the potential to solve many difficult
and unanswered questions about microbial activities in the environment at
the physiological and molecular levels. This review describes the use of PCR
for the detection of specific microbes in environmental samples and discusses
how PCR may be used to answer future questions in molecular microbial
ecology. The first two sections of the review will discuss preparation of nucleic
acids from environmental microorganisms and PCR methodology specific to
environmental microbiology. Subsequent sections present information on
applying these methods to environmental problems such as: detection of
genetically engineered microbes, detection of indigenous microorganisms
in the environment and indicator microorganisms in water, detection of
waterborne microbial pathogens and viable but nonculturable microorganisms,
and environmental monitoring with multiplex PCR.
4.16 Environmental Biotechnology

Purification of nucleic acids from environmental microorganisms

for PCR amplification
Extraction and extensive purification of nucleic acids from environmental
microorganisms is necessary for successful PCR amplification. Some of the
general environmental contaminants that can inhibit the PCR reaction are the
presence of humic materials, clay, and organics. For example, the addition
of as little as 0.001 mg of montomorillite humic material in the PCR reaction
inhibits the amplification process.

Purification of Nucleic Acids from Soil and Sediment

Much effort has been devoted to developing convenient methods for
removing humic materials from environmental samples to achieve successful
PCR amplification of the target nucleic acids. Either direct lysis or isolation
of bacterial cells followed by lysis can be used for extraction of DNA
from microorganisms present in the environmental soil or sediment. ~61
Purification of the released DNA can be performed by applying a combination
of the various standard purification methods such as phenol-chloroform
extraction followed by ammonium acetate-ethanol precipitation, repeated
polyvinylpolypyrrolidone (PVPP) treatment or dialysis, hydroxylapatite
or affinity chromatography, and multiple CsC1- EtBr density gradient
centrifugation. All of these methods produce positive PCR amplification from
the environmental samples. Recently, Tsai and O1- son (7~ have described a
direct extraction method using lysozyme followed by freeze-thaw disruption
of the cells. The released DNAs were then purified by standard phenol-
chloroform extraction and chromatography. In another study, bacterial cells
were differentially separated from soil colloids on the basis of their buoyant
densities. In this method, a modified sucrose gradient centrifugation protocol
is used to separate most of the soil colloids from the bacterial cells in the sample
for PCR amplification of the target DNA. To isolate and purify RNA from
environmental microorganisms, the samples can be treated with guanidinium
hydrochloride, and phenolchloroform-isoamyl alcohol extraction followed
by ethanol precipitation. Although significantly higher amounts of nucleic
acids can be recovered by following the direct lysis method, the presence of
eukaryotic DNA in the sample is a possibility.

Purification of Nucleic Acids from Microorganisms in Water

Nucleic acids for PCR amplification from microorganisms present in the
aquatic environment can be isolated and purified more easily than those from
soil and sediment. A simple method for isolating nucleic acids from aquatic
samples has been demonstrated by Sommersville and consists of collecting
cells, followed by lysis and separation of plasmid DNA, chromosomal DNA,
and RNA in a single filter cartridge. The dissolved and particulate DNA then
can be conveniently purified for PCR analysis. In various other studies, the
Environmental Microbiology—Methods and Applications 4.17

following procedures were found to be essential to yield sufficiently pure

DNA for various molecular biological analyses and perhaps PCR analyses:
CsCI-EtBr density gradient centrifugation/for all environmental DNAs,
multiple PVPP treatment for rRNA studies, (and repeated phenol-chloroform
extractions for total planktonic DNA t14,1s~ or cyanobacterial DNA. Microbial
cells can be collected on a filter, lysed by repeated freeze-thaw methods, and
used for PCR amplification without removing the filter from the reaction tube.
Alternatively, filtered cells can be lysed directly on the filter by lysozyme, and
the released DNA can be purified by phenol-choroform extraction followed by
ethanol precipitation to yield adequately purified DNA for PCR amplification
and analysis. Although the methods described above can remove the majority
of the environmental contaminants and are useful for various molecular
biological studies, there is no standard protocol for removing all possible
inhibitors that can be applied for all types of environmental samples.

PCR Methodology for Environmental Applications

Basically, PCR is the in vitro enzymatic amplification of a DNA fragment
that is performed by using two flanking oligonucleotide primers at the two
ends of the target DNA. This methodology has several advantages, as well
as disadvantages, over conventional methodologies used in environmental
microbiology. Some of the recent advancements in PCR technology that may
be very useful in environmental applications and that we would like to discuss
in some detail are: (1) “hot start,” (2) removal of PCR carryover contaminants,
and (3) thermostable DNA polymerase from various thermotolerent
microorganisms, some of them with additional activities such as reverse
transcriptase.

Hot Start
The specificity of oligonucleotide primers for the amplification of a specific
target for detecting a specific microbial pathogen or a released genetically
engineered microorganism (GEM) in the environment can be determined by
testing varieties of microorganisms. However, because of the vast diversity
of microorganisms in any environmental sample, it is possible that one may
get non-specific PCR amplification. Thus, increased specificity is an important
issue for environmental PCR. Methods such as increasing or decreasing MgC12
concentrations in the PCR reaction, high Tm value of the primers with an
approximately 50% GC content, and a random base distribution minimum of
5-6 bases at the 3’ end of each of the primers can be used to remove non-specific
amplified DNA. Also effective is the “hot start” method. This method is based
on the fact that non-specific priming and subsequent production of unwanted
amplified DNA bands generally result due to the retention of considerable
enzymatic activity at temperatures below the optimum for DNA synthesis.
Therefore, during the initial heating step of the PCR reaction, primers that
anneal nonspecifically to a partially single-stranded template region can be
4.18 Environmental Biotechnology

extended and stabilized before the reaction reaches the 72~ temperature for
extension of specifically annealed primers. If the DNA polymerase is activated
only after the reaction has reached high temperatures, nontarget amplification
can be minimized by manual addition of an essential reagent, e.g., DNA
polymerase, to the reaction tube at elevated temperatures. This approach
improves specificity and minimizes the formation of “primer dimers.”

Removal of PCR Carryover Contamination

Another potential problem is contamination of the PCR amplification reaction
with products of a previous PCR reaction, i.e., product carryover, cross-
contamination between samples, or contamination with exogenous nucleic
acids from the laboratory environment, all of which can create false-positive
results. Although some general precautions and good laboratory practice
will reduce the possibility of such contamination problems, other simple and
more effective preventive measures can be taken by exposing the reaction
mixture to ultraviolet light, treating the reaction mixture with multiple
restriction enzymes or DNase I, followed by inactivation of these enzymes,
or photochemical modification of the contaminating DNA with psoralen
or isopsoralen. Another effective approach is to make contaminating PCR
products susceptible to degradation by substituting dUTP for dTTP in every
PCR reaction and treating the subsequent PCR reaction mix with uracil DNA
glycosylase, which will selectively eliminate dU-containing DNA by cleaving
the uracil. This enzyme is active for both double- and single-stranded DNAs
containing a basic polynucleotide (dUTP), but does not react on RNA template.
Thus, when a PCR reaction is contaminated with RNA, this approach will
not be useful and false amplification may result. Another problem with this
approach is that the amplified DNA must be stored at high temperature until
it is transferred to a freezer to prevent degradation of the newly amplified
DNA product from any residual activity of the enzyme.

Thermostable DNA Polymerase

Native Taq DNA polymerase and recombinant AmpliTaq DNA polymerase
of Thermus aquaticus are commonly used for DNA amplification by PCR.
Recently, several other thermostable DNA polymerases have been introduced
and these may have potential advantages for environmental PCR technology.
The Stoffel fragment, a modified version of the Taq DNA polymerase in which
289 amino acids are deleted from the amino-terminal end of the enzyme, is
approximately two-fold more thermostable, exhibits optimal activity over a
broader range of magnesium ion concentrations (2-10 mM), and lacks any
intrinsic 5‘→3‘ exonuclease activity compared to the complete Taq DNA
polymerase. As a result, when using the Stoffel fragment, the denaturation
temperature can be raised several degreesf this is useful for templates that are
Environmental Microbiology—Methods and Applications 4.19

G+C-rich or that contain complex secondary structures. Manv environmental

microbes are identified by amplifying ribosomal RNA, which may have such
complex secondary structure, as targets. In addition, when using the Stoffel
fragment, the primer can be selected with higher annealing temperature
for better specificity and the entire PCR reaction can be performed at an
elevated temperature without losing much of the enzyme activity. Another
thermostable DNA polymerase isolated from Thermus thermophilus (Tth) has
significant reverse transcriptase activity, especially in the presence of MnC12
Tth polymerase can be useful for detecting gene expression and viable but
nonculturable microorganisms in the environmental samples by using
mRNA-cDNA-PCR amplification in a single reaction. Recently, several other
thermostable DNA polymerases have been isolated and tested for their fidelity,
stability at high temperatures, and exonuclease activities. The fidelity of the
DNA polymerase isolated from Thennococcus litoralis (Vent DNA polymerase)
has been studied and compared with other DNA polymerases such as Taq,
Klenow, T4, and T7 DNA polyrnerases by Eckert and Kunkel. The advantage
of using Vent DNA polymerase is that it has 3’~5’ exonuclease activity, which
increases the fidelity of the reaction about six-fold as compared to the Taq
DNA polymerase.
It is noteworthy to mention that Vent DNA polymerase has a base
substitution error rate of 1/31,000 in a reaction containing 1 mM dNTPs, which
is similar to that observed for the Klenow DNA polymerase. Interestingly,
T. litoralis grows at a temperature of 98~ in thermal vents on the ocean
floor, and Vent DNA polyrnerase, isolated from this organism, retains its
activity for over 2 hr at 100~ with a primer extension capacity of up to 13
kb. Recently, another therrnostable DNA polymerase has been isolated from
a thermophilic, anaerobic, marine archaebacterium, Pyrococcus fitriosus. The
pill DNA polymerase is a monomeric, 92-kD protein with both 5 ‘~ 3‘ and 3
‘~ 5‘ exonuclease, activities has better thermostability at 95~ than some other
thermostable DNA polymerases and has a 12-fold lower mutation frequency
than Taq DNA polyrnerase. Eckert and Kunkel/have shown in a comparison
of the fidelities of the various thermostable DNA polymerases that Tth and the
Thermus flavis Replinase are lO-fold less accurate than the T4 or native T7 DNA
polymerases. Although, the use of various newly introduced thermostable
DNA polymerases with proofreading activities and lower misincorporation
rates during synthesis may be an advantage to PCR users for direct cloning
and sequencing of PCR products, PCR-based methods for mutagenesis, or
detection of point mutations, the remaining concern is the degradation of
primers in the PCR reaction mixtures by these enzymes.
From a comparative study of the exonuclease activities and temperature
profiles of Vent and pfu DNA polymerases, it has been claimed that pfu has
a much lower primer degradation activity than the Vent DNA polymerase
(E. Mathur, pers. comm.).
4.20 Environmental Biotechnology

Detection of Released GEMS in The Environment By PCR

There are many promising applications of GEMs in industry, agriculture, and
medicine, but their use has been limited thus far because of a lack of sensitive
methods for the monitoring and detection of GEMs after their release in the
environment. Cultural methods and methods such as colony hybridization,
which depend on the ability to recover and culture the organism from
an environmental sample, lack sensitivity due to the limited efficiency of
recovering bacteria from natural environments. Extraction of microbial DNA
from the environmental sample, either directly or after recovery of microbial
cells, followed by gene probe hybridization, though more sensitive than
cultural methods, still lacks the level of sensitivity required to determine
the ultimate fate of GEMs because of the limited relative numbers of target
gene sequences, that may be present in the sample PCR amplification of the
engineered genes from the released microorganisms to several million-fold,
can potentially increase the sensitivity of detection of released GEMs in the
environment. The PCR method was first applied to monitor GEMs by Steffan
and Atlas. They detected by PCR amplification a portion of the 1.3-kb repeat
sequence from Pseudomonas cepacia ACllO0, a herbicide (2,4,5-T)—degrading
bacterium, after the organism was released in the soil. Their sensitivity of
detection was 100 GEMs in 100 grams of sediment against a background of
1011 diverse non-target microorganisms, at least 103-fold higher than the
sensitivity of nonamplified conventional dot-blot hybridization detection. In
another example, a 0.3-kb unique DNA sequence from Pennisetum purpureum
(napier grass) was cloned into pRCIO, a derivative of 2,4-dichlorophenoxyacetic
acid-degrading plasmid, and transferred into Escherichia coll. This genetically
altered microbe was then released into filter-sterilized lake and sewage-water
samples at a concentration of 104 cells per milliliter. The microbe was detected
by PCR at a sensitivity several-fold higher than the conventional plating
technique, even after 10-14 days of incubation, using the unique cloned DNA
sequence as a target.
These studies show that the PCR method can be used for monitoring
released GEMs in an environment consisting of a complex habitat of
diverse microorganisms, in which it may be tedious and time-consuming to
discriminate the GEMs from the indigenous microorganisms. In another study,
a single copy of the transposon Tn5 was transferred into the genomic DNA of
Rhizobium leguminosarum, which was released into the soil. These GEMs were
detected by “double” PCR amplification using the transposon Tn5 as target to
a sensitivity of 1-10 cfu/gram of soil. Although it is adequate to use Tn5, which
contains an antibiotic resistance gene as a model target for PCR detection, this
may not be an appropriate marker for releasing GEMs into the environment
because of Tn5’s ability to be transferred into indigenous microorganisms,
making them antibiotic resistant also. Detection of the indigenous Tn5
sequence by PCR may give false-positive results in such cases.
Environmental Microbiology—Methods and Applications 4.21

Detection of Indigenous Microorganisms in the Environment by

PCR
Detection of Degrading Microorganisms: One of the potential ways to
eliminate various pollutants and toxic wastes in the environment is efficient
biodegradation and bioremediation by various indigenous microorganisms at
the polluted sites. Sensitive detection of such degrading microorganisms in
the polluted and toxic waste sites may be possible by using PCR. Although
there might be variations in the genes of the same or different groups of
microorganisms for the degradation of one or several types of pollutants, it
may be possible to identify these microorganisms by detecting the conserved
regions of these genes by PCR amplification. One such study used the
nucleotide sequence information of a chlorocatechol dioxygenase-degrading
gene from Alcaligenes eutrophus JMP134 (pJP4); oligonucleotide primers were
designed for the detection of various chloro-aromatic-degrading bacteria
by PCR amplification. PCR amplication using such oligonucleotide primers
provides information quickly on the variations, similarities, and functional
aspects of various pollutantde-grading genes present in closely or distantly
related microorganisms in the environment. When such a polluted site
is identified, it is important to investigate the possibility of the presence of
various degrading microbes at that site. In many instances, conventional
microbiological techniques do not detect all the pesticide-degrading
indigenous microorganisms, as some of them may be in a viable but non-
culturable condition. In another study, specific detection of a herbicide
(2,4-dichlorophenoxyacetic acid)-degrading bacteria was achieved by PCR
amplification of a region of/gene from pJP4 and its derivative plasmid pRO103
(I. Pepper, pers. comm.). In this study, by using direct PCR amplified DNA
analysis, it was possible to detect approximately 3000 cfu or 15.6 pg of plasmid
DNA.The sensitivity of such detection was onefold higher when DNA-DNA
hybridization was performed with an oligonucleotide probe internal to the
amplified DNA. Using such oligonucleotide primers and PCR amplification, it
is possible to detect the specific microorganisms carrying the degrading gene
from a complex-mixed microbial population in the environment.

Identification of Microorganisms in Biofilms

Formation of biofilms on various surfaces by microorganisms in the
environment can be beneficial or detrimental. For example, microbial
aggregation or attachment is required for various water treatments; on the
other hand, extensive corrosion and biodeterioration can be caused due to
the formation of such microbial biofilms. Characterization and ecology of
microbial populations in biofilms has been hindered because the available
determinative techniques require culture of microorganisms in selective media.
These methods eliminate many of the important microbes from the biofilms
since they survive only in a mixed culture and live on the cometabolism
4.22 Environmental Biotechnology

(the gratuitous metabolic transformation of a substance by a microorganism

growing on another substance).
PCR amplification of specific targets makes it possible to identify a group
of microbes in such a biofilm that may have been missed by the conventional
techniques. To determine the feasibility of PCR, a sulfidogenic biofilm has
been established in an anaerobic fixed-bed bioreactor. PCR amplification was
performed for the detection of the population architecture of all the gram-
negative sulfate-reducing bacteria using a region of the 16S ribosomal RNA
conserved in the resident sulfate-reducing bacteria.

Detection of Indicator Microorganisms in Water

The bacteriological safety of water supplies is tested by monitoring coliform
bacteria whose presence in the water indicates potential human fecal
contamination and the possibility of the presence of enteric pathogens.
Coliform bacteria are traditionally detected by culturing on media such as
Mac-Conkey, m-Endo, eosin methylene blue, or brilliant-green-lactose-bile
media. The culture method for monitoring E. coli in environmental and potable
waters has several problems associated with it. The conventional confirmative
tests for the detection of E. coli, all of which require culturing of the organism,
are time-consuming. Moreover, they do not detect viable but non-culturable
bacteria, which may occur due to chlorine injury during the process of water
purification and treatment. Also, the cells may die between the time of
collection and the test. A colorimetric test, the Colilert test, for the detection
of E. coli is based on the detection of β-D-glucuronidase enzyme produced
by uidA gene. This method requires culturing of bacteria. In addition, this
method fails to detect β-D-glucuronidase-negative E. coli.
Bej and group have developed a PCR gene probe-based method for the
detection of coliform bacteria. Amplification of a portion of the lacZ gene detects
E. coli and other coliform bacteria, including Shigella spp. Amplification of part
of the lamB gene detects E. coli, Salmonela, and Shigella spp. In another study,
Bej and group developed a method for the detection of E. coli and Shigella spp.
using four different regions of the uidA gene, which codes for the [β-glueruron
dass glucuronidase enzyme, and part of the uidR gene, which is the regulatory
region of the uidA gene, as targets. Besides being less time consuming and
having higher specificity and sensitivity, the most important advantage of this
method over conventional and other commercially available methods is that
it can detect the uidA-negative E. coli that do not show a positive signal with
the conventional tests because they lack the 13-glucuronidase enzyme. The
sensitivity of the method is 1-10 fg of genomic DNA and 1-5 viable E. coli cells.
Similarly, Cleuziat and Baudouy-Robert have used a large region of the
uid gene of E. coli as a target for PCR amplification and gene probe detection of
E. coli and Shigella spp. This PCR gene probe-based method has the specificity
and sensitivity required for monitoring coliforms as indicator organisms in
Environmental Microbiology—Methods and Applications 4.23

environmental and potable waters. A field evaluation of PCR detection of

enteric pathogens and indicator microorganisms has been reported using uidA
and lacZ as targets./Although these targets for PCR amplification detection
show great promise, the targets for specific detection of E. coli, Shigella spp.,
and Salmonella spp. are yet to be described.

Detection of Water-borne Microbial Pathogens by PCR

Apart from the detection and monitoring of indicator microorganisms for
water quality assessment, it is also important to detect with high sensitivity
and specificity various water-borne microbial pathogens.

PCR Detection of Legionella

Legionella spp. is a water-borne microbial pathogen and can cause Legionnaires’
disease in humans via aerosol. Starnbach reported the detection of Legionella
pneumophila by amplification of a fragment of DNA of unknown function
from Legionella using PCR. Their sensitivity of detection was equivalent to
35 colony forming units detected by viable plating. Mahbubani et al. have
developed a method based upon PCR and gene probes for detecting Legionella
in environmental water sources. All species of Legionella, including all
15 serogroups of L. pneumophila, were detected by PCR amplification of a
104-bp DNA sequence that codes for a region of 5S rRNA followed by
radiolabeled oligoprobe hybridization to an internal region of the amplified
DNA. Strains of L. pneumophila (all serogroups) were specifically detected
based upon amplification of a portion of the coding region of the macrophage
infectivity potentiator (mip) gene. Pseudomonas spp. that exhibit antigenic
cross-reactivity in serological detection methods did not produce positive
signals in the PCR gene probe method using Southern blot analyses. Single-
cell, single-gene Legionella detection was achieved with the PCR gene probe
methods.

PCR Detection of Giardia

Another microbial pathogen, Giardia lamblia, causes defined waterborne
diarrhea in the United States and in many other parts of the world. Diagnosis
of G. lamblia from environmental samples is performed by concentrating
100 gallons of water followed by microscopic examination using fluorescent
dye. Using PCR amplification of different segments of the giardin gene
of G. lamblia, it was possible to differentiate G. lamblia from G. muris. Also,
a single Giardia cyst was detected by PCR amplification after separating the
cyst by a micromanipulator. Although the specificity and sensitivity of the
detection of Giardia shows great promise for rapid and reliable monitoring
of this pathogen in water, application of this method for the detection of this
pathogen in concentrates of 100 gallons of environmental water sample needs
to be demonstrated.
4.24 Environmental Biotechnology

Multiplex PCR Amplification for Environmental Monitoring of

Microorganisms
It is possible that environmental samples and drinking water may contain
more than one type of microbial pathogen in addition to the indicator
microorganism. Use of multiplex PCR for amplification and detection of
more than one target in a single PCR reaction can be useful for monitoring
multiple microbial pathogens in a single environmental or water sample. This
method was first described by Chamberlain for detecting human genes. A
modification of this approach of simultaneous PCR amplification of multiple
targets associated in different bacteria in the environmental samples has been
demonstrated. Multiplex amplification of two different Legionella genes, one
specific for Legionella pneumophila (mip) and the other for the genus Legionella
(5S rRNA), was achieved by staggered addition of two different sets of primers
at two different concentrations. This method can detect genus Legionella and
L. pneumophila should they be present in one sample. Using the same target
genes, mip and 5S rRNA, a multiplex PCR assay for genus Legionella and
L. pnemnophila was described. In this study, equal amplified products were
achieved by adding equimolar quantities of each of the primers. This may be
due to the fact that the two target sequences were closer in length than in the
system, developed and described previously.
In a field study of water quality monitoring, simultaneous PCR
amplification was performed using lacZ and uidA as targets. In this study, it
was posssible to detect in one sample total coliform bacteria by amplification
of the lacZ gene, the indicator microorganism E. coli, and a pathogen Shigella
spp. bv the amplification of the uidA gene. Also, in this study the lacZ
PCR detection method gave results statistically equivalent to those of the
conventional plate count and defined substrate methods accepted by the U.S.
Environmental Protection Agency for water quality monitoring. The uidA PCR
method was more sensitive than the 4-methylumbelliferyl-13-D-glucuronide-
based defined substrate test for the specific detection of E. coli. In another
study multiplex amplification of five different targets in a single PCR reaction
has been achieved for the detection of non-pneumophila Legionella spp., L.
pneumophila, total coliforms, E. coli and Shigella spp., and total eubacterial
species. It may be desirable in future studies to group certain microbial
pathogens and indicators in the environmental samples and design the
primers for specific targets. For example, one can group all the environmental
and water-borne respiratory pathogens and amplify all the specific target
genes by PCR in a single reaction for their detection. When several GEMs
are released together for the degradation of complex hazardous wastes and
pollutants, they can be monitored together, possibly both qualitatively and
quantitatively, in a single PCR reaction by amplifying a unique segment of the
DNA of each of the GEMs, as well as by amplifying a common segment of all
the GEMs that is not present in other eubacterial species.
Environmental Microbiology—Methods and Applications 4.25

Detection of Viable But Non-culturable Microorganisms in the

Environment
There are several reports on the existence of many microorganisms, including
human pathogens, in the environment in a viable but non-culturable, i.e.,
dormant, stage. These microbial pathogens are shown to be potentially
infectious when suitable conditions prevail. One obvious difficulty in
elucidating this potential hazard is the inability to detect these viable but non-
culturable cells in the environment because routine microbiological methods
will not allow them to grow (on agar media) or will not distinguish them from
the dead cells (by microscopic technique). Recognizing that the terms “alive”
and “viable” are subject to different definitions, the reasonably acceptable
definition would be that the live cells are considered those capable of cell
division, metabolism (respiration), or gene transcription (mRNA production).
To detect those microbial cells that are in a viable but non-culturable state in
the environment, it is desirable to target the mRNA rather than the DNA first
for cDNA synthesis followed by PCR amplification. The potential problem of
this approach is that most of the prokaryotic mRNAs have half-lives of only
few minutes.

PCR Detection of L. pneumophila

Mahbubani have shown that the mRNA of the mip gene of L. pneumophila
can be stabilized simply by growing the cells for 10-15 min in the presence of
chloramphenicol before harvesting. They have shown that the PCR amplification
of the mip mRNA could be a potential means for the detection of metabolically
active L. pneumophila cells. The use of chloram-phenicol for increasing the
stability of bacterial mRNA is yet to be tested in other microorganisms. Another
perplexing issue that may create additional problems in such an approach is
the efficiency of gene expression of these dormant microbial pathogens. It is
possible that the transcriptional or regulatory systems of the target genes in
these microbial pathogens are inhibited by various environmental factors and
inhibitors when they are present in the natural environment. Therefore, in this
situation, the quantity of the target mRNA level may be so low that it may
remain undetected even by a method as sophisticated as PCR. However, it
has been shown that, targeting DNA for PCR amplification may be sufficient
for the detection of culturable and nonculturable microbial pathogens. Both
viable culturable and viable non-culturable cells of L. pneumophila, formed
during exposure to hypochlorite, showed positive PCR amplification, whereas
nonviable cells did not. Field verification of this approach for the detection
of metabolically active (viable vs. dead) L. pneumophila from contaminated
environmental samples is yet to be done.
4.26 Environmental Biotechnology

PCR Detection of Vibrio vulnificus

Besides L. pneumophila, another important marine water-borne microbial
pathogen, Vibrio vulnificus, which can cause fatal infections in humans who
ingest contaminated raw oysters, has been found to enter in a viable but
non-culturable state during the colder months and resuscitate from the
non-culturable state when a suitable environment prevails. Using PCR
amplification of the hemolysin gene, Scientists detected DNA from culturable
and from non-culturable cells.

Difficulties in Detection of Non-culturable Cells by PCR

Although the decreased sensitivity of detection of non-culturable cells by PCR
is not well understood at this time, several possible explanations have been
described. Among these possibilities, the important criteria that may be of
concern in applying PCR methodology for the detection of viable but non-
culturable microorganisms are: (1) less DNA content per cell, (2) difficulty in
breaking open the cell because of the change in the cell wall that may occur due
to carbon or nitrogen starvation or changes in the environmental conditions,
and (3) modification of the target gene due to genetic rearrangement.
However, Brauns did not attempt to use hemolysin mRNA as a target for
PCR amplification from the non-culturable cells, a method that could have
determined the exact nature of the ceils (i.e., alive or dead), and the gene
expression of the target.
A study by Mahbubani has shown that mRNA-PCR alone is not
sufficient to distinguish live Giardia cysts from dead ones, since cysts killed
by heat treatment or monochloramination also give positive mRNA-PCR
amplification. Therefore, in this organism, if the giardin mRNA is used as a
target for PCR amplification, it is necessary to include an mRNA induction step
in the procedure to determine the viability of the cysts. Since in the viable but
non-culturable stage there may be changes in the structure as well as in gene
expression in many microorganisms, a modified version of the PCR method
may need to be developed for the detection of such microbial pathogens in the
environment.

Detection of Gene Expression in the Environment by PCR

An important issue in environmental microbial molecular genetics is how
various genes are regulated and expressed under various environmental
conditions. One known fact is that some of the environmental microbial
pathogens such as L. pneumophila and V. vulnificus alter their gene expression
and remain in a dormant stage as non-culturable organisms in the environment.
It has also been predicted that several biodegradative microorganisms may
not express their degrading genes in the environment. As a result, one may
not be sure whether the released GEMs or indigenous microorganisms are
degrading the pollutants at a contaminated site. Using specific mRNA as a
Environmental Microbiology—Methods and Applications 4.27

target for PCR amplification and developing a quantitative assay for such
a method, it is possible to detect the level of mRNA production with high
sensitivity in the environmental samples.
A promising method for extraction of specific mRNA from soil seeded
with naphthalene-degrading and mercury-resistant bacterial cells has been
described. This method can be completed within a few hours; approximately
17 mg of total RNA per gram (wet weight) of soil containing 8.0 × 108 bacterial
cells can be purified with a DNA-RNA hybridization detection sensitivity
of 160 ng of specific target mRNA. Although, this method has potential for
studying in situ gene expression, the humic acid compounds may precipitate
with samples containing high-cation-exchange capacity, e.g., some sediments,
which will greatly reduce the total RNA recovery efficiency and sensitivity
of detection. Application of PCR for detecting specific mRNA extracted from
various environmental samples by this method has yet to be evaluated.
Although the application of PCR in the area of environmental microbiology
has not progressed as much as applications in diagnostics, medicine, and
molecular biology several studies have shown great promise in solving various
difficult problems. One of the most important problems in environmental
microbiology, is the detection and monitoring of released GEMs in the
environment with high sensitivity. It has been shown that the application of
the PCR method can detect 1-100 GEMs per gram of soil or sediment, which is
a level of sensitivity several orders of magnitude higher than the conventional
DNA-DNA hybridization method. One of the drawbacks of this approach
is that PCR requires purified nucleic acid, which must be achieved from the
environmental samples through several rigorous methodological steps.
The application of PCR technology to monitoring pathogens and indicator
microorganisms has reached a stage where it is safe to say that this method
can be used, with greater specificity and sensitivity, as an alternative to
the conventional methods. The most important criterion in applying PCR
technology in this area of environmental microbiology is the removal of
inhibitors and contaminants from the samples. Although several inhibitors
from various environmental samples have been identified with possible
removal procedures, unlimited numbers of such inhibitors may exist that
have not yet been identified. For detection of microorganisms from soil and
sediments, it has been found that the humic and fulvic acid compounds
inhibit the polymerase activities and reduce the sensitivity of detection.
Although, several procedures such as diluting the samples, ion-exchange
chromatography, gel filtration chromatography, PVPP treatment, sucrose
gradient purification, and so forth, have been used to remove humic and
fulvic acids and other inhibitors from the samples, none of these methods
seems to remove them totally. The application of PCR technology to various
environmental samples for detection of pathogens and other microorganisms
may be affected severely if a relatively universal method for removal of
inhibitors from the environmental samples is not developed.
4.28 Environmental Biotechnology

Quantitation of microbial populations by conventional methods has

several drawbacks. Although there are several reports on the quantitation
of PCR-amplified products that can give information about the starting
number of cells, this approach needs to be developed for the quantitation of
a microbial population in a given environment. This will permit microbial
succession, competition, and community structure in an ecosystem to be
studied in the environment, including microbes living in many extreme
environments. Another potential application of PCR methodology in the
area of environmental microbiology is distinguishing the live cells from
the dead ones in a given environmental sample. However, more research
must be done before this approach can be applied to actual environmental
samples. Alteration of gene expression in many microbial pathogens and
other pollutant-degrading microbes due to various environmental conditions
is a growing concern to human health, and detection of specific mRNA in the
environment by PCR will provide information on the in situ activity of these
microorganisms. PCR shows promise for cloning genes from environmentally
important microorganisms, including those organisms that have not been
cultured yet. In the near future, technological improvements and subsequent
new development of the PCR method will solve many unanswered questions in
the area of microbial ecology, microbial community structure, environmental
health, and environmental analyses of molecular microbiology.

4.4 PESTICIDES AND OTHER POLLUTANTS DEGRADATION

BY MICROORGANISMS AND GENETICALLY ENGINEERED
MICROBES
In the last few decades, highly toxic organic compounds have been synthesized
and released into the environment for direct or indirect application over a long
period of time. Pesticides, fuels, polycyclic aromatic hydrocarbons (PAHs),
polychlorinated biphenyls (PCBs), chlorophenols, and dyes are some of these
types of compounds. The paramount pollution in our environment is a dire
consequence of continually expanding population along with an exponential
development in the industrial field.
Biodegradation: According to the definition by the International Union
of Pure and Applied Chemistry, the term biodegradation is “Breakdown of a
substance catalyzed by enzymes in vitro or in vivo.
This may be characterized for the purpose of hazard assessment such as:
• Primary. Alteration of the chemical structure of a substance resulting in
loss of a specific property of that substance.
• Environmentally acceptable. Biodegradation to such an extent as to
remove undesirable properties of the compound. This often corresponds
to primary biodegradation but it depends on the circumstances under
which the products are discharged into the environment.
Environmental Microbiology—Methods and Applications 4.29

• Ultimate. Complete breakdown of a compound to either fully oxidized

or reduced simple molecules (such as carbon dioxide/methane, nitrate/
ammonium and water). It should be noted that the biodegradation
products can be more harmful than the substance degraded.”
Microbial degradation of chemical compounds such as pesticides and
other pollutants in the environment is an important route for the removal of
these compounds. Microbes are ubiquitous in nature and are being exposed to
the continuous release of more and more recalcitrant xenobiotic compounds
into the environment. No wonder, these microbes, inhabiting polluted
environments, are armed with various resistance and catabolic potentials. The
catalytic potential of microbes in nature is enormous and this is advantageous
to mankind for a cleaner and healthier environment through bioremediation.
In general, potential microbes with broad spectrum of activities from
their native habitat have been screened, characterized, genetically modified
and released back to their native habitat for better performance. By such
studies, the core problem of pollution is tactfully attacked and benefits of
decontamination add healthy atmosphere to mankind. The purified degrading
enzymes, Nitrilase, Azoreductases and Oragnophosphate hydrolases could
be effectively used in industry for the treatment of effluents. The systems
developed are eco-friendly and economical and hence could effectively be
integrated with physico-chemical methods for pollution control.
The index of xenobiotic compounds released into the environment
increases due to industrialization and combating pollution by the release of
these compounds is essential for the sustenance of the future generation. In
this context, microbes such as algae, fungi and bacteria, play an important role
by giving us a helping hand in bioremediation of these xenobiotic compounds.
Degradation of pesticides by different bacterial population proves to be
the best example for citing the role of microbes in bioremediation of xenobiotic
compounds. A large number of pesticides and insecticides like morpholine,
methyl parathion, organophosphorous compounds and benzimidazoles are
widely used to increase the agricultural output and has also contributed to the
pollution load, as many of these man-made chemicals are non-biodegradable.
The pollution control strategies involving physico-chemical methods many a
time aggravate the problem, rather than eliminating it. Microbes play a very
important role in the mineralization of pollutants either by natural selection
or through recombinant DNA technology making bioremediation process an
extension of normal microbial metabolism. Xenobiotic compounds are also
widely employed in our day-to-day life. Microbes also mediate degradation
of xenobiotic compounds like dyes and plastics.
Understanding the molecular biology of the microorganisms, and the
ability to genetically manipulate the microorganisms and infuse engineering
principles into biology have led to novel strategies for combating environmental
problems.
4.30 Environmental Biotechnology

Construction of strains with broad spectrum of catabolic potential

with heavy metal resistant traits makes them ideal for bioremediation of
polluted environments in both aquatic and terrestrial ecosystems. The
transfer of genetic traits from one organism to another paves way in creating
Genetically Engineered Organisms (GEMs) for combating pollution in
extreme environments making it a boon to mankind to cleanup the mess that
has created in nature. Therefore, bioremediation protocols for treatment of
industrial wastewaters like distillery effluent, textile mill effluent, tannery
effluent and pharmaceutical effluent have been devised and managed by the
author for commercial applications.
Biodegradation of Wastewater and industrial effluents: Micro-organisms
in sewage treatment plants remove the more common pollutants from waste
water before it is discharged into rivers or the sea. Increasing industrial and
agricultural pollution has led to a greater need for processes that remove
specific pollutants such as nitrogen and phosphorus compounds, heavy
metals and chlorinated compounds. New methods include aerobic, anaerobic
and physico-chemical processes in fixed-bed filters and in bioreactors in which
the materials and microbes are held in suspension. The costs of wastewater
treatment can be reduced by the conversion of wastes into useful products.
• For example, heavy metals and sulphur compounds can be removed
from waste streams of the galvanisation industry by the aid of sulphur
metabolising bacteria and reused.
• Another example is the production of animal feed from the fungal
biomass which remains after the production of penicillin. Most
anaerobic waste watertreatment systems produce useful biogas.
Drinking and Process Water: Abundant supplies of water are vital for
modern urban and industrial development. By the turn of this century, it
is estimated that two-thirds of the world’s nations will be water stressed –
using clean water faster than it is replenished in aquifers or rivers. A very
important aspect of biotechnology is therefore its potential for the reclamation
and purification of waste waters for re-use. Public concern has also increased
over the quality of drinking water. Not only does water need to be recycled
in the development of sustainable use of resources, overall quality must
also be improved to satisfy consumers. In many agricultural regions of the
world, animal wastes and excess fertilisers result in high levels of nitrates in
drinking water. Biotechnology has provided successful methods by which
these compounds can be removed from processed water before it is delivered
to customers.
Air and waste gases: Originally, industrial waste gas treatment systems
were based on cheap compost-filled filters that removed odours. Such systems
still exist. However, slow processing rates and the short life of such filters drove
research into better methods such as bioscrubbers, in which the pollutants
are washed out using a cell suspension and biotrickling filters, in which the
Environmental Microbiology—Methods and Applications 4.31

pollutant is degraded by micro-organisms immobilised on an inert matrix

and provided with an aqueous nutrient film trickling through the device. The
selection of micro-organisms that are more efficient at metabolising pollutants
has also led to better air and gas purifying biofilters.
• Examples are a bioscrubber based system for the simultaneous removal
of nitrogen and sulphur oxides from the flue gas of blast furnaces
which has been developed as an alternative to the classical limestone
gypsum process, and the elimination of styrene from the waste gas of
polystyrene-processing industries by a biofilter containing fungi.
Soil and land treatment: Both in situ (in its original place) and ex situ
(somewhere else) methods are commercially exploited for the cleanup
of soil and the associated groundwater. In situ treatments may include the
introduction of micro-organisms (bioaugmentation), ventilation and/or adding
nutrient solutions (biostimulation). Ex situ treatment involves removing the
soil and groundwater and treating it above ground. The soil may be treated as
compost, in soil banks, or in specialised slurry bioreactors.
Groundwater is treated in bioreactors and either pumped back into the
ground or drained into surface water. Bioremediation of land (biorestoration) is
often cheaper than physical methods and its products are harmless if complete
mineralisation takes place. Its action can however, be time-consuming, tying
up capital and land. The in situ bioremediation of the ground under petrol
stations has already become common practice but also for chlorinated solvents
like tri- and tetrachloroethylene in situ bioremediation is possible. The
applicability of in situ bioremediation is and probably will remain dependent
on the physical parameters of the soil, mainly its transport properties.
Bioremediation using plants is called phytoremediation. This technique
is presently already used to remove metals from contaminated soils and
groundwater and is being further explored for the remediation of other
pollutants. The combined use of plants and bacteria may also be possible.
Certain bacteria live closely associated with the roots of plants and depend
on substances excreted by the roots. Such rhizobacteria, whose numbers are
much higher than those of other soil bacteria, may be genetically modified to
break down pollutants.
Detection and monitoring of microorganisms used for bioremediation:
When laboratory grown micro-organisms are inoculated into a bioremediation
site (bioaugmentation), it often becomes necessary to monitor their presence
and/or multiplication to check the progress of the process. This is especially true
and even required when genetically modified micro-organisms are involved.
The traditional technique to detect the presence of micro-organisms in soil
is direct plating on selective media. This is greatly facilitated if the organism
contains a marker which can be selected for. Newer techniques include the
above mentioned immunological and light-based bioreporter techniques. The
4.32 Environmental Biotechnology

spatial distribution of specific microorganisms in a sample can be determined

microscopically and non-invasively by using fluorescent in situ hybridisation
(FISH) of micro-organisms. The most sensitive and specific technique is the
direct isolation and amplification of DNA from soil, which is increasingly
being used.
Genetic Engineering: Recombinant DNA technology has had amazing
repercussions in the last few years. Molecular biologists have mapped entire
genomes, many new medicines have been developed and introduced and
agriculturists are producing plants with novel types of disease resistance
that could not be achieved through conventional breeding. Several of the
previously mentioned examples like the amylose-free potato and the indigo-
producing bacterium also involve the use of organisms genetically modified
by recombinant DNA technology. Many enzymes are routinely produced by
genetically modified organisms too.
• Given the overwhelming diversity of species, biomolecules and
metabolic pathways on this planet, genetic engineering can, in
principle, be a very powerful tool in creating environmentally
friendlier alternatives for products and processes that presently pollute
the environment or exhaust its non-renewable resources. Politics,
economics and society will ultimately determine which scientific
possibilities will become reality.
• Nowadays organisms can also be supplemented with additional genetic
properties for the biodegradation of specific pollutants if naturally
occurring organisms are not able to do that job properly or not quickly
enough. By combining different metabolic abilities in the same micro-
organism, bottlenecks in environmental cleanup may be circumvented.
Until now this has not been done on any significant scale. The main
reason being the fact that, in most cases, naturally occurring organisms
can be found or selected for, which are able to clean up a polluted site.
Examples have been found where soil bacteria have developed new
properties in response to the introduction of xenobiotics (that is, man-
made chemicals, that are normally not found in nature).
• In some cases, they even appear to have acquired properties from other
species. In the USA, some genetically modified bacteria have been
approved for bioremediation purposes but large scale applications
have not yet been reported. In Europe, only controlled field tests have
been authorized. Because new organisms can be created by genetic
engineering that may never be produced by spontaneous or selection
driven evolution, concerns exist about the unpredictability of their
possible interactions with the eco-system.
• Genetically modified organisms which are properly kept within the
confines of their approved production facilities are much less a concern
Environmental Microbiology—Methods and Applications 4.33

than genetically modified organisms which are meant to be released

into the environment like disease-resistant plants or soil bacteria for
bioremediation. The possible ecological effects of the latter are even
more difficult to evaluate due to the fact that it is well known that soil
bacteria frequently exchange genetic material (also between species).
This together with the fact that we know little about the great majority
of soil-inhabiting bacterial species, makes it almost impossible to
predict the fate of every DNA copy of a newly introduced genetic
property in a soil bacterium. If the extra DNA is derived from another
soil bacterium, it may on the other hand be reasonable to argue that the
genetically modified bacterium might also have evolved spontaneously
some day due to the frequent exchange of genetic material in the soil.
Use of genetic engineering and genetic manipulations for more efficient
bioremediation: In recent years, efforts have been made to create genetically
engineered microorganisms (GEMs) to enhance bioremediation. This is done
to overcome some of the limitations and problems in bioremediation. These
problems are:
• Sometimes the growth of microorganisms gets inhibited or reduced by
the xenobiotics.
• No single naturally-occurring microorganisms has the capability of
degrading all the xenobiotics present in the environmental pollution.
• The microbial degradation is a very slow process.
• Sometimes, certain xenobiotics get adsorbed on to the particulate
matter of soil and thus, become unavailable for microbial degradation.
As the majority of genes responsible for the synthesis of enzymes
with biodegradation capability are located on the plasmids, the genetic
manipulations of plasmids can lead to the creation of new strains of bacteria
with different degradative pathways. In 1970s, Chakrabarty and his team of co-
workers reported the development of a new strain of bacterium Pseudomonas
by manipulations of plasmid transfer which they named as “superbug”.
This superbug had the capability of degrading a number of hydrocarbons
of petroleum simultaneously such as camphor, octane, xylene, naphthalene,
etc. In 1980, United States granted the patent to this superbug making it the
first genetically engineered microorganism to be patented. In certain cases,
the process of plasmid transfer was used. E.g., The bacterium containing
CAM (camphor, degrading) plasmid was conjugated with another bacterium
with OCT (octane, degrading) plasmid. Due to non-compatibility, these
plasmids cannot coexist in the same bacterium. However, due to the presence
of homologous regions of DNA, recombination occurs between these two
plasmids which results in a single CAM-OCT plasmid giving the bacterium
the capacity to degrade both camphor as well as octane.
4.34 Environmental Biotechnology

4.5 DEGRADATION OF OIL BY MICROORGANISMS FOR THE

PRODUCTION OF USEFUL PRODUCTS
Brief Introduction to Oil: Crude oil (or petroleum): a liquid mixture of a variety
of hydrocarbon compounds derived from ancient algal and plant remains and
found in reservoirs under the Earth’s surface. Nitrogen and sulfur-containing
molecules (“resins”) are common constituents of some crude oils.

Crude oil components

• Volatile compounds—low molecular weight compounds, like methane
(natural gas) or propane, that are normally gaseous or evaporate very
quickly at room temperature.
• Saturated hydrocarbons—compounds with carbon and hydrogen
atoms connected only by single bonds. Saturated hydrocarbons can
be arranged in straight or branched chains of up to about 25 carbon
atoms. Saturated hydrocarbons are readily biodegraded although
degradability decreases with chain length.
• Aromatic compounds—compounds that contain rings of carbon
atoms held together with double bonds between the carbon atoms. The
smallest aromatic compounds in petroleum have six carbons in such a
ring structure (e.g. benzene and toluene), but other compounds contain
multiple rings. These are known as polycyclic aromatic hydrocarbons,
often abbreviated ‘PAH’. Most aromatic molecules in petroleum have
multiple attached hydrocarbon chains.
The smallest aromatic molecules (one- and two-rings) are both volatile
and readily biodegraded, even with attached side-chains, but four-ring and
larger aromatic compounds, are more resistant to biodegradation. They are,
however, susceptible to photooxidation. Some larger PAHs are of concern
because they are potentially carcinogenic; 16 different PAHs are designated as
priority pollutants by the EPA.
The percentage of PAHs in crude oil varies, but the ‘priority pollutants’
are present at low levels in crude oils; they are much more common as a
byproduct of burning carbonaceous materials such as fuel, coal, wood, tobacco
and other materials. Asphaltenes (used in making roads and roofing products)
are examples of high molecular weight (heavy) PAHs that have additional
chemical side chains attached to their aromatic rings. Asphaltenes are not
soluble in water and most organic solvents.
Oil-degrading bacteria: Bacteria usually are the dominant hydrocarbon
degraders in aquatic systems such as oceans. They also posess diverse
metabolic pahtways that are not present in fungi which allows them to utilize
most recalcitrant petroleum hydrocarbons.
Bacterial degradation of aromatic compounds can be divided into three
steps:
Environmental Microbiology—Methods and Applications 4.35

• modification and conversion of the many different compounds into a

few central aromatic intermediates (ring-fission substrates); this step is
referred as peripheral pathway and involves considerable modification
of the ring and/or perhaps elimination of substituent groups;
• oxidative ring cleavage by dioxygenases, which are responsible for the
oxygenolytic ring cleavage of dihdyroxylated aromatic compounds
(catechol, protocatechuate, gentisate);
• further degradation of the non-cyclic, non-aromatic ring-fission
products to intermediates of central metabolic pathways.
Long-chain hydrocarbons (C10-C18) can be used rapidly by many high G+C
gram-positive bacteria. Only a few bacteria can oxidize C2-C8 hydrocarbons.
Degradation of n-alkanes requires activation of the inert substrates by
molecular oxygen with help of oxygenases by three possible ways that are
associated with membranes:
• Monooxygenase attacks at the end producing alkan-1-ol:
R–CH3 + O2 + NAD(P)H + H+ → R–CH2OH _ NAD(P)+ + H2

Aerobic pathways for the degradation of alkanes by terminal

and subterminal oxidation
4.36 Environmental Biotechnology

• Dioxygenase attack produces the hydroperoxides, which are reduced

to yield also alkan-1-ol:
R–CH3 + O2 → R–CH2OOH + NAD(P)H + H+ → R–CH2OH + NAD(P)+ +
H2O
•
Rarely, subterminal oxidation at C2 by monooxygenase yields
secondary alcohols.
It is important to keep in mind that many strains within one species of
bacteria usually exist. Usually, only some of strains are capable of hydrocarbon
degradation and some of strains can cause opportunistic infections in humans
and animals.
Biotechnology and Control of Oil Spillage: Microorganisms can now
be genetically engineered for use in oil recovery, pollution control, mineral
leaching and recovery.
•
In the petroleum industry, microorganisms can also be genetically
engineered to produce chemicals useful for enhanced oil recovery .
•
Cleaning up oil spills could, in the future, be left to genetically-
engineered bacteria. In the mining industries, micro-organisms with
the property of enhanced leaching ability could be designed.
•
Micro-organisms can bind metals to their surfaces and concentrate
them internally. As a result of this, genetically improved strains can be
used to recover valuable metals or remove polluting metals from dilute
solution as in industrial waste.
•
Research is already being carried out to improve the naturally-
occurring bacteria that can ‘eat oil’, for use following an oil spill.
•
By applying bacteria to oilcovered beaches, the complex oil molecules
would be broken down into harmless sugars. Many micro-organisms
can degrade various kinds of environmental pollutants into relatively
harmless materials before the death of the micro-organisms. This
property could also be used in overcoming the environmental hazards
of DDT, lead and other environmental pollutants like toxic wastes,
globally.
•
Strains of bacteria which can degrade fuel hydrocarbons have been
designed and the use of genetically engineered micro-organisms
to clean up oil spillages or treat sewages has been proposed and is
undergoing production/manufacturing.
Oil Biodegradation in Marine Systems: Petroleum oil is toxic for most
life forms and, episodic and chronic pollution of the environment by oil,
causes major ecological perturbations. Marine environments are especially
vulnerable since oil spills of coastal regions and the open sea are poorly
containable and mitigation is difficult. In addition to pollution through
human activities, millions of tons of petroleum enter the marine environment
Environmental Microbiology—Methods and Applications 4.37

every year from natural seepages. Despite its toxicity, a considerable fraction
of petroleum oil entering marine systems is eliminated by the hydrocarbon-
degrading activities of microbial communities, in particular, by a remarkable
recently discovered group of specialists, the so-called hydrocarbonoclastic
bacteria (HCB). Alcanivorax borkumensis, a paradigm of HCB and probably
the most important global oil degrader, was the first to be subjected to a
functional genomic analysis. This analysis has yielded important new insights
into its capacity for
(i) n-alkane degradation including metabolism, biosurfactant production
and biofilm formation,
(ii) scavenging of nutrients and cofactors in the oligotrophic marine
environment, as well as
(iii) coping with various habitat-specific stresses.
The understanding, thereby gained, constitutes a significant advance in
efforts towards the design of new knowledge-based strategies for the mitigation
of ecological damage caused by oil pollution of marine habitats. HCB also
have potential biotechnological applications in the areas of bioplastics and
biocatalysis.

4.6 DEGRADATION OF PLASTICS BY MICROORGANISMS FOR

PRODUCTION OF USEFUL PRODUCTS
Microorganisms such as bacteria and fungi are involved in the degradation of
both natural and synthetic plastics. The biodegradation of plastics proceeds
actively under different soil conditions according to their properties, because
the microorganisms responsible for the degradation differ from each other
and they have their own optimal growth conditions in the soil. Polymers,
especially plastics, are potential substrates for heterotrophic microorganisms.
Biodegradation is governed by different factors that include: polymer
characteristics, type of organism, and nature of pretreatment.
• The polymer characteristics such as its mobility, tacticity, crystallinity,
molecular weight, the type of functional groups and substituents present
in its structure, and plasticizers or additives added to the polymer all
play an important role in its degradation. During degradation, the
polymer is first converted to its monomers, then these monomers are
mineralized.
• Most polymers are too large to pass through cellular membranes, so
they must first be depolymerized to smaller monomers before they can
be absorbed and biodegraded within microbial cells.
• The initial breakdown of a polymer can result from a variety of physical
and biological forces. Physical forces, such as heating/cooling, freezing/
thawing, or wetting/drying, can cause mechanical damage such as the
cracking of polymeric materials.
4.38 Environmental Biotechnology

• The growth of many fungi can also cause small-scale swelling and
bursting, as the fungi penetrate the polymer solids. Synthetic polymers,
such as poly (caprolactone), are also depolymerized by microbial
enzymes, after which the monomers are absorbed into microbial cells
and biodegraded. Abiotic hydrolysis is the most important reaction for
initiating the environmental degradation of synthetic polymers like
polycarboxylates, poly (ethylene terephthalate), polylactic acids and
their copolymers, poly (α-glutamic acids), and polydimethylsiloxanes,
or silicones.
Generally, an increase in molecular weight results in a decline of
polymer degradability by microorganisms. In contrast, monomers, dimers,
and oligomers of a polymer’s repeating units are much easily degraded and
mineralized. High molecular weights result in a sharp decrease in solubility
making them unfavorable for microbial attack because bacteria require the
substrate to be assimilated through the cellular membrane and then further
degraded by cellular enzymes. At least two categories of enzymes are actively
involved in biological degradation of polymers: extracellular and intracellular
depolymerases.
During degradation, exoenzymes from microorganisms break down
complex polymers yielding smaller molecules of short chains e.g., oligomers,
dimers, and monomers, that are smaller enough to pass the semi-permeable
outer bacterial membranes, and then to be utilized as carbon and energy
sources. The process is called depolymerization.
When the end products are CO2, H2O, or CH4, the degradation is called
mineralization. It is important to note that biodeterioration and degradation of
polymer substrate can rarely reach 100% and the reason is that a small portion
of the polymer will be incorporated into microbial biomass, humus and other
natural products. Dominant groups of microorganisms and the degradative
pathways associated with polymer degradation are often determined by the
environmental conditions.
When O2 is available, aerobic microorganisms are mostly responsible for
destruction of complex materials, with microbial biomass, CO2, and H2O as
the final products. In contrast, under anoxic conditions, anaerobic consortia
of microorganisms are responsible for polymer deterioration. The primary
products will be microbial biomass, CO2, CH2 and H2O under methanogenic
(anaerobic) conditions (e.g. landfills/ compost).

4.7 RECOVERY OF MINERALS BY MICROBES

The recovery of copper from the drainage water of mines was probably
a widespread practice in the Mediterranean basin as early as 1000 B.C.
Although such mining operations are difficult to document, it is known that
Environmental Microbiology—Methods and Applications 4.39

the leaching of copper on a large scale was well established at the Rio Tinto
mines in Spain by the 18th century. What none of the miners engaged in this
traditional method of mineral extraction realized until about 25 years ago is,
that bacteria take an active part in the leaching process. They help to convert
the copper into a water-soluble form that can be carried off by the leach water
Today, bacteria are being deliberately exploited to recover millions of pounds
of copper from billions of tons of low-grade ore. Copper obtained in this way
accounts for more than 10 per cent of the total U.S. production. In recent years,
bacterial leaching has also been applied to the recovery of another non-ferrous
metal: uranium. 
Bioleaching: Bioleaching is leaching where the extraction of metal from
solid minerals into a solution is facilitated by the metabolism of certain
microbes—bioleaching microbes. Bioleaching is a process described as “the
use of microorganisms to transform elements so that the elements can be
extracted from a material when water is filtered through it”.

Schematic representation of the Bioleaching process

Bioleaching involves the use of micro-organisms to extract metals from
low grade ores and has been performed successfully on earth to obtain gold,
copper and uranium.
• About 20% of the world’s copper is produced by bioleaching.
•
Bioleaching of nickel, zinc and cobalt can be done with thermophyllic
bacteria.
4.40 Environmental Biotechnology

Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, Thiobacillus thiooxidans,

Sulfolobus species and others have been used for bioleaching. Acidiphilium,
Sulfobacillus, Ferroplasma, Sulfolobus, Metallosphaera, and Acidianus have also
been used. These bacteria tolerate acids and metabolize sulfur. Weak solutions
of acids are dripped through the ore and a bacterial liquor forms that is then
electrolytically or chemically processed. Sometimes, this requires water and
organic substrate like potato peels as well as solvents to extract the metals
from the bacterial mass. Chaff from crops may be used for bioleaching rather
than livestock feed. Precious water will be recycled. If bioleaching becomes
a major industrial activity on the Moon we will be pressed to conserve our
vital water and hydrogen resources for this instead of wasting them in the
form of rocket fuel. Only ores containing sulfur can be bioleached because
the bacteria feed on sulfur. Bioleaching does not require lots of energy but it
is slow. High temperature roasting and smelting is not required, so there are
decided benefits in addition to the fact that bioleaching can get metals from
low-grade ores.

Factors Influencing Bioleaching

•
Temperature (optimum between 30° & 50°
•
pH (2.3-2.5)
•
Iron supply
•
Oxygen
•
Availability of other nutrients required for growth
Recent progress in the genetic manipulation of microorganisms for
industrial purposes promises to revitalize not only the bacterial leaching
of metal-bearing ores but also the microbiological treatment of metal-
contaminated wastewater. The enthusiasm of the microbiologists working on
the development of the new “biomining” techniques is matched by a need in
the minerals industry to find alternatives to conventional methods of mining,
ore-processing and wastewater treatment. The need arises from recent trends
in the industry: the continued depletion of high-grade mineral resources,
the resulting tendency for mining to be extended deeper underground, the
growing awareness of environmental problems associated with the smelting
of sulfide minerals and the burning of sulfur-rich fossil fuels and the rising cost
of the prodigious amounts of energy required in the conventional recovery
methods. The current methods will surely prevail for many years to come, but
biological processes are generally less energy-intensive and less polluting than
most non-biological ones, and so the role of biological technology in mining,
ore processing and wastewater treatment is likely to become increasingly
important. 
The bacteria involved in the leaching of metals from ores are among
the most remarkable life forms known. The microorganisms are said to be
Environmental Microbiology—Methods and Applications 4.41

chemolithotrophic (“rock-eating”); they obtain energy from the oxidation

of inorganic substances. Many of them are also autotrophic, that is, they
capture carbon for the synthesis of cellular components not from organic
nutrients but from carbon dioxide in the atmosphere. The leaching bacteria
live in environments that would be quite inhospitable to other organisms;
for example, the concentration of sulfuric acid and of soluble metals is often
very high. Some thermophilic, or heat-loving, species require temperatures
above 50°C (122°F), and a few strains have been found at temperatures near
the boiling point of water. 
For many years, the only microorganism thought to be important in
the leaching of metals from ores was the rod-shaped bacterium Thiobacillus
ferrooxidans. This microorganism was discovered in the acidic water-draining
coal mines; it was not until 1957 that a correlation was recognized between the
presence of the bacterium and the dissolution of metals in copper-leaching
operations. Since then, a great deal of information has been amassed on T.
ferrooxidans and on its vital role in the leaching of metals. 
T. ferrooxidans is acidophilic, or acid-loving; it tends to live in environments
such as hot springs, volcanic fissures and sulfide ore deposits that have a high
concentration of sulfuric acid. It is also moderately thermophilic, thriving
in the temperature range between 20 and 35°C. The bacterium gets energy
for growth from the oxidation of either iron or sulfur. The iron must be in
the ferrous, or bivalent, form (Fe++), and it is converted by the action of the
bacterium into the ferric, or trivalent, form (Fe++). Several forms of sulfur can
be attacked. They include both soluble and insoluble sulfides (compounds
containing the bivalent sulfur ion S- -), elemental sulfur and soluble compounds
that incorporate either the thiosulfate ion (S2O3- -) or the tetrathionate ion
(S4O6- -). In each case, the product of the transformation is a substance in which
the sulfur atom has fewer valence electrons, culminating in the formation
of the sulfate ion (SO4- -). T. ferrooxidans obtains carbon autotrophically from
atmospheric carbon dioxide. 
Although T. ferrooxidans is essential to the bacterial leaching of metals, it
is by no means the only microorganism with an important role in the process.
Among the other microorganisms taking part is T. thioxidans, a rod-shaped
bacterium, not unlike T. ferrooxidans, that grows on elemental sulfur and some
soluble sulfur compounds. Studies by Donovan P. Kelly and his associates
at the University of Warwick have confirmed the importance of mixed
cultures of bacteria in the extraction of metals from ores. T. ferrooxidans and T.
thiooxidans combined, for example, are more effective in leaching certain ores
than either organism is alone. Similarly, the combination of Leptospirillium
ferrooxidans and T. organoparus can degrade pyrite (FeS2) and chalcopyrite
(CuFeS2), a feat neither species can accomplish alone. 
In acidic environments supporting leaching bacteria, one can often isolate a
number of heterotrophic microorganisms: bacteria and fungi that scavenge the
4.42 Environmental Biotechnology

small amounts of organic matter present in these environments or that survive

on the organic by-products of other organisms’ autotrophic metabolism. The
role of the heterotrophic microorganisms in the leaching process is largely
undetermined. Thiobacilli, that attack some sulfide minerals and certain
soluble sulfur compounds under neutral conditions (that is, neither acidic nor
alkaline), are often found in sulfide ore deposits and in other habitats where
sulfur is available. Thiobacilli of this type may be responsible for the initial
increase in acidity that establishes an environment conducive to the growth of
the more acidophilic-leaching bacteria. 
At temperatures between 60 and 75°C, and under neutral conditions the
filamentous bacterium  Thermothrix thiopara  oxidizes sulfhydryl ions (HS-),
sulfite ions (SO3-), thiosulfate ions and elemental sulfur to form sulfate ions.
There is increasing evidence of the widespread existence of Thermothrix species
and similar filamentous, sulfur-oxidizing bacteria, in thermal springs and near
volcanic fissures. Few leaching sites have been tested for the presence of these
bacteria. Their existence in sulfur-bearing springs, however, suggests they
could colonize sulfidic ores and thereby, prepare such environments for the
more acidophilic species. 
The most robust of the leaching microorganisms are the extremely
thermophilic and acidophilic species of the genus Sulfolobus. These bacteria
flourish in acidic hot springs and volcanic fissures at temperatures that
can exceed 60 degrees C. Some strains of Sulfolobus have been observed
in springs at temperatures near the boiling point of water. The cell wall of
the  Sulfolobus  bacteria has a different structure from that of most bacteria.
Microorganisms of this type are thought to belong to the Archaebacteria, a
group of unusual bacteria proposed as a separate kingdom of life forms.
Sulfolobus acidocaldarius and S. brierleyi oxidize sulfur and iron for energy,
relying on either carbon dioxide or simple organic compounds for carbon.
Ordinarily, oxygen is required by Sulfolobus, — as in other aerobic organisms,
the oxygen serves as the ultimate acceptor of the electrons removed in the
process of chemical oxidation.  Sulfolobus  bacteria can also grow anaerobically,
however. It has been demonstrated that molybdenum (Mo6+) and ferric iron
can serve as electron acceptors in the absence of air. Minerals that resist most
microorganisms, such as chalcopyrite and molybdenite (MoS2), are readily
attacked by Sulfolobus, and the resulting soluble metals are not toxic to the
organism. Molybdenum, which is extremely toxic even to the metal-tolerant
thiobacilli, is readily endured by S. brierleyi in concentrations as high as 750
milligrams per liter. Sulfolobus has not been isolated from commercial leaching
operations, but laboratory studies confirm the ability of the organism to
proliferate in such environments. The potential of Sulfolobus species to leach
metals from ores is only now being recognized: because of the extraordinary
ability of these organisms to attack resistant mineral structures, however,
they are certain to be among the leaching bacteria that will be successfully
exploited in the future. 
Environmental Microbiology—Methods and Applications 4.43

Indirect leaching, in contrast, does not proceed through a frontal attack,

by the bacteria on the atomic structure of the mineral. Instead, the bacteria
generate ferric iron by oxidizing soluble, ferrous iron; ferric iron in turn is a
powerful oxidizing agent that reacts with other metals, transforming them into
the soluble, oxidized form in a sulfuric acid solution. In this reaction, ferrous
iron is again produced and is rapidly reoxidized by the bacteria. Indirect
leaching is usually referred to as bacterially-assisted leaching. In an acidic
solution without the bacteria, ferrous iron is stable, and leaching mediated
by ferric iron would be slow. T ferrooxidans can accelerate such an oxidation
reaction by a factor of more than a million. 
Direct and indirect leaching by bacteria are difficult to differentiate
quantitatively because most minerals include some iron. Even if leaching were
to begin with the direct process exclusively, the iron would be released from
the mineral and would establish an indirect leaching cycle. Direct leaching by
thiobacilli has been demonstrated in the laboratory with iron-free synthetic
metal sulfides. 
In practice, the leaching of metals is far more complex than the above
analysis might suggest; there are numerous processes in addition to direct
enzymatic oxidation and bacterial generation of ferric iron. Some chemical
reactions between ferric iron and metal-sulfide minerals result in the
formation of secondary minerals and elemental sulfur which can “blind,” or
inactivate the reactive surfaces. When sulfur is formed, T. thiooxidans plays an
indispensable role in oxidizing the sulfur to sulfuric acid, thus exposing the
metal for further leaching. 
The control of acidity is of utmost importance in leaching, because an
acidic environment must be maintained in order to keep ferric iron and other
metals in solution. Acidity is controlled by the oxidation of iron, sulfur and
metal sulfides, by the dissolution of carbonate ions and by the decomposition
of ferric iron through reaction with water. The last reaction promotes leaching
by generating hydrogen ions (which make the solution more acidic), but it may
also be detrimental because precipitates of basic ferric sulfates may inactivate
the surfaces of metal-sulfide minerals, and in some cases, may even prevent the
flow of the leaching solution. The chemical and biological processes are part
of a complex system whose functioning depends on elements of hydrology,
geology, physics and engineering. 
The dumps are not inoculated with the leaching bacteria. The organisms
are ubiquitous, and when conditions in the rock pile become suitable for their
growth, they proliferate. Rock samples collected near the top of a leach pile
typically harbor more than a million bacteria of the species T. ferrooxidans per
gram;  T thiooxidans bacteria are present in somewhat smaller numbers. The
leach solution percolates through the leach dump, and the “pregnant,” or
metal-laden, solution is collected in catch basins or reservoirs at the foot of the
dump. The copper is removed from solution either by a cementation reation, in
4.44 Environmental Biotechnology

which ferrous iron replaces the copper in solution, or by solvent extraction, in

which the copper is concentrated by transferring it from the aqueous leaching
solution to an organic solution. The “barren,” or copper-free solution, is then
recycled to the top of the dump. 
In a large dump-leaching operation, the ore is processed for many years
to recover as much of the copper as possible. Because of the construction
methods employed and the volume of the solid material treated, dump
leaching is a crude operation. The placement of the dump in a natural valley
can impede the flow of air to the interior of the pile. The large size of some
of the rocks limits contact among the metal-sulfide minerals, the oxidizing
solution and the bacteria. During the dumps construction, large ore haulers
compact the surface, creating impermeable zones in the pile. Gypsum (CaSO4),
ferric hydroxide [Fe(OH)3] and basic ferric sulfate precipitates also decrease
permeability, further reducing the contact of the solution with the sulfide
rocks. 
Although, studies of bacteria in the dump environment have barely
begun, some factors that may adversely affect the populations are known.
They include high metal concentrations, particularly of ions such as silver
and mercury that are known to be toxic to the organisms, lack of air, and
temperatures higher than those tolerated by the organisms. Because the
limitations of dump leaching are more clearly defined today than they were
in the past, considerable forethought now goes into the construction of the
leaching piles. Special “finger” dumps allow greater air circulation, and
haulage is controlled to minimize compaction. From a biological viewpoint,
however, dump leaching remains an essentially uncontrolled process. 
Extractive methods other than dump leaching offer somewhat more
regulation of biological, chemical and engineering factors. Heap leaching, for
example, is used to extract metals from sulfide and oxide minerals in ores
of a somewhat higher grade than those subjected to dump leaching. In heap
leaching, the rocks are often crushed to avoid the solution-contact problems
encountered when leaching large boulders, and the heaps are built up on
impermeable pads to prevent loss of the solution into the underlying soil.
Aeration systems have been installed to increase the flow of air in the piles. 
In-place leaching is a promising technique for the recovery of metals
from low-grade ores in inaccessible sites. This technology, which has minimal
impact on the environment, is currently employed to extract residual minerals
from abandoned mine workings and to recover uranium from low-grade
deposits. To leach metals from depleted mine workings, the leaching solution
is applied directly to the walls and the roof of an intact stope (an underground
excavation from which ore has been removed) or to the rubble of fractured
workings. In-place leaching techniques have been successful in the recovery
of both copper and uranium. 
Environmental Microbiology—Methods and Applications 4.45

In the extraction of uranium, the bacteria do not directly attack the

uranium mineral; instead, they generate ferric iron from pyrite and soluble
ferrous iron. Ferric iron readily attacks minerals incorporating quadrivalent
uranium (U4+), converting this ion into hexavalent uranium (U6+), which is
soluble in dilute sulfuric acid. 
The bacterially–assisted leaching of uranium could, in principle, be applied
not only to the recovery of residual metals but also to the in-place leaching
of low-grade uranium ore bodies. There are a number of low-grade deposits
in the western and southwestern U.S., but they are low in pyritic material
and include rocks that tend to neutralize acids. Such mineralogical conditions
are not conducive to bacterial leaching, and an exclusively chemical method
of leaching has been adopted instead. Wells are drilled into the isolated ore
bodies at depths ranging from tens to hundreds of feet. A carbonate solution
containing an oxidant is then injected into the mineralized zone, where the
uranium is solubilized. The uranium-bearing solution is withdrawn from the
formation through a precisely engineered pattern of recovery wells. 
Although bacterial leaching is currently exploited only for the recovery of
copper and uranium, the appetite of the leaching bacteria is fairly nonspecific.
The organisms readily degrade other sulfide minerals, yielding zinc from
sphalerite (ZnS) and lead from galena (PbS). The leaching bacteria can therefore
be considered for the extraction of many other metals. The bacteria readily
catalyze the dissolution of inorganic sulfur from coal, and recent advances
indicate that organic sulfur may also be vulnerable to microbiological attack. 
The microbiological processes currently exploited by the minerals industry
are fairly simple in engineering design, and their effectiveness is sensitive
to seasonal changes and sudden alterations in the chemistry of the system.
Recent developments in the study of the uptake of metals by S. cerevisiae, 
R. arrhizus and P. aeruginosa make it probable that these microorganisms can
be utilized in precisely engineered processes for the recovery of metals from
wastewater streams. The new tools of genetic engineering may well lead to the
creation of modified organisms with, greater effectiveness in metal removal.
It was noted above that only 44 per cent of the P. aeruginosa cells take part in
the uranium-uptake process; Shumate and Strandberg speculate that if the
factor or factors controlling the uptake can be identified, the bacteria may be
genetically altered to increase the population that accumulates the metal. 
The accumulation of metals by microorganisms, whether the process is
intracellular uptake or surface accumulation, is fairly nonspecific: negatively
charged groups of atoms on the surface of microorganisms attract any positively
charged ions in the solution. Many organisms have cellular components
that are highly metal-specific. One of the best-understood metal-binding
agents is the protein metallothionein. Structural studies of metallothionein
indicate there is a high concentration of sulfur-containing amino acid units,
which, when they are brought into juxtaposition by the folding of the protein
4.46 Environmental Biotechnology

chain, form a sulfhydryl (HS-) chelation site. In the marine blue-green

alga Synechococcus, a comparatively small cadmium-binding metallothionein
can bind an average of 1.28 atoms of cadmium per molecule of protein.
The identification of the gene or the genes that specify the structure of the
metallothionein of this organism or any other may enable geneticists to isolate
and clone the genes in selected microorganisms. Cells carrying the cloned
genes could be directed to synthesize massive quantities of metallothionein
with a specific metal-binding capacity. The small protein could be immobilized
on an inert carrier and wastewater contaminated with metals could be passed
over the fixed protein. Further studies of metallothioneins with the ability to
bind specific metals may provide clues for the laboratory synthesis of simple
compounds with an increased metal-binding capacity. Based on nature’s
own workings, on controlled laboratory studies done by many workers and
on current applications in the field, it is clear that microorganisms and their
versatile activities will help man to lay claim to mineral wealth buried deep
in the ground or available in amounts not economically feasible to recover at
present. These small servants of man promise to help in cleaning the air and
water while retrieving valuable metal resources.

4.8 BIOINDICATORS OF HAZARDOUS POLLUTANTS

The term ‘bioindicator’ is used for organisms or organism associations which
respond to pollutant load with changes in vital functions, or which accumulate
pollutants. Information about specific biological effects, supplements data on
air pollutions generated by technical analysis methods. The most important
reasons for using bioindicators are:
•
the direct determination of biological effects,
•
the determination of synergetic and antagonistic effects of multiple
pollutants on an organism,
•
the early recognition of pollutant damage to plants as well as toxic
dangers to humans and
•
relatively low cost compared to technical measuring methods.
The great potential of bioindicators for environmental monitoring is often
confronted with difficult questions of methodology resulting from the use of
“living measuring instruments”. The effects of environmental load cannot
always be clearly differentiated from natural stress factors. Lack of practical
experience with certain bioindicators sometimes makes clear interpretation of
findings more difficult, especially, if no comparable pollutant measurements
are available.
Intensive research over the last decades has resulted in the availability
of numerous bioindicators which satisfy the requirements of convenience,
standardization, cost, and evaluative capability. Bioindicators are commonly
grouped into accumulation indicators and response indicators. Accumulation
Environmental Microbiology—Methods and Applications 4.47

indicators store pollutants without any evident changes in their metabolisms.

Response indicators react with cell changes or visible symptoms of damage
when taking up even small amounts of harmful substances.
Biomonitoring is divided into passive and active:
• Passive biomonitoring is the use of organisms, organism associations,
and parts of organisms which are a natural component of the ecosystem
and appear there spontaneously.
• Active biomonitoring includes all methods which insert organisms
under controlled conditions into the site to be monitored.

Aquifer (Underground Water) Indicators

The organisms used as bioindicators must be characterized by much higher
sensitivity than the best chemical indicators. Aquatic organisms accumulating
pollutants allow us to detect them even when their water concentrations are
too low to be detected. An example may be determination of radioisotope
activity in plankton, which is several times higher than in water. Sometimes,
the level of toxic substances in the abiotic part of a given area is low and does
not suggest any threat to the environment, even in the case of further pollutant
leakage. Analysis based on bioindicators, may at the same time, show that
the concentration of toxic substances in living organisms is so high that its
further increase may result in irreversible damage to particular populations
or the whole organic world in the biotope examined. To make global analyses
uniform, international organizations have established a set of principles to
be followed during toxicity determination, and compiled a list of indicatory
organisms. Bioindicators should be selected according to the following criteria:
• sedentary life,
• abundance, wide distribution,
• simple procedure of identification and sampling,
• high tolerance for the pollutants analyzed,
• population stability,
• high accumulating capacity.
The water purity state should be determined using organisms sensitive to
pollution, characterized by a narrow range of tolerance. The following tests
and bioindicators can be applied to analysis of water and sewage toxicity:
• test based on Chlorella vulgaris – a unicellular green alga, widespread in
fresh waters. Diluted sewage solutions are introduced into laboratory
algal cultures, then absorbance is measured with a spectrophotometer
in the visible range;
• test based on Daphnia magna Straus – a crustacean living in fresh waters.
Young organisms are placed in crystallizers with sewage solutions of
different concentrations. The count of bioindicators showing the test
4.48 Environmental Biotechnology

effect (organism immobilization) is determined after 24 and 48 hours.

These data allow to determine sample toxicity;
• test Spirotox, based on the protozoan Spirotostomum ambiguum, present
in clean rivers and lakes. Ciliates are placed in the sample and observed
under slight magnification. The cells of these very sensitive organisms
undergo dissolution (lysis) when affected by toxicants. Sample toxicity
is determined by its dilution, causing lysis of 50% of the population;
• test Microtox, which consists in measurement of the natural
luminescence of bacteria Vibrio fischeri, suspended in the solution of
the sample, analyzed. Toxic chemical compounds inhibit the activity
of bacterial enzymes, which reduces the intensity of luminescence. The
measurement is performed by the spectrophotometric method.
One of the criteria of water cleanliness is the fecal pollution index,
referred to as the coli index, showing the degree of pollution with intestinal
pathogenic bacteria. Also the so-called saprobiotic index is applied to evaluate
running water purity. Water quality is determined on the basis of the count of
indicatory organisms in a given site, and the catalogue value of the saprobiotic
index. The suitability of particular animal species as bioindicators depends on
their specific requirements towards the environment. Following table presents
selected indicatory organisms typical of different water purity classes.
Occurrence of selected bioindicators depending on water purity class
Oligosaprobiotic b-mesosaprobiotic a-mesosaprobiotic Polysaprobiotic
zone zone zone zone
Diatoms Snails Fungi Bacteria
Ceratoneis arcus Planorbis comeus Leptomitus lapteus Spherotilus nataus
Meridion cerculare Viparus viparus Single diatoms Zoogla ramigera
Chrysophyte Hydrulus Lymne stagualis Navicula viridula Bacterhim cynusii
foetidus Common mayflies Thiothrix nivea
Zoobenthos Beggioata
Red algae Ephemera vulgata Asselus aquaticus
Betrachospermum vagum Diatoms Erpobdella actoculata Zoobenthos
Melosira gramirata Dipteran’s larvae
Zoobenthos Bivalves Chironomus phomosus
Perla sp. Melostra variens Sphertum corneian Eristalomya
Caddis-flies Blue-green algae
Molanna angustata Microcistis aeruginoza
Bivalves
Pisidhon amnicum
The oligosaprobiotic zone is characterized by the presence of all
systematic groups, corresponds to the first water purity class and is
suitable for Salmonidae breeding. The most common bioindicators here are
dipteran’s larvae, hemipterans and caddis-flies. The β-mesosaprobiotic zone
(second water purity class) is suitable for breeding fish other than the family
Salmonidae. The most popular bioindicators here are snails and diatoms. In
the α-mesosaprobiotic zone (third water purity class) bioindicators are first
of all fungi, whereas in the polysaprobiotic zone (fourth water purity class) –
bacteria.
Environmental Microbiology—Methods and Applications 4.49

Today the indices of water cleanliness are also determined on the basis
of the species composition and count of different organisms (e.g. plankton,
periphytons, benthos), as well as analysis of matter production and destruction
processes. In clean waters, a state of equilibrium is maintained between these
processes. An increase in organic matter supply results in the domination of
destruction over production and macroconsumption. The only consumers left
in the ecosystem are destructors. The presence or absence of certain indicatory
species (algae, insects, crustaceans, fish) may provide detailed information on
the purity or pollution state of aquatic ecosystems.

Use of Whole Organism for Detection of Pesticides and Heavy Metals

A wide range of biological methods are already in use to detect pollution
incidents and for the continuous monitoring of pollutants such as pesticides
and heavy metals. Long established measures include:
•
counting the number of plants,
•
animal and microbial species,
•
counting the numbers of individuals in those species or analysing the
levels of oxygen, methane or other compounds in water.
More recently, biological detection methods, using biosensors and
immunoassays, have been developed and are now being commercialised.
Most biosensors are a combination of biological and electronic devices—often
built onto a microchip. The biological component might be simply an enzyme
or antibody, or even a colony of bacteria, a membrane, neural receptor,
or an entire organism. Immobilised on a substrate, their properties change
in response to some environmental effect in a way that is electronically or
optically detectable. It is then possible to make quantitative measurements of
pollutants with extreme precision or to very high sensitivities. The sensors can
be designed to be very selective, or sensitive to a broad range of compounds.
•
For example, a wide range of herbicides can be detected in river water
using algal-based biosensors; the stresses inflicted on the organisms
being measured as changes in the optical properties of the plant’s
chlorophyll.
Microbial biosensors are micro-organisms which produce a reaction upon
contact with the substance to be sensed. Usually they produce light but cease
to do so upon contact with substances which are toxic to them. Both naturally-
occurring light-emitting microorganisms as well as specially developed ones
are used. Positively acting bacterial biosensors have been constructed which
start emitting light upon contact (and subsequent reaction) with a specific
pollutant. In the USA, such a light emitting bacterium has been approved for
the detection of polyhalogenated aromatic hydrocarbons in field tests.
Immunoassays use labelled antibodies (complex proteins produced in
biological response to specific agents) and enzymes to measure pollutant
4.50 Environmental Biotechnology

levels. If a pollutant is present, the antibody attaches itself to it; the label making
it detectable either through colour change, fluorescence or radioactivity.
Immunoassays of various types have been developed for the continuous,
automated and inexpensive monitoring of pesticides such as dieldrin and
parathion. The nature of these techniques, the results of which can be as
simple as a colour change, make them particularly suitable for highly sensitive
field testing where the time and large equipment needed for more traditional
testing is impractical. Their use is however limited to pollutants which can
trigger biological antibodies. If the pollutants are too reactive, they will either
destroy the antibody or suppress its activity and so also the effectiveness of
the test.
Detection and monitoring of microorganisms used for bioremediation:
When laboratory grown micro-organisms are inoculated into a bioremediation
site (bioaugmentation), it often becomes necessary to monitor their presence
and/or multiplication to check the progress of the process. This is especially
true and even required when genetically modified microorganisms are
involved.
The traditional technique to detect the presence of micro-organisms in soil
is direct plating on selective media. This is greatly facilitated if the organism
contains a marker which can be selected for. Newer techniques include the
above mentioned immunological and light-based bioreporter techniques. The
spatial distribution of specific microorganisms in a sample can be determined
microscopically and non-invasively by using fluorescent in situ hybridisation
(FISH) of micro-organisms. The most sensitive and specific technique is the
direct isolation and amplification of DNA from soil, which is increasingly
being used.
Detection and monitoring of ecological effects: Bioremediation is aimed
at improving the quality of the environment by removing pollutants. However,
the disappearance of the original pollutant is not the only criterion by which
the success of a bioremediation operation is determined. (Even more) toxic
metabolites may be produced from the pollutant or the biodegrading bacterium
may cause diseases or produce substances that are harmful to useful micro-
organisms, plants, animals or humans. All these negative effects, are of course,
excluded as much as possible in advance by getting as familiar as possible
with the organism through extensive literature searches and microcosm
studies in which the bioremediation process is simulated in the laboratory.
To avoid unexpected effects, especially after the release of new member of
the eco-system like a genetically modified organism, the monitoring of the
ecological effects of a bioremediation operation may be required. The problem
with monitoring ecological effects is, what to monitor. Numerous ecological
effects are possible but not all of them may be relevant or permanent or even
the result of the bioremediation operation. The parameters to be monitored are
usually determined case-by-case. Monitoring techniques may include all of
those mentioned in the two previous subsections on detection and monitoring.
Environmental Microbiology—Methods and Applications 4.51

Biogeochemical Methods
Biogeochemistry integrates several disciplines, including biology, geology,
geography, and chemistry. Biogeochemical studies can be diverse and may
include topics such as nutrient cycling, biotic and abiotic weathering processes,
carbon cycling, microbiological-macrobiological interactions, among others.
•
The world is complex and Earth systems do not work independently
of one another; that is, the biotic world is inherently tied to the abiotic
world. Therefore, a more wholistic understanding of the Earth system
can be achieved by integrating biological, geological, and geographic
studies, among others.

Ion Chromatography
Ion chromatography is used for water chemistry analysis. Ion chromatographs
are able to measure concentrations of major anions, such as fluoride, chloride,
nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium,
ammonium, potassium, calcium, and magnesium in the parts-per-billion
(ppb) range. Concentrations of organic acids can also be measured through
ion chromatography.
How Does Ion Chromatography Work? Ion chromatography, a form
of liquid chromatography, measures concentrations of ionic species by
separating them based on their interaction with a resin. Ionic species separate
differently, depending on species type and size. Sample solutions pass through
a pressurized chromatographic column where ions are absorbed by column
constituents. As an ion extraction liquid, known as eluent, runs through the
column, the absorbed ions begin separating from the column. The retention
time of different species determines the ionic concentrations in the sample.
Applications: Some typical applications of ion chromatography include:
•
Drinking water analysis for pollution and other constituents.
•
Determination of water chemistries in aquatic ecosystems.
•
Determination of sugar and salt content in foods.
•
Isolation of select proteins.

How to – Sample Collection, Preparation and Concerns

Liquid Samples: Liquid samples should be filtered prior to evaluation with
an ion chromatograph to remove sediment and other particulate matter as
well as to limit the potential for microbial alteration before the sample is run.
Aqueous samples should be collected using a sterile syringe or bottle rinsed
three times with sample water and then filtered through 0.45 um (or smaller)
filters. The collection vial should, likewise, be rinsed three times with filtrate
before being filled brimfull of sample filtrate. Samples should be stored cold
until they can be processed. The minimum sample required for analysis is
approximately 5 mL, with no maximum limits.
4.52 Environmental Biotechnology

Solid samples and Organic Liquids

Solid samples can be extracted with water or acid (cations) to remove ions
from the sample surface. Liquid samples must also be filtered and stored cold
until analysis can be performed. The minimum sample required for a solid
sample is approximately 2-3 cm2 for solids, with no maximum limits.

Measuring Primary Production Using 14C Radiolabeling

Primary Productivity: Primary productivity is the process by which
organisms make their own food from inorganic sources. The majority of
primary producers are terrestrial plants and microbial life, such as algae.
These organisms are known as autotrophs, since they can use inorganic
substrates and solar energy to carry out metabolic processes and build cellular
material. Primary productivity due to photosynthesis is commonly measured
by quantifying oxygen production or CO2 assimilation.
How Does This Method Work?
The theory behind using 14C to measure productivity involves using a
labeled tracer to quantify assimilated carbon. The 14C method estimates the
uptake and assimilation of dissolved inorganic carbon (DIC) by planktonic
algae in the water column. The method is based on the assumption that
biological uptake of 14C-labelled DIC is proportional to the biological uptake
of the more commonly found 12C-DIC. In order to determine uptake, one
must know the concentration of DIC naturally occurring in the sample water,
the amount of 14C-DIC added, and the amount of 14C retained in particulate
matter (14C-POC) at the end of the incubation experiment. A 5% metabolic
discrimination factor may be applied to the data as well, since organisms
preferentially take up lighter isotopes. Carbon uptake can be measured by the
following equation:
C uptake = (naturally occurring DIC × 14C-POC × 1.05)/( 14C-DIC added)
Applications: Aquatic primary productivity generally governs the
biological activity of a lake. Since primary productivity makes up the bottom-
most trophic level, it provides essential nutrients and energy to higher trophic
levels and higher organisms. For instance, primary productivity in a lake may
supply oxygen to aerobic organisms such as insects and fish, and so, times
of stressed productivity may result in a decline in the fish population. Low
primary productivity may thus hinder other lake biota by limiting available
nutrients. Conversely, extremely high primary productivity may result in
algal blooms, which may eventually lead to mass kills at other trophic levels
due to nutrient depletion or by high turbidity from massive concentrations of
plankton. So, relatively high rates of primary productivity may support the
most diverse and largest amount of biomass, but productivity can also become
too high for a system to handle.
Environmental Microbiology—Methods and Applications 4.53

How To- Sample Collection, Protocol, Analysis, and Considerations

Protocols and Analyses: There are several protocols for measuring rates of
primary productivity via 14C. The basic idea remains the same: before dawn,
water samples are collected in a series of bottles—generally, two clear and one
amber/dark-colored bottle through which light is not able to penetrate. The
water samples are inoculated with 14C, capped, and placed in the environment
they were collected from, for a day (this time may vary). Following the
incubation, the samples are collected under low light conditions and filtered
through a 0.2 um filter. The filter is placed into a liquid scintillation vial,
acidified to purge excess 14C, and kept cold until it can be analyzed in a
scintillation counter.
The following resources provide step-by-step protocols for measuring
primary productivity using 14C.
• Limnological Methods for the McMurdo Long Term Ecological Research
Program,
• Primary Productivity.

Considerations
The 14C method for measuring productivity has several important
considerations, including:
This method assumes that 14C will be taken up proportionally to the
•
more naturally prevalent 12C species.
•
Bottles should be inoculated and collected in low-light conditions (pre-
dawn or post-sunset) in order to ensure that samples collected from
subsurface waters will not receive sunlight they would not naturally
receive.
•
Samples should be filtered as soon as possible after collection. Excess
(non-incorporated) 14C can be purged from the filter, using a small
amount of 1M HCl (this will drive off excess 14C as CO2.
•
When running a scintillation count, dissolving the filter in the
scintillation cocktail is not necessarily bad, since it ensures that all 14C
will be in solution, and thus, be counted.
Results Analysis: Rates of primary productivity will vary by environment.
Factors influencing rates of primary productivity include:
•
Availability of nutrients,
•
Availability of Photosynthetically Active Radiation (PAR), or available
light. This can be influenced by lake depth, turbidity/suspended
sediment load, and shading by macrophytes, or plants,
•
Biota dynamics in the system (e.g. population composition, number of
primary producing organisms, competition, population establishment
time vs. disturbance).
4.54 Environmental Biotechnology

Rates of primary productivity can range from 0-9000 kcal/m2/yr, with

desert regions having lower rates and estuaries having higher rates. More
constrained rates of primary productivity for specific environments may be
researched by looking at primary literature for a specific site.

Measuring Dissolved and Particulate Organic Carbon (DOC and POC)

Dissolved organic carbon (DOC) is defined as the organic matter that is able
to pass through a filter (filters generally range in size between 0.7 and 0.22
um). Conversely, particulate organic carbon (POC) is that carbon, that is too
large and is filtered out of a sample. If you have ever seen a body of water that
appears straw, tea, or brownish in color, it likely has a high organic carbon
load. This color comes from the leaching of humic substances from plant
and soil organic matter. This organic matter contributes acids to the stream,
resulting in the yellow-brown coloration as well as weathering of the soils.
Organic carbon can be allochthonous, or sourced from outside the system (e.g.
by atmospheric deposition, or transported long distances via stream flow)
or it can be autochthonous, or sourced from the immediate surroundings of
the system (e.g. plant and microbial matter and sediments/soils within the
catchment). High amounts of organic matter are common in low-oxygen
areas, such as bogs and wetlands.
• Dissolved and particulate organic carbon are important components
in the carbon cycle and serve as a primary food sources for aquatic
food webs. In addition, DOC alters aquatic ecosystem chemistries
by contributing to acidification in low-alkalinity, weakly buffered,
freshwater systems. Furthermore, DOC forms complexes with trace
metals, creating water-soluble complexes which can be transported
and taken up by organisms. Finally, organic carbon, as well as other
dissolved and particulate matter, can affect light penetration in aquatic
ecosystems, which is important for the ecosystem’s phototrophs that
need light to subsist.
• Dissolved organic carbon can be measured via several different
techniques. High temperature combustion and UV/persulfate
oxidation methods are discussed in detail below, but both methods
share the same sample preparation protocol:
• The sample is collected in a glass container that has been baked in
the laboratory at 550°C for 2-4 hours (the baking process removes
any residual carbon, in or on the collection vessel, that may cause
contamination).
• The sample is then filtered with a glass filtration device. Commonly
used filters include glass fiber filters (GF/F), silver membrane filters,
or a nitrocellulose/polypro filters and range between 0.7-0.25 mm in
pore size. The nitrocellulose/polypro filters are the least expensive of
these filters, but may leach DOC, so they should be cleaned by passing
deionized water through them, before collection.
Environmental Microbiology—Methods and Applications 4.55

•
Once collected, samples should be stored cold (e.g. in the refrigerator
or on ice) until they can be processed. They should be processes as
soon as possible to prevent post-filtering sample alteration.
• Measuring DOC By High Temperature Combustion: The high
temperature combustion method for measuring DOC involves
conversion of inorganic carbon to dissolved CO2, and purging this
from the sample. The remaining (organic) carbon is then oxidized at
a high temperature to CO2 which can be detected by the instrument’s
nondispersive infrared (NDIR) sensor and directly correlated to total
organic carbon (TOC) content.
• Measuring DOC By UV/Persulfate Oxidation: This method combines
the sample with an acid, lowering the sample pH to 2.0. This process
converts inorganic carbon to dissolved CO2, which is then purged from
the sample. A persulfate reagent is then added to the sample and the
remaining carbon is oxidized by UV radiation to form CO2, which can
be detected by the NDIR sensor and directly correlated to total organic
carbon (TOC) content.
Particulate organic carbon is measured by determining mass lost, upon
combustion of a sample. In aqueous samples, this can be done by measuring
the dry mass of a filter that had a known amount of water passed through
it before and after it is subjected to combustion via heating the filter to
550° C. This method requires that the filter is purged of extraneous POC
before filtration (by combusting it at 550°C for 2 hours), and that the filter and
sample are dry (this can be done by putting them in a warm oven) at their pre-
combustion weight measurement. The method also requires that the sample
has a measurable amount of organic carbon present. POC in soil samples can
also be measured by mass loss by measuring the dry weight of a given volume
of sample before and after combustion. These methods assume that the mass
loss is attributable solely to carbon, rather than any other sample component.
In addition to measuring DOC concentrations in a sample, DOC can be
characterized to determine its reactivity (including quality and composition),
source, and potential importance in its ecosystem. Characterization via
absorbance and fluorescence are discussed below.
Absorbance: Terrestrial and microbial-derived humic substances differ in
their carbon to nitrogen ratio (C:N ratio).
•
Terrestrially-derived humic acids have high C:N ratios because they
are derived from lignin, and lignin does not contain nitrogen. These
humics contain large amounts of carbon in the form of aromatic
carbons and phenols.
•
Microbially-derived humic substances have high N contents, relative
to terrestrial sources, along with a low aromatic carbon and phenolic
content.
4.56 Environmental Biotechnology

Utilizing these differences, UV absorbance can provide an estimate of the

aromaticity of the DOC in a sample, and thereby, determine its source.
Fluorescence: DOC can be characterized by differences in fluorescence
spectra also, which is associated with different sources of organic carbon in
aquatic systems. This method involves excitation of a sample and looking at
its 3-d excitation-emission matrix for fulvic acids. Basically, a sample is excited
and its corresponding emission intensity is used to determine its source. The
emission intensity ratio is generally higher for microbially-derived fulvic acids
than it is for terrestrially-derived acids.
Results Analysis: As stated above, DOC is an important component
in an ecosystem. It provides a primary food source for aquatic food webs,
suggesting that high DOC is beneficial to an ecosystem. However, DOC
can also contribute to the acidity of a water body and can increase light
attenuation, thus, detrimentally affecting phototrophic organisms in an
aquatic environment. Therefore, as with most things, moderation is key for
DOC content. Depending on factors such as buffering capacity, or the ability of
an aquatic system to stabilize its acidity/alkalinity, biomass composition and
amount, and water depth, DOC necessary to support an ecosystem varies by
area. Typical DOC values for various environments are commonly reported in
scientific literature such as peer-reviewed journal articles and textbooks.
 CHAPTER

5 Beneficial and Effective Microorganisms

for a Sustainable Agriculture
and Environment

The uniqueness of microorganisms and their often unpredictable nature and

biosynthetic capabilities, given a specific set of environmental and cultural
conditions, has made them likely candidates for solving particularly difficult
problems in the life sciences and other fields, as well. The various ways in
which microorganisms have been used over the past 50 years to advance
medical technology, human and animal health, food processing, food safety
and quality, genetic engineering, environmental protection, agricultural
biotechnology, and more effective treatment of agricultural and municipal
wastes provide a very impressive record of achievement. Many of these
technological advances would not have been possible using straightforward
chemical and physical engineering methods, or if they were, they would not
have been practically or economically feasible.
Nevertheless, while microbial technologies have been applied to various
agricultural and environmental problems with considerable success in recent
years, they have not been widely accepted by the scientific community
because it is often difficult to consistently reproduce their beneficial effects.
Microorganisms are effective only when they are presented with suitable and
optimum conditions for metabolizing their substrates including available
water, oxygen (depending on whether the micro-organisms are obligate
aerobes or facultative anaerobes), pH and temperature of their environment.
Meanwhile, the various types of microbial cultures and inoculants
available in the market today have increased rapidly because of these new
technologies. Significant achievements are being made in systems where
technical guidance is coordinated with the marketing of microbial products.
Since microorganisms are useful in eliminating problems associated with
the use of chemical fertilizers and pesticides, they are now widely applied
in nature farming and organic agriculture Environmental pollution, caused
by excessive soil erosion and the associated transport of sediments, chemical
fertilizers and pesticides to surface waters and groundwater, and improper
5.2 Environmental Biotechnology

treatment of human and animal wastes has caused serious environmental

and social problems throughout the world. Often, engineers have attempted
to solve these problems using established chemical and physical methods.
However, they have usually found that such problems cannot be solved
without using microbial methods and technologies in coordination with
agricultural production.
For many years, soil microbiologists and microbial ecologists have tended
to differentiate soil microorganisms as beneficial or harmful according to their
functions and how they affect soil quality, plant growth and yield, and plant
health. As shown in following Table 1, beneficial microorganisms are those that
can fix atmospheric nitrogen, decompose organic wastes and residues, detoxify
pesticides, suppress plant diseases and soil-borne pathogens, enhance nutrient
cycling, and produce bioactive compounds such as vitamins, hormones and
enzymes that stimulate plant growth. Harmful microorganisms are those
that can induce plant diseases, stimulate soil-borne pathogens, immobilize
nutrients, and produce toxic and putrescent substances that adversely affect
plant growth and health.
Some common functions of beneficial and harmful soil micro-organisms
as they affect soil quality, crop production, and plant health
Functions of Beneficial Functions of Harmful
Microorganisms Microorganisms
• Fixation of atmospheric nitrogen • Induction of plant diseases
• Decomposition of organic wastes • Stimulation of soil-borne
and residues pathogens
• Suppression of soil-borne • Immobilization of plant nutrients
pathogens
• Recycling and increased • Inhibition of seed germination
availability of plant nutrients
• Degradation of toxicants • Inhibition of plant growth and
including pesticides development
• Production of antibiotics and • Production of phytotoxic
other bioactive compounds substances
• Production of simple organic
molecules for plant uptake
• Complexation of heavy metals to
limit plant uptake
• Solubilization of insoluble
nutrient sources
• Production of polysaccharides to
improve soil aggregation
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.3

A more specific classification of beneficial microorganisms has been

suggested by Higa (1991; 1994; 1995) which he refers to as “Effective
Microorganisms” or EM. This report presents some new perspectives on the
role and application of beneficial microorganisms, including EM, as microbial
inoculants for shifting the soil microbiological equilibrium in ways that can
improve soil quality, enhance crop production and protection, conserve
natural resources, and ultimately create a more sustainable agriculture
and environment. The report also discusses strategies on how beneficial
microorganisms, including EM, can be more effective after inoculation into
soils.

5.1 THE CONCEPT OF EFFECTIVE MICROORGANISMS: THEIR

ROLE AND APPLICATION
The concept of Effective Microorganisms (EM) was developed by Professor
Teruo Higa, University of the Ryukyus, Okinawa, Japan. EM consists of mixed
cultures of beneficial and naturally-occurring microorganisms that can be
applied as inoculants to increase the microbial diversity of soils and plants.
Research has shown that the inoculation of EM cultures to the soil/plant
ecosystem can improve soil quality, soil health, and the growth, yield, and
quality of crops.
EM contains selected species of microorganisms including predominant
populations of lactic acid bacteria and yeasts, and smaller numbers of
photosynthetic bacteria, actinomycetes and other types of organisms. All of
these are mutually compatible with one another and can coexist in liquid
culture.
EM is not a substitute for other management practices. It is, however, an
added dimension for optimizing our best soil and crop management practices
such as crop rotations, use of organic amendments, conservation tillage, crop
residue recycling, and biocontrol of pests. If used properly, EM can significantly
enhance the beneficial effects of these practices.
Throughout the discussion which follows, we will use the term “beneficial
microorganisms” in a general way to designate a large group of often unknown
or ill-defined microorganisms that interact favorably in soils and with plants
to render beneficial effects which are sometimes difficult to predict. We use the
term “Effective Microorganisms” or EM to denote specific mixed cultures of
known, beneficial microorganisms that are being used effectively as microbial
inoculants.

5.2 UTILIZATION OF BENEFICIAL MICROORGANISMS IN

AGRICULTURE
What Constitutes an Ideal Agricultural System: Conceptual design is
important in developing new technologies for utilizing beneficial and Effective
5.4 Environmental Biotechnology

Microorganisms for a more sustainable agriculture and environment. The

basis of a conceptual design is simply to first conceive an ideal or model and
then to devise a strategy and method for achieving the reality. However, it
is necessary to carefully coordinate the materials, the environment, and
the technologies constituting the method. Moreover, one should adopt a
philosophical attitude in applying microbial technologies to agricultural
production and conservation systems.
There are many opinions on what an ideal agricultural system is. Many
would agree that such an idealized system should produce food on a long-
term sustainable basis. Many would also insist that it should maintain and
improve human health, be economically and spiritually beneficial to both
producers and consumers, actively preserve and protect the environment, be
self-contained and regenerative, and produce enough food for an increasing
world population.
Efficient Utilization and Recycling of Energy: Agricultural production
begins with the process of photosynthesis by green plants, which requires solar
energy, water, and carbon dioxide. It occurs through the plant’s ability to utilize
solar energy in “fixing” atmospheric carbon dioxide into carbohydrates. The
energy obtained is used for further biosynthesis in the plant including essential
amino acids and proteins. The materials used for agricultural production are
abundantly available with little initial cost. However, when it is observed as
an economic activity, the fixation of carbon dioxide by photosynthesis has
an extremely low efficiency mainly because of the low utilization rate of
solar energy by green plants. Therefore, an integrated approach is needed to
increase the level of solar energy utilization by plants so that greater amounts
of atmospheric carbon dioxide can be converted into useful substrates.
Although the potential utilization rate of solar energy by plants has been
estimated theoretically at between 10 and 20%, the actual utilization rate is
usually less than 1%. Even some C 4 plants, such as sugarcane, with very high
photosynthetic efficiencies, will seldom exceed a utilization rate of 6 or 7%
during the maximum growth period. The utilization rate is normally less than
3% even for optimum crop yields.
Past studies have shown that photosynthetic efficiency of the chloroplasts
of host crop plants cannot be increased much further; this means that their
biomass production has reached a maximum leve1. Therefore, the best
opportunity for increasing biomass production is to somehow utilize the
visible light, which chloroplasts cannot presently use, and the infrared
radiation; together, these comprise about 80% of the total solar energy. Also,
we must explore ways of recycling organic energy contained in plant and
animal residues through direct utilization of organic molecules by plants.
Thus, it is difficult to exceed the existing limits of crop production unless
the efficiency of utilizing solar energy is increased, and the energy contained
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.5

in existing organic molecules (amino acids, peptides and carbohydrates) is

utilized either directly or indirectly by the plant. This approach could help
to solve the problems of environmental pollution and degradation caused by
the misuse and excessive application of chemical fertilizers and pesticides to
soils. Therefore, new technologies that can enhance the economic-viability of
farming systems with little or no use of chemical fertilizers and pesticides are
urgently needed and should be a high priority of agricultural research, both
now, and in the immediate future.
Preservation of Natural Resources and the Environment: The excessive
erosion of topsoil from farmland caused by intensive tillage and row-crop
production has caused extensive soil degradation and also contributed to
the pollution of both surface waters and groundwater. Organic wastes from
animal production, agricultural and marine processing industries, and
municipal wastes (e.g., sewage and garbage), have become major sources
of environmental pollution in both developed and developing countries.
Furthermore, the production of methane from paddy fields and ruminant
animals and, of carbon dioxide from the burning of fossil fuels, land clearing
and organic matter decomposition have been linked to global warming as
“green house gases”.
Chemical-based, conventional systems of agricultural production have
created many sources of pollution that, either directly or indirectly, can
contribute to degradation of the environment and destruction of our natural
resource base. This situation would change significantly if these pollutants
could be utilized in agricultural production as sources of energy.
Therefore, it is necessary that future agricultural technologies be
compatible with the global ecosystem and with solutions to such problems
in areas different from those of conventional agricultural technologies. An
area that appears to hold the greatest promise for technological advances
in crop production, crop protection, and natural resource conservation is
that of beneficial and Effective Microorganisms applied as soil, plant and
environmental inoculants.

5.3 BENEFICIAL AND EFFECTIVE MICROORGANISMS FOR A

SUSTAINABLE AGRICULTURE
Integration of Essential Components for Optimum Crop and Livestock
Production: Agriculture, in a broad sense, is not an enterprise which leaves
everything to nature without intervention. Rather, it is a human activity in
which the farmer attempts to integrate certain agro-ecological factors and
production inputs for optimum crop and livestock production. Thus, it is
reasonable to assume that farmers should be interested in ways and means of
controlling beneficial soil microorganisms as an important component of the
agricultural environment. Nevertheless, this idea has often been rejected by
naturalists and proponents of nature farming and organic agriculture. They
5.6 Environmental Biotechnology

argue that beneficial soil microorganisms will increase naturally when organic
amendments are applied to soils as carbon, energy and nutrient sources. This
indeed may be true where an abundance of organic materials are readily
available for recycling which often occurs in small-scale farming. However,
in most cases, soil microorganisms, beneficial or harmful, have often been
controlled advantageously when crops in various agro ecological zones are
grown and cultivated in proper sequence (i.e., crop rotations) and without
the use of pesticides. This explains why scientists have long been interested in
the use of beneficial microorganisms as soil and plant inoculants to shift the
microbiological equilibrium in ways that would enhance soil quality and the
yield and quality of crops.
Most would agree that a basic rule of agriculture is to ensure that
specific crops are grown according to their agro climatic and agro ecological
requirements. However, in many cases the agricultural economy is based
on market forces that demand a stable supply of food, and land to its full
productive potential throughout the year.
The purpose of crop breeding is to improve crop production, crop
protection, and crop quality. Improved crop cultivars along with improved
cultural and management practices have made it possible to grow a wide
variety of agricultural and horticultural crops in areas where it once would
not have been culturally or economically feasible. The cultivation of these
crops in such diverse environments has contributed significantly to a stable
food supply in many countries. However, it is somewhat ironic that new
crop cultures are almost never selected with consideration of their nutritional
quality or bioavailability after ingestion.
To enhance the concept of controlling and utilizing beneficial
microorganisms for crop production and protection, one must harmoniously
integrate the essential components for plant growth and yield including
light (intensity, photoperiod, and quality), carbon dioxide, water, nutrients
(organic-inorganic), soil type, and the soil microflora. Because of these vital
interrelationships, it is possible to envision a new technology and a more
energy-efficient system of biological production.

5.4 BENEFICIAL MICROORGANISMS FOR SOIL QUALITY AND A

MORE SUSTAINABLE AGRICULTURE:
As will be discussed later, crop growth and development are closely related
to the nature of the soil microflora, especially those in close proximity to
plant roots, i.e., the rhizosphere. Thus, it will be difficult to overcome the
limitations of conventional agricultural technologies without controlling
soil microorganisms. This particular tenet is further reinforced because the
evolution of most forms of life on earth and their environments are sustained
by microorganisms. Most biological activities are influenced by the state of
these invisible, minuscule units of life. Therefore, to significantly increase
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.7

food production, it is essential to develop crop cultivars with improved

genetic capabilities (i.e., greater yield potential, disease resistance, and
nutritional quality) and with a higher level of environmental competitiveness,
particularly under stress conditions (i.e., low rainfall, high temperatures,
nutrient deficiencies, and aggressive weed growth).
Low agricultural production efficiency is closely related to a poor
coordination of energy conversion which, in turn, is influenced by crop
physiological factors, the environment, and other biological factors including
soil microorganisms. The soil and rhizosphere microflora can accelerate the
growth of plants and enhance their resistance to disease and harmful insects
by producing bioactive substances.
These microorganisms maintain the growth environment of plants, and
may have primary effects on both soil quality and crop quality. A wide range
of results are possible depending on their predominance and activities at
anyone time. Nevertheless, there is a growing consensus that it is possible to
attain maximum economic crop yields of high quality, at higher net returns,
without the application of chemical fertilizers and pesticides.
Until recently, this was not thought to be a very likely possibility using
conventional agricultural methods. However, it is important to recognize that
the best soil and crop management practices to achieve a more sustainable
agriculture will also enhance the growth, numbers and activities of beneficial
soil microorganisms that, in turn, can improve the growth, yield and quality
of crops. In essence, soil quality is the very foundation of a more sustainable
agriculture.

5.5 CONTROLLING THE SOIL MICROFLORA: PRINCIPLES AND

STRATEGIES
Principles of Natural Ecosystems and the Application of Beneficial and
Effective Microorganisms: The misuse and excessive use of chemical
fertilizers and pesticides have often adversely affected the environment and
created many problems associated with, a) food safety and quality and b)
human and animal health. Consequently, there has been a growing interest in
nature farming and organic agriculture by consumers and environmentalists
as possible alternatives to chemical-based, conventional agriculture.
Agricultural systems which conform to the principles of natural
ecosystems are now receiving a great deal of attention in both developed and
developing countries. A number of books and journals have recently been
published which deal with many aspects of natural farming systems. New
concepts such as alternative agriculture, sustainable agriculture, soil quality,
integrated pest management, integrated nutrient management and even
beneficial microorganisms, are being explored by the agricultural research
establishment. Although, these concepts and associated methodologies hold
5.8 Environmental Biotechnology

considerable promise, they also have limitations. For example, the main
limitation in using microbial inoculants is the problem of reproducibility and
lack of consistent results.
Unfortunately, certain microbial cultures have been promoted by their
suppliers as being effective for controlling a wide range of soil-borne plant
diseases, when in fact, they were effective only on specific pathogens under
very specific conditions. Some suppliers have suggested that their particular
microbial inoculant is akin to a pesticide that would suppress the general soil
microbial population while increasing the population of a specific beneficial
microorganism. Nevertheless, most of the claims for these single-culture
microbial inoculants are greatly exaggerated and have not proven to be
effective under field conditions. One might speculate that if all of the microbial
cultures and inoculants that are available as marketed products were applied
at the same time, some degree of success might be achieved because of the
increased diversity of the soil microflora and stability that is associated with
mixed cultures. While this, of course, is a hypothetical example, the fact
remains that there is a greater likelihood of controlling the soil microflora by
introducing mixed cultures of compatible microorganisms, rather than single,
pure cultures.
Even so, the use of mixed cultures in this approach has been criticized
because it is difficult to demonstrate conclusively which microorganisms are
responsible for the observed effects, how the introduced microorganisms
interact with the indigenous species, and how these new associations affect
the soil plant environment. Thus, the use of mixed cultures of beneficial
microorganisms as soil inoculants to enhance the growth, health, yield, and
quality of crops has not gained widespread acceptance by the agricultural
research establishment because conclusive scientific proof is often lacking.
The use of mixed cultures of beneficial microorganisms as soil inoculants
is based on the principles of natural ecosystems which are sustained by their
constituents; that is, by the quality and quantity of their inhabitants and
specific ecological parameters, i.e., the greater the diversity and number of
the inhabitants, the higher the order of their interaction and the more stable
the ecosystem. The mixed culture approach is simply an effort to apply
these principles to natural systems such as agricultural soils, and to shift the
microbiological equilibrium in favor of increased plant growth, production
and protection.
It is important to recognize that soils can vary tremendously as to their types
and numbers of microorganisms. These can be both beneficial and harmful
to plants, and often, the predominance of either one depends on the cultural
and management practices that are applied. It should also be emphasized that
most fertile and productive soils have a high content of organic matter and,
generally, have large populations of highly diverse microorganisms (i.e., both
species and genetic diversity). Such soils will also usually have a wide ratio of
beneficial to harmful microorganisms.
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.9

5.6 CONTROLLING THE SOIL MICROFLORA FOR OPTIMUM

CROP PRODUCTION AND PROTECTION
The idea of controlling and manipulating the soil microflora through the use
of inoculants, organic amendments, and cultural and management practices
to create a more favorable soil microbiological environment for optimum crop
production and protection is not new. For almost a century, microbiologists
have known that organic wastes and residues, including animal manures,
crop residues, green manures, municipal wastes (both raw and composted),
contain their own indigenous populations of microorganisms, often with
broad physiological capabilities.
It is also known that when such organic wastes and residues are applied
to soils, many of these introduced microorganisms can function as biocontrol
agents by controlling or suppressing soil-borne plant pathogens through their
competitive and antagonistic activities. While this has been the theoretical
basis for controlling the soil microflora, in actual practice, the results have
been unpredictable and inconsistent, and the role of specific microorganisms
has not been well-defined. For many years, microbiologists have tried to
culture beneficial microorganisms for use as soil inoculants to overcome the
harmful effects of phyto-pathogenic organisms, including bacteria, fungi, and
nematodes.
Such attempts have usually involved single applications of pure cultures
of microorganisms which have been largely unsuccessful for several reasons.
First, it is necessary to thoroughly understand the growth and survival
characteristics of each particular beneficial microorganism, including their
nutritional and environmental requirements. Second, we must understand
their ecological relationships and interactions with other microorganisms,
including their ability to coexist in mixed cultures, both before and after
application to soils.
There are other problems and constraints that have been major obstacles
to controlling the microflora of agricultural soils. First and foremost is the
large number of types of microorganisms that are present at any one time,
their wide range of physiological capabilities, and the dramatic fluctuations
in their populations that can result from man’s cultural and management
practices applied to a particular farming system. The diversity of the total soil
microflora depends on the nature of the soil environment and those factors
which affect the growth and activity of each individual organism including
temperature, light, aeration, nutrients, organic matter, pH and water. While
there are many microorganisms that respond favorably to these factors, or a
combination thereof, there are some that do not. Microbiologists have actually
studied relatively few of the microorganisms that exist in most agricultural
soils, mainly because we don’t know how to culture them; i.e., we know very
little about their growth, nutritional, and ecological requirements.
5.10 Environmental Biotechnology

The diversity and population factors associated with the soil microflora
have discouraged scientists from conducting research to develop control
strategies. Many believe that, even when beneficial microorganisms are
cultured and inoculated into soils, their number is relatively small compared
with the indigenous soil inhabitants, and they would likely be rapidly
overwhelmed by the established soil microflora. Consequently, many would
argue that even if the application of beneficial microorganisms is successful
under limited conditions (e.g., in the laboratory), it would be virtually
impossible to achieve the same success under actual field conditions. Such
thinking still exists today, and serves as a principal constraint to the concept of
controlling the soil microflora.
It is noteworthy that most of the microorganisms encountered in any
particular soil are harmless to plants with only a relatively few that function
as plant pathogens or potential pathogens. Harmful microorganisms become
dominant if conditions develop that are favorable to their growth, activity
and reproduction. Under such conditions, soilborne pathogens (e.g., fungal
pathogens) can rapidly increase their populations with devastating effects on
the crop. If these conditions change, the pathogen population declines just as
rapidly to its original state. Conventional farming systems that tend toward
the consecutive planting of the same crop (i.e., monoculture) necessitate
the heavy use of chemical fertilizers and pesticides. This, in turn, generally
increases the probability that harmful, disease-producing, plant pathogenic
microorganisms will become more dominant in agricultural soils.
Chemical-based conventional farming methods are not unlike symptomatic
therapy. Examples of this are, applying fertilizers when crops show symptoms
of nutrient-deficiencies, and applying pesticides whenever crops are attacked
by insects and diseases. In efforts to control the soil microflora some scientists
feel that the introduction of beneficial microorganisms should follow a
symptomatic approach. However, we do not agree. The actual soil conditions
that prevail at any point in time may be most unfavorable to the growth and
establishment of laboratory-cultured, beneficial microorganisms.
To facilitate their establishment, it may require that the farmer make
certain changes in his cultural and management practices to induce conditions
that will: (a) allow the growth and survival of the inoculated microorganisms
and (b) suppress the growth and activity of the indigenous plant pathogenic
microorganisms.
An example of the importance of controlling the soil microflora and
how certain cultural and management practices can facilitate such control is
useful here. Vegetable cultivars are often selected on their ability to grow and
produce over a wide range of temperatures. Under cool, temperate conditions
there are generally few pest and disease problems. However, with the onset of
hot weather, there is a concomitant increase in the incidence of diseases and
insects making it rather difficult to obtain acceptable yields without applying
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.11

pesticides. With higher temperatures, the total soil microbial population

increases as do certain plant pathogens such as Fusarium, which is one of the
main putrefactive, fungal pathogens in soil. The incidence and destructive
activity of this pathogen can be greatly minimized by adopting reduced tillage
methods and by shading techniques to keep the soil cool during hot weather.
Another approach is to inoculate the soil with beneficial, antagonistic,
antibiotic-producing microorganisms such as actinomycetes and certain fungi.

5.7 APPLICATION OF BENEFICIAL AND EFFECTIVE

MICROORGANISMS
A New Dimension for a Sustainable Agriculture and Environment: Many
microbiologists believe that the total number of soil microorganisms can
be increased by applying organic amendments to the soil. This is generally
true because most soil microorganisms are heterotrophic, i.e., they require
complex organic molecules of carbon and nitrogen for metabolism and
biosynthesis. Whether the regular addition of organic wastes and residues
will greatly increase the number of beneficial soil microorganisms in a short
period of time, is questionable. However, we do know that heavy applications
of organic materials, such as seaweed, fish meal, and chitin from crushed crab
shells, not only helps to balance the micronutrient content of a soil but also
increases the population of beneficial antibiotic-producing actinomycetes.
This can transform the soil into a disease-suppressive state within a relatively
short period.
The probability that a particular beneficial microorganism will become
predominant, even with organic farming or nature farming methods, will
depend on the ecosystem and environmental conditions. It can take several
hundred years for various species of higher and lower plants to interact
and develop into a definable and stable ecosystem. Even if the population
of a specific microorganism is increased through cultural and management
practices, whether it will be beneficial to plants is another question. Thus,
the likelihood of a beneficial, plant-associated microorganism becoming
predominant under conservation-based farming systems is virtually
impossible to predict. Moreover, it is very unlikely that the population of
useful anaerobic microorganisms, which usually comprise only a small part of
the soil microflora, would increase significantly even under natural farming
conditions.
This information then emphasizes the need to develop methods for
isolating and selecting different microorganisms for their beneficial effects
on soils and plants. The ultimate goal is to select microorganisms that are
physiologically and ecologically compatible with one another and that can
be introduced as mixed cultures into soil where their beneficial effects can be
realized.
Principles and Fundamental Considerations: Microorganisms are
utilized in agriculture for various purposes; as important components of
5.12 Environmental Biotechnology

organic amendments and composts, as legume inoculants for biological

nitrogen fixation, as a means of suppressing insects and plant diseases to
improve crop quality and yield, and for reduction of labor. All of these are
closely related to one another. An important consideration in the application
of beneficial microorganisms to soils is the enhancement of their synergistic
effects. This is difficult to accomplish if these microorganisms are applied
to achieve symptomatic therapy, as in the case of chemical fertilizers and
pesticides.
If cultures of beneficial microorganisms are to be effective after inoculation
into soil, it is important that their initial populations be at a certain critical
threshold level. This helps to ensure that the amount of bioactive substances
produced by them will be sufficient to achieve the desired positive effects
on crop production and/or crop protection. If these conditions are not met,
the introduced microorganisms, no matter how useful they are, will have
little, if any, effect. At present, there are no chemical tests that can predict the
probability of a particular soil inoculated microorganism to achieve a desired
result. The most reliable approach is to inoculate the beneficial microorganism
into soil as part of a mixed culture, and at a sufficiently high inoculum density
to maximize the probability of its adaptation to environmental and ecological
conditions.
The application of beneficial microorganisms to soil can help to define the
structure and establishment of natural ecosystems. The greater the diversity
of the cultivated plants that are grown and the more chemically complex
the biomass, the greater the diversity of the soil microflora as to their types,
numbers and activities. The application of a wide range of different organic
amendments to soils can also help to ensure a greater microbial diversity.
For example, combinations of various crop residues, animal manures, green
manures, and municipal wastes applied periodically to soil will provide
a higher level of microbial diversity than when only one of these materials
is applied. The reason for this is that each of these organic materials has its
own unique indigenous microflora which can greatly affect the resident soil
microflora after they are applied, at least for a limited period.

5.8 CLASSIFICATION OF SOILS BASED ON THEIR

MICROBIOLOGICAL PROPERTIES
Most soils are classified on the basis of their chemical and physical properties;
little has been done to classify soils according to their microbiological
properties. The reason for this is that a soil’s chemical and physical properties
are more readily defined and measured than their microbiological properties.
Improved soil quality is usually characterized by increased infiltration,
aeration, aggregation and organic matter content and by decreased bulk
density, compaction, erosion and crusting. While these are important indicators
of potential soil productivity, we must give more attention to soil biological
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.13

properties because of their important relationship (though poorly understood)

to crop production, plant and animal health, environmental quality, and food
safety and quality. Research is needed to identify and quantify reliable and
predictable biological/ecological indicators of soil quality. Possible indicators
might include total species diversity or genetic diversity of beneficial soil
microorganisms as well as insects and animals.
The basic concept here is not to classify soils for the study of microorganisms
but for farmers to be able to control the soil microflora so that biologically-
mediated processes can improve the growth, yield, and quality of crops as
well as the tilt, fertility, and productivity of soils. The ultimate objective is to
reduce the need for chemical fertilizers and pesticides.

5.8.1 Functions of Microorganisms: Putrefaction, Fermentation,

and Synthesis
Soil microorganisms can be classified into decomposer and synthetic
microorganisms. The decomposer microorganisms are subdivided into groups
that perform oxidative and fermentative decomposition. The fermentative
group is further divided into useful fermentation (simply called fermentation)
and harmful fermentation (called putrefaction). The synthetic microorganisms
can be subdivided into groups having the physiological abilities to fix
atmospheric nitrogen into amino acids and/or carbon dioxide into simple
organic molecules through photosynthesis. Fermentation is an anaerobic
process by which facultative microorganisms (e.g., yeasts) transform complex
organic molecules (e.g., carbohydrates) into simple organic compounds that
often can be absorbed directly by plants. Fermentation yields a relatively
small amount of energy compared with aerobic decomposition of the same
substrate by the same group of microorganisms. Aerobic decomposition
results in complete oxidation of a substrate and the release of large amounts
of energy, gas, and heat with carbon dioxide and water as the end products.
Putrefaction is the process by which facultative heterotrophic microorganisms
decompose proteins anaerobically, yielding malodorous and incompletely
oxidized metabolites (e.g., ammonia, mercaptans and indole) that are often
toxic to plants and animals.
The term “synthesis”, as used here, refers to the biosynthetic capacity of
certain microorganisms to derive metabolic energy by “fixing” atmospheric
nitrogen and/or carbon dioxide. In this context, we refer to these as “synthetic”
microorganisms, and if they should become a predominant part of the soil
microflora, then the soil would be termed a “synthetic” soil. Nitrogen-fixing
microorganisms are highly diverse, ranging from “free-living”, autotrophic
bacteria of the genus Azotobacter to symbiotic, heterotrophic bacteria of the
genus Rhizobium, and blue-green algae (now, mainly classified as blue-green
bacteria), all of which, function aerobically. Photosynthetic microorganisms
fix atmospheric carbon dioxide in a manner similar to that of green plants.
5.14 Environmental Biotechnology

They are also highly diverse, ranging from blue-green algae and green algae,
that perform complete photosynthesis aerobically to photosynthetic bacteria,
which perform incomplete photosynthesis anaerobically.

5.8.2 Relationships Between Putrefaction, Fermentation, and

Synthesis
The processes of putrefaction, fermentation, and synthesis proceed
simultaneously according to the appropriate types and numbers of
microorganisms that are present in the soil. The impact on soil quality
attributes and related soil properties is determined by the dominant process.
The production of organic substances by microorganisms results from the
intake of positive ions, while decomposition serves to release these positive
ions. Hydrogen ions play a pivotal role in these processes. A problem occurs
when hydrogen ions do not recombine with oxygen to form water, but are
utilized to produce methane, hydrogen sulfide, ammonia, mercaptans and
other highly reduced putrefactive substances, most of which are toxic to
plants and produce malodors. If a soil is able to absorb the excess hydrogen
ions during periods of soil anaerobiosis and if synthetic microorganisms such
as photosynthetic bacteria are present, they will utilize these putrefactive
substances and produce useful substrates from them which helps to maintain
a healthy and productive soil.
The photosynthetic bacteria, which perform incomplete photosynthesis
anaerobically, are highly desirable, beneficial soil microorganisms because
they are able to detoxify soils by transforming reduced, putrefactive
substances such as hydrogen sulfide into useful substrates. This helps to
ensure efficient utilization of organic matter and to improve soil fertility.
Photosynthesis involves the photo-catalyzed splitting of water which yields
molecular oxygen as a by-product. Thus, these microorganisms help to
provide a vital source of oxygen to plant roots. Reduced compounds, such as
methane and hydrogen sulfide are often produced, when organic materials are
decomposed under anaerobic conditions. These compounds are toxic and can
greatly suppress the activities of nitrogen-fixing microorganisms. However,
if synthetic microorganisms, such as photosynthetic bacteria that utilize
reduced substances, are present in the soil, oxygen deficiencies are not likely
to occur. Thus, nitro nitrogen-fixing microorganisms, coexisting in the soil
with photosynthetic bacteria, can function effectively in fixing atmospheric
nitrogen even under anaerobic conditions.
Photosynthetic bacteria not only perform photosynthesis but can also fix
nitrogen. Moreover, it has been shown that, when they coexist in soil with
species of Azotobacter, their ability to fix nitrogen is enhanced. This then is
an example of a synthetic soil. It also suggests that by recognizing the role,
function, and mutual compatibility of these two bacteria and utilizing them
effectively to their full potential, soils can be induced to a greater synthetic
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.15

capacity. Perhaps, the most effective synthetic soil system results from the
enhancement of zymogenic and synthetic microorganisms; this allows
fermentation to become dominant over putrefaction and useful synthetic
processes to proceed.

5.9 CLASSIFICATION OF SOILS BASED ON THE FUNCTIONS

OF MICROORGANISMS
As discussed earlier, soils can be characterized according to their indigenous
microflora which perform putrefactive, fermentative, synthetic and
zymogenic reactions and processes. In most soils, these functions are going on
simultaneously, with the rate and extent of each, determined by the types and
numbers of associated microorganisms, that are actively involved at any time.
A simple diagram, showing a classification of soils based on the activities and
functions of their predominant microorganisms, is presented in Figure 5.1.
Disease-Inducing Soils. In this type of soil, plant pathogenic
microorganisms such as Fusarium fungi can comprise 5 to 20 per cent of the
total microflora. If fresh organic matter with a high nitrogen content is applied
to such a soil, incompletely oxidized products can arise that are malodorous
and toxic to growing plants. Such soils tend to cause frequent infestations of
disease-causing organisms, and harmful insects. Thus, the application of fresh
organic matter to these soils is often harmful to crops. Probably more than
90 per cent of the agricultural land devoted to crop production worldwide
can be classified as having disease-inducing soils. Such soils generally have
poor physical properties, and large amounts of energy are lost as “greenhouse
gases,” particularly in the case of rice fields. Plant nutrients are also subject to
immobilization into unavailable forms.
Disease-Suppressive Soils. The microflora of disease-suppressive
soils is usually dominated by antagonistic microorganisms that produce
copious amounts of antibiotics. These include fungi of the genera Penicillium,
Trichoderma, and Aspergillus, and actinomycetes of the genus Streptomyces. The
antibiotics they produce can have biostatic and biocidal effects on soil-borne
plant pathogens, including Fusarium which would have an incidence in these
soils of less than 5 per cent. Crops planted in these soils are rarely affected
by diseases or insect pests. Even if fresh organic matter with a high nitrogen
content is applied, the production of putrescent substances is very low and the
soil has a pleasant earthy odor after the organic matter is decomposed. These
soils generally have excellent physical properties; for example, they readily
form water-stable aggregates and they are well-aerated, and have a high
permeability to both air and water. Crop yields in the disease-suppressive
soils are often slightly lower than those in synthetic soils. Highly acceptable
crop yields are obtained whenever a soil has a predominance of both disease-
suppressive and synthetic microorganisms.
5.16 Environmental Biotechnology

Zymogenic Soils. These soils are dominated by a microflora that can

perform useful kinds of fermentations, i.e., the breakdown of complex organic
molecules into simple organic substances and inorganic materials. The
organisms can be either obligate or facultative anaerobes. Such fermentation-
producing microorganisms often comprise the microflora of various organic
materials, i.e., crop residues, animal manures, green manures and municipal
wastes including composts. After these amendments are applied to the soil,
their numbers and fermentative activities can increase dramatically and
overwhelm the indigenous soil microflora for an indefinite period. While these
microorganisms remain predominant, the soil can be classified as a zymogenic
soil which is generally characterized by: a) pleasant, fermentative odors,
especially, after tillage, b) favorable soil physica1 properties (e.g., increased
aggregate stability, permeability, aeration and decreased resistance to tillage),
c) large amounts of inorganic nutrients, amino acids, carbohydrates, vitamins
and other bioactive substances which can directly or indirectly enhance the
growth, yield and quality of crops, d) low occupancy of Fusarium fungi which
is usually less than 5 per cent, and e) low production of greenhouse gases (e.g.,
methane, ammonia, and carbon dioxide) from croplands, even where flooded
rice is grown.
Synthetic Soils. These soils contain significant populations of
microorganisms which are able to fix atmospheric nitrogen and carbon dioxide
into complex molecules such as amino acids, proteins and carbohydrates. Such
microorganisms include photosynthetic bacteria which perform incomplete
photosynthesis anaerobically, certain Phycomycetes (fungi, that resemble
algae), and both green algae and blue-green algae which function aerobically.
All of these are photosynthetic organisms that fix atmospheric nitrogen. If the
water content of these soils is stable, their fertility can be largely maintained
by regular additions of only small amounts of organic materials. These soils
have a low Fusarium occupancy, and they are often of the disease-suppressive
type. The production of gases from fields where synthetic soils are present is
minimal, even for flooded rice. This is a somewhat simplistic classification of
soils based on the functions of their predominant types of microorganisms,
and whether they are potentially beneficial or harmful to the growth and yield
of crops. While these different types of soils are described here in a rather
idealized manner, the fact is that, in nature, they are not always clearly defined
because they often tend to have some of the same characteristics. Nevertheless,
research has shown that a disease-inducing soil can be transformed into
disease-suppressive, zymogenic and synthetic soils by inoculating the
problem soil with mixed cultures of Effective Microorganisms (EM). Thus, it
is somewhat obvious that the most desirable agricultural soil for optimum
growth, production, protection, and quality of crops would be the composite
soil indicated in Figure 2, i.e., a soil that is highly zymogenic and synthetic,
and has an established disease-suppressive capacity. This, then is the principal
reason for seeking ways and means of controlling the microflora of agricultural
soils.
Beneficial and Effective Microorganisms for a Sustainable Agriculture 5.17

Classification of soils based on the activities and

functions of their predominant microorganisms
Disease-inducing soils can be transformed into diesease-suppressing, zymogenic
and synthetic soils that are more conductive growth and health of plants by
introducing beneficial microorganisms as microbial inoculate and following best
management practices. The ideal soil for a more sustainable agriculture is a
composite of the other three soil types and contains associative groups of beneficial
microorganisms to enhance the optimum growth, yield and quality of crops.
Controlling the soil microflora to enhance the predominance of beneficial
and Effective Microorganisms can help to improve and maintain the soil
chemical and physical properties. The proper and regular addition of organic
amendments are often an important part of any strategy to exercise such
control.
Previous efforts to significantly change the indigenous microflora of a soil
by introducing single cultures of extrinsic microorganisms have largely been
unsuccessful. Even when a beneficial microorganism is isolated from a soil,
cultured in the laboratory, and reinoculated into the same soil at a very high
population, it is immediately subject to competitive and antagonistic effects
from the indigenous soil microflora, and its numbers, soon decline. Thus,
the probability of shifting the “microbiological equilibrium” of a soil and
controlling it to favor the growth, yield and health of crops is much greater
if mixed cultures of beneficial and Effective Microorganisms are introduced
that are physiologically and ecologically compatible with one another. When
these mixed cultures become established, their individual beneficial effects
are often magnified in a synergistic manner. Actually, a disease-suppressive
microflora can be developed rather easily by selecting and culturing certain
types of gram-positive bacteria that produce antibiotics and have a wide range
of specific functions and capabilities; these organisms include facultative
anaerobes, obligate aerobes, acidophilic and alkalophilic microbes. These
microorganisms can be grown to high populations in a medium consisting of
5.18 Environmental Biotechnology

rice bran, oil cake and fish meal and then applied to soil along with well-cured
compost that also has a large stable population of beneficial microorganisms,
especially facultative anaerobic bacteria. A soil can be readily transformed into
a zymogenic/synthetic soil with disease-suppressive potential if mixed cultures
of Effective Microorganisms with the ability to transmit these properties are
applied to that soil.
The desired effects from applying cultured beneficial and Effective
Microorganisms to soils can be somewhat variable, at least, initially. In some
soils, a single application (i.e., inoculation) may be enough to produce the
expected results, while for other soils, even repeated applications may appear
to be ineffective. The reason for this is that, in some soils, it takes longer
for the introduced microorganisms to adapt to a new set of ecological and
environmental conditions and to become well-established as a stable, effective
and predominant part of the indigenous soil microflora. The important
consideration here is the careful selection of a mixed culture of compatible,
Effective Microorganisms, properly cultured, and provided with acceptable
organic substrates. Assuming that repeated applications are made at regular
intervals during the first cropping season, there is a very high probability that
the desired results will be achieved. There are no meaningful or reliable tests
for monitoring the establishment of mixed cultures of beneficial and Effective
Microorganisms after application to a soil. The desired effects appear only
after they are established and become dominant, and remain stable and active
in the soil. The inoculum densities of the mixed cultures and the frequency
of application serve only as guidelines to enhance the probability of early
establishment. Repeated applications, especially during the first cropping
season, can markedly facilitate early establishment of the introduced Effective
Microorganisms. Once the “new” microflora is established and stabilized,
the desired effects will continue indefinitely and no further applications
are necessary unless organic amendments cease to be applied, or the soil is
subjected to severe drought or flooding.
Finally, it is far more likely that the microflora of a soil can be controlled
through the application of mixed cultures of selected beneficial and Effective
Microorganisms than by the use of single or pure cultures. If the microorganisms
comprising the mixed culture can coexist and are physiologically compatible
and mutually complementary, and if the initial inoculum density is sufficiently
high, there is a high probability that these microorganisms will become
established in the soil and will be effective as an associative group, whereby
such positive interactions would continue. If so, then it is also highly probable
that they will exercise considerable control over the indigenous soil microflora
which, in due course, would likely be transformed into or replaced by a “new”
soil microflora.
 CHAPTER

6 Phytoremediation

Phytoremediation combines the Greek word “phyton” (plant), with the Latin
word “remediare” (to remedy) to describe a system whereby certain plants,
working together with soil organisms, can transform contaminants into
harmless and often, valuable forms. This practice is increasingly used to
remediate sites contaminated with heavy metals and toxic organic compounds.
Phytoremediation can be defined as “the efficient use of plants to remove,
detoxify or immobilize environmental contaminants in a growth matrix (soil,
water or sediments) through the natural, biological, chemical or physical
activities and processes of the plants”.
Plants are unique organisms equipped with remarkable metabolic
and absorption capabilities, as well as transport systems that can take up
nutrients or contaminants selectively from the growth matrix, soil or water.
Phytoremediation involves growing plants in a contaminated matrix, for a
required growth period, to remove contaminants from the matrix, or facilitate
immobilization (binding/containment) or degradation (detoxification) of the
pollutants. The plants can be subsequently harvested, processed and disposed.
Plants evolved a great diversity of genetic adaptations to handle the
accumulated pollutants that occur in the environment. Growing and, in some
cases, harvesting plants on a contaminated site as a remediation method is
a passive technique that can be used to clean up sites with shallow, low to
moderate levels of contamination. Phytoremediation can be used to clean up
metals, pesticides, solvents, explosives, crude oil, polyaromatic hydrocarbons,
and landfill leachates. It can also be used for river basin management
through the hydraulic control of contaminants. Phytoremediation has
been studied extensively in research and small-scale demonstrations, but
full-scale applications are currently limited to a small number of projects.
Further research and development will lead to wider acceptance and use of
phytoremediation.
6.2 Environmental Biotechnology

6.1 PRINCIPAL MECHANISM OF PHYTOREMEDIATION

There are several ways in which plants are used to clean up, or remediate,
contaminated sites. To remove pollutants from soil, sediment and/or water,
plants can break down, or degrade, organic pollutants or contain and stabilize
metal contaminants by acting as filters or traps. The uptake of contaminants
in plants occurs primarily through the root system, in which the principal
mechanisms for preventing contaminant toxicity are found. The root system
provides an enormous surface area that absorbs and accumulates the water
and nutrients essential for growth, as well as other non-essential contaminants.
Researchers are finding that the use of trees (rather than smaller plants) is
effective in treating deeper contamination because tree roots penetrate more
deeply into the ground. In addition, deep-lying contaminated ground water
can be treated by pumping the water out of the ground and using plants to
treat the contamination.
Plant roots also cause changes at the soil-root interface as they release
inorganic and organic compounds (root exudates) in the rhizosphere.
These root exudates affect the number and activity of the microorganisms,
the aggregation and stability of the soil particles around the root, and the
availability of the contaminants. Root exudates, by themselves, can increase
(mobilize) or decrease (immobilize) directly or indirectly the availability
of the contaminants in the root zone (rhizosphere) of the plant through
changes in soil characteristics, release of organic substances, changes in
chemical composition, and/or increase in plant-assisted microbial activity.
Phytoremediation is an alternative or complimentary technology that can be
used along with or, in some cases, in place of mechanical conventional clean-
up technologies that often require high capital inputs and are labour and
energy-intensive. Phytoremediation is an in situ, remediation technology that
utilizes the inherent abilities of living plants. It is also an ecologically friendly,
solar energy-driven clean-up technology, based on the concept of—using
nature to cleanse nature.

6.2 PHYTOREMEDIATION PROCESSES

Depending on the underlying processes, applicability, and type of contaminant,
phytoremediation can be broadly categorised as:
Phytoremediation includes the following processes and
mechanisms of contaminant removal
S.No. Process Mechanism Contaminant
1 Rhizofiltration Rhizosphere Organics/
accumulation Inorganics
2 Phytostabilization Complexation Inorganics
3 Phytoextraction Hyper-accumulation Inorganics
Phytoremediation 6.3

4 Phytovolatilization Volatilization by leaves Organics/

Inorganics
5 Phytotransformation Degradation in plants Organics

6.2.1  Rhizofiltration
Rhizofiltration is similar in concept to Phytoextraction but is concerned with
the remediation of contaminated groundwater rather than the remediation
of polluted soils. The contaminants are either adsorbed onto the root surface
or are absorbed by the plant roots. Plants used for rhizofiltration are not
planted directly in situ, but are acclimated to the pollutant first. Plants are
hydroponically grown in clean water rather than soil, until a large root
system has developed. Once a large root system is in place, the water supply
is substituted for a polluted water supply to acclimatize the plant. After the
plants become acclimatized, they are planted in the polluted area, where the
roots uptake the polluted water and the contaminants along with it. As the
roots become saturated, they are harvested and disposed of safely. Repeated
treatments of the site can reduce pollution to suitable levels as was exemplified
in Chernobyl where sunflowers were grown in radioactively contaminated
pools.

6.2.2  Phytostabilization
Phytostabilization is the use of certain plants to immobilise soil and water
contaminants. Contaminant are absorbed and accumulated by roots, adsorbed
onto the roots, or precipitated in the rhizosphere. This reduces or even prevents
the mobility of the contaminants preventing migration into the groundwater
or air, and also reduces the bioavailability of the contaminant, thus preventing
spread through the food chain. This technique can also be used to re-establish
a plant community on sites that have been denuded due to the high levels
of metal contamination. Once a community of tolerant species has been
established the potential for wind erosion (and thus, spread of the pollutant)
is reduced and leaching of the soil contaminants is also reduced.

6.2.3  Phytoextraction
Phytoextraction (or Phytoaccumulation) uses plants or algae to remove
contaminants from soils, sediments or water into harvestable plant biomass
(organisms that take larger-than-normal amounts of contaminants from the
soil are called hyperaccumulators). Phytoextraction has been growing rapidly
in popularity worldwide for the last twenty years or so. In general, this process
has been tried more often for extracting heavy metals than for organics. At the
time of disposal, contaminants are typically concentrated in the much smaller
volume of the plant matter than in the initially contaminated soil or sediment.
‘Mining with plants’, or phytomining, is also being experimented with. The
main advantage of phytoextraction is environmental friendliness. Traditional
6.4 Environmental Biotechnology

methods that are used for cleaning up heavy metal-contaminated soil disrupt
soil structure and reduce soil productivity, whereas phytoextraction can clean
up the soil without causing any kind of harm to soil quality. Another benefit
of phytoextraction is that it is less expensive than any other clean-up process.
Disadvantages of this process is that this process is controlled by plants, it
takes more time than anthropogenic soil clean-up methods.
There are two versions of phytoextraction:
• natural hyper-accumulation, where plants naturally take up the
contaminants in soil unassisted, and
• induced or assisted hyper-accumulation, in which a conditioning fluid
containing a chelator or another agent is added to soil to increase metal
solubility or mobilization so that the plants can absorb them more
easily. In many cases, natural hyperaccumulators are metallophyte
plants that can tolerate and incorporate high levels of toxic metals.

Enzymes in plant roots break (degrade) organic contaminant.

The fragments are incorporate into new plant material.

6.2.4  Phytovolatilisation
Phytovolatilization is the process where plants uptake contaminants which
are water soluble and release them into the atmosphere as they transpire the
water. The contaminant may become modified along the way, as the water
travels along the plant’s vascular system from the roots to the leaves, whereby
the contaminants evaporate or volatilize into the air surrounding the plant.
There are varying degrees of success with plants as phytovolatilizers, with
one study showing poplar trees to volatilize up to 90% of the TCE they absorb.

6.2.5  Phytotransformation or Phytostimulation or Rhizodegradation

Rhizodegradation (also called enhanced rhizosphere biodegradation,
phytostimulation, and plant-assisted bioremediation) is the breakdown of
organic contaminants in the soil by soil-dwelling microbes which is enhanced
by the rhizosphere’s presence. Certain soil-dwelling microbes digest organic
pollutants such as fuels and solvents, producing harmless products through
Phytoremediation 6.5

a process known as Bioremediation. Plant root exudates such as sugars,

alcohols, and organic acids act as carbohydrate sources for the soil microflora
and enhance microbial growth and activity. Some of these compound may
also act as chemotactic signals for certain microbes. The plant roots also loosen
the soil and transport water to the rhizosphere, thus, additionally enhancing
microbial activity.

Enzymes in plant roots break down (degrade) organic contaminants.

The fragments are incorporated into new plant material.

6.3 PHYTOREMEDIAITON OF ORGANIC POLLUTANTS

To avoid the toxicity associated with hazardous chemicals that are present
in the environment, researchers have developed strategies that employ
plants to degrade, remove or stabilize a range of different compounds from
polluted soils. These environmental pollutants may include metals such
as lead, zinc, cadmium, selenium, chromium, cobalt, copper, nickel and
mercury; inorganic compounds such as arsenic, sodium, nitrate, ammonia
and phosphate; radioactive compounds like uranium, cesium and strontium;
or organic compounds, including chlorinated solvents like trichloroethylene
(TCE), explosives such as trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-
hexahydrotriazine (RDX), petroleum hydrocarbons such as benzene, toluene
and xylene (BTX), polycyclic aromatic hydrocarbons (PAHs), and pesticides
such as atrazine and bentazon.
While some organic compounds can be metabolized (remediated) by soil
bacteria in the absence of plants, this process is often slow and inefficient. This
notwithstanding, the field of bacterial bioremediation has been expanding.
Contaminant-degrading bacteria have been isolated from a wide range of
impacted soils. In addition, it has been suggested that these contaminant-
degrading bacteria may be found in virtually all soils. Following isolation and
characterization of contaminant-degrading bacteria, attempts have been made
to inoculate contaminated field soils with the isolates; however, as indicated
above, this strategy has generally proven to be unsuccessful. This lack of
success may be attributed to:
(i) the inability of introduced bacterial isolates to compete with existing
microflora and microfauna in the soil;
6.6 Environmental Biotechnology

(ii) the inability of the bacteria to reach sub-surface contaminants;

(iii) the lack of sufficient nutrients in contaminated soils to support bacterial
growth;
(iv) the low bioavailability of many contaminants;
(v) the preferential utilization by the degradative bacteria of carbon
compounds other than the contaminant of interest; and
(vi) the presence of other toxicants within the soil that may inhibit bacterial
growth.
However, in the area around plant roots (the rhizosphere), some organic
soil contaminants can be completely degraded and mineralized by plant
enzymes through the process of phytodegradation. This process occurs
because many plants produce, and secrete to the environment, enzymes that
can degrade a wide range of organic compounds. Phytoremediation of organic
compounds may occur by phytostabilization (stabilizing pollutants in the soil
to make them less bioavailable and therefore less hazardous); phytostimulation
(the stimulation of microbial biodegradation in the rhizosphere—sometimes
called rhizodegradation); or by phytotransformation—the absorption and
degradation of organic contaminants by the plant.
The biodegradation of recalcitrant organic compounds in the soil is
often enhanced around the roots of plants. This is a direct consequence of
the high level of nutrients (including sugars, amino acids and organic acids)
that most plants release (exude) into the soil as root exudates, nutrients that
typically support a bacterial concentration in the rhizosphere that is often
100- to 1000-fold greater than the bacterial concentration in the bulk soil.
Some rhizosphere bacteria are directly involved in the degradation of the
organic soil contaminants, while others (plant growth-promoting bacteria)
can positively affect plant growth and health, enhancing root development
or increasing plant tolerance to various environmental stresses. As a direct
consequence of their interaction with plant growth-promoting bacteria, plants
grow larger and healthier, and are better able to phytoremediate a range of
organic soil contaminants.
Unfortunately, inorganic environmental pollutants cannot readily be
degraded. They must either be stabilized in the soil to make them less
bioavailable and thereby reduce their spread in the environment; extracted,
transported, accumulated and concentrated from the soil into plant roots and/
or shoots (phytoextraction); removed from liquid effluents via the use of plant
roots (rhizofiltration); or transformed into volatile forms (phytovolatilization).
Following phytoextraction, plants may be harvested, dried and converted to
ash to recover the concentrated metal. A serious impediment to more effective
phytoextraction of metals is the tight binding of metals to soil particles so
that often only a small fraction of the metal that is present in the soil can be
mobilized and taken up by plant roots.
Phytoremediation 6.7

As a result of the testing of numerous plants, several, that are naturally

able to accumulate large amounts of metal per unit of plant biomass, have
been identified and are being studied for possible use in the phytoremediation
of metallic contaminants. These plants are called hyperaccumulators and are
often found growing in soils with elevated metal concentrations. A practical
limitation of using hyperaccumulators is that many of the plants that are
most effective at removing metals from the soil, such as Thlaspi caerulescens
(Alpine pennycress) and Alyssum bertolonii, are small, containing only a low
level of biomass, and they are slow-growing, thus reducing their potential
for metal phytoextraction from soil (on a large scale) in the field. Moreover,
the growth of metal-resistant, metal-accumulating plants, that are capable of
hyperaccumulating metals, can be severely inhibited when the concentration
of available metal in the contaminated soil is very high. This results in a
decrease in plant biomass and, thereby, in the efficiency of phytoremediation.
To be effective for the remediation of metal polluted soils, plants must
be tolerant to one or more metals, highly competitive, fast-growing, and
produce a high aboveground biomass. Because of their high biomass and
extensive root system, some species of trees (e.g. poplar) have been considered
to be attractive for phytoremediation; however, metal accumulation by
trees is generally low. Finally, a convergence of phytoremediation and
bacterial bioremediation strategies has led to a more successful approach
to remediation of contaminants, particularly, organic compounds. Bacteria-
assisted phytoremediation, both with bacteria already present in the soil
and with bacteria deliberately introduced by seed inoculation, has been
investigated in a number of laboratory, greenhouse and field studies. In this
regard, phytoremediation is most effective when the introduced bacteria can
both degrade the soil contaminant(s) and promote the growth of plants.
Given the above mentioned considerations, it is currently possible to
develop phytoremediation strategies to clean up a large number of the sites
contaminated with organic compounds (this process may require several field
seasons depending on the particular plant, soil, bacteria, contaminants and
climate involved). On the other hand, phytoremediation is not yet a practical
approach for the removal of inorganic compounds from contaminated soil
environments.

6.4 PLANTS’ RESPONSE TO HEAVY METALS

Heavy metals are elements having atomic weight between 63.54 and 200.59,
and a specific gravity greater than 6. Trace amount of some heavy metals are
required by living organisms, however, any excess amount of these metals
can be detrimental to the organisms. Non-essential heavy metals include
arsenic, antimony, cadmium, chromium, mercury, lead, etc; these metals are
of particular concern to surface water and soil pollution. Heavy metals exist
in colloidal, ionic, particulate and dissolved phase. Metals also have a high
6.8 Environmental Biotechnology

affinity for humic acids, organic clays, and oxides coated with organic matter.
The soluble forms are generally ions or unionized organometallic chelates or
complexes. The solubility of metals in soil and groundwater is predominantly
controlled by pH amount of metal, cation exchange capacity, organic carbon
content, the oxidation state of the mineral components, and the redox potential
of the system. In general, soil pH seems to have the greatest effect of any single
factor on the solubility or retention of metals in soils with a greater retention
and lower solubility of metal cations occurring at high soil pH .
Under the neutral to basic conditions, typical of most soils, cationic metals
are strongly adsorbed on the clay fractions and can be adsorbed by hydrous
oxides of iron, aluminium, or manganese present in soil minerals. Elevated
salt concentration creates increased competition between cations and metals
for binding sites. Also competitive adsorption between various metals has
been observed in experiments involving various solids with oxide surfaces;
in several experiments, Cd adsorption was decreased by the addition of Pb or
Cu.
Plants have three basic strategies for growth on metal-contaminated soil.

6.4.1  Metal excluders

They prevent metal from entering their aerial parts or maintain low and
constant metal concentration over a broad range of metal concentration in soil,
they mainly restrict metal in their roots. The plant may alter its membrane
permeability, change metal-binding capacity of cell walls or exude more
chelating substances.

6.4.2  Metal indicators

Species which actively accumulate metal in their aerial tissues and generally
reflect metal level in the soil. They tolerate the existing concentration level of
metals by producing intracellular metal binding compounds (chelators), or
alter metal compartmentalisation pattern by storing metals in non-sensitive
parts.

6.4.3  Metal accumulator plant species

They can concentrate metal in their aerial parts, to levels, far exceeding than
soil. Hyperaccumulators are plants that can absorb high levels of contaminants
concentrated either in their roots, shoots and/or leaves. Baker and Brooks
have defined metal hyperaccumulator as plants that contain more than, or
up to, 0.1% i.e. more than (1000 mg/g) of copper, cadmium, chromium, lead,
nickel cobalt or 1% (>10,000 mg/g ) of zinc or manganese in the dry matter. For
cadmium and other rare metals, it is > 0.01% by dry weight.
Researchers have identified hyperaccumulator species by collecting
plants from the areas where soil contains greater than usual amount of
metals as in case of polluted areas or, geographically rich in a particular
element. Approximately, 400 hyper accumulator species from 22 families
Phytoremediation 6.9

have been identified. The Brassicaceae family contains a large number of

hyperaccumulating species with widest range of metals, these include 87
species from 11 genera.

Conceptual response strategies of metal concentrations in plant tops in

relation to increasing total metal concentrations in the soil.

6.5 HYDRAULIC CONTROL OF POLLUTANTS

Hydraulic control is the term given to the use of plants to control the migration
of subsurface water through the rapid uptake of large volumes of water
by the plants. The plants are effectively acting as natural hydraulic pumps
which when a dense root network has been established near the water table
can transpire up to 300 gallons of water per day. This fact has been utilized
to decrease the migration of contaminants from surface water into the
groundwater (below the water table) and drinking water supplies. There are
two such uses for plants:

6.5.1  Riparian corridors

Riparian corridors and buffer strips are the applications of many aspects of
phytoremediation along the banks of a river or the edges of groundwater
plumes. Phytodegradation, phytovolatilization, and rhizodegradation are
used to control the spread of contaminants and to remediate polluted sites.
Riparian strips refer to these uses along the banks of rivers and streams,
whereas buffer strips are the use of such applications along the perimeter of
landfills.

6.5.2  Vegetative cover

Vegetative cover is the name given to the use of plants as a cover or cap
growing over landfill sites. The standard caps for such sites are usually plastic
or clay. Plants used in this manner are not only more aesthically pleasing they
may also help to control erosion, leaching of contaminants, and may also help
to degrade the underlying landfill.
6.10 Environmental Biotechnology

6.6 ADVANTAGES AND DISADVANTAGES OF PHYTOREMEDIATION

As with most new technologies phytoremediation has many pros and
cons. When compared to other more traditional methods of environmental
remediation, it becomes clearer what the detailed advantages and
disadvantages actually are.
Advantages of phytoremediation compared to classical remediation
• It is more economically viable, using the same tools and supplies as
agriculture.
• It is less disruptive to the environment and does not involve waiting for
new plant communities to recolonize the site.
• Disposal sites are not needed.
• It is more likely to be accepted by the public as, it is more aesthetically
pleasing then traditional methods.
• It avoids excavation and transport of polluted media, thus reducing the
risk of spreading the contamination.
• It has the potential to treat sites polluted with more than one type of
pollutant.
Disadvantages of phytoremediation compared to classical remediation:
• It is dependent on the growing conditions required by the plant (i.e.,
climate, geology, altitude, temperature).
• Large scale operations require access to agricultural equipment and
knowledge.
• Success is dependant on the tolerance of the plant to the pollutant.
• Contaminants collected in senescing tissues may be released back into
the environment in autumn.
• Contaminants may be collected in woody tissues used as fuel.
• Time taken to remediate sites far exceeds that of other technologies.
• Contaminant solubility may be increased leading to greater
environmental damage and the possibility of leaching.
The low cost of phytoremediation (up to 1000 times cheaper than
excavation and reburial) is the main advantage of phytoremediation; however
many of the pros and cons of phytoremediation applications depend greatly
on the location of the polluted site, the contaminants in question, and the
application of phytoremediation.

6.7 PHYTOREMEDIATION & BIOTECHNOLOGY

The first goal in phytoremediation is to find a plant species which is resistant to
or, tolerates a particular contaminant with a view to maximizing it’s potential
for phytoremediation. Resistant plants are usually located growing on soils
Phytoremediation 6.11

with underlying metal ores or, on the boundary of polluted sites. Once a
tolerant plant species has been selected, traditional breeding methods are used
to optimize the tolerance of a species to a particular contaminant. Agricultural
methods such as the application of fertilizers, chelators, and pH adjusters can
be utilized to further improve the potential for phytoremediation.
Genetic modification offers a new hope for phytoremediation as GM
approaches can be used to overexpress the enzymes involved in the existing
plant metabolic pathways or to introduce new pathways into plants. Richard
Meagher and colleagues introduced a new pathway into Arabidopsis to
detoxify methylmercury, a common form of environmental pollutant, to
elemental mercury, which can be volatilized by the plant.
• The genes originated in gram-negative bacteria.
• MerB encodes a protein, organomercurial lyase converts methylmercury
to ionic mercury.
• MerA encodes mercuric reductase, which reduces ionic mercury to the
elemental form.
• Arabidopsis plants were transformed with either MerA or MerB coupled
with a constitutive 35S promoter.
• The MerA plants were more tolerant to ionic mercury, volatilized
elemental mercury, and were unaffected in their tolerance of
methylmercury.
• The MerB Plants were significantly more tolerant to methylmercury
and other organomercurials and could also convert methylmercury to
ionic mercury, which is approximately 100 times less toxic to plants.
• MerA MerB double transgenics were produced in an F2 generation.
These plants not only showed a greater resistance to organic mercury
when compared to the MerA, MerB, and wildtype plants but also were
capable of volatilizing mercury when supplied with methylmercury.
• The same MerA/MerB inserts have been used in other plant species
including tobacco (Nicotiana tabacum), yellow poplar (Liriodendron
tulipifera).
• Wetland species (bulrush and cat-tail) and water tolerant trees (willow
and poplar) have also been targeted for transformation.

6.7.1 Risk Assessment

The use of phytoremediation in the field is subject to many environmental
concerns, especially, in the light of the recent public hysteria about the release
of GM crops into the environment. Even if non GM strains of plants are used,
there are still many concerns:
• It is unknown what ecological effects hyperaccumulator plants may
have if ingested by animals.
6.12 Environmental Biotechnology

• Fallout from senescing tissues in autumn may also re-enter the food
chain.
• Do volatilized contaminants remain at ‘safe’ levels in the atmosphere?
• Exposure of the ecosystem to contaminants is prolonged as
phytoremediation is a relatively slow process.
• However there are other issues that affect the risk assessment for the
use of transgenic organisms as phytoremediators. Not only do such
organisms have the same risks as wild type remediates, but they also
have the same risks as releasing any GM organism into the field have:
• The potential genetic pollution of native species.
• Potential for the gene to recombine with other genes possibly leading
to the hyperaccumulation of non-contaminant compounds.
• Reporter/marker genes may also escape into the environment.
• The GM plants may revert to a wild type genotype.

6.7.2 Future of Phytoremediation

One of the key aspects to the acceptance of phytoextraction pertains to the
measurement of its performance, ultimate utilization of by-products and its
overall economic viability. To date, commercial phytoextraction has been
constrained by the expectation that site remediation should be achieved
in a time comparable to other clean-up technologies. So far, most of the
phytoremediation experiments have taken place in the lab scale, where plants
grown in hydroponic setting are fed heavy metal diets. While these results are
promising, scientists are ready to admit that solution culture is quite different
from that of soil. In real soil, many metals are tied up in insoluble forms, and
they are less available and that is the biggest problem.
The future of phytoremediation is still in research and development
phase, and there are many technical barriers which need to be addressed.
Both agronomic management practices and plant genetic abilities need to be
optimized to develop commercially useful practices. Many hyperaccumulator
plants remain to be discovered, and there is a need to know more about their
physiology. Optimization of the process, proper understanding of plant heavy
metal uptake and proper disposal of biomass produced is still needed.
Phytoremediation is a fast developing field; since last ten years; lot of field
application were initiated all over the world—it includes Phytoremediation
of Organic, Inorganic and Radionuclides. This sustainable and inexpensive
process is fast emerging as a viable alternative to conventional remediation
methods, and will be most suitable for a developing country like India. Most of
the studies have been done in developed countries and knowledge of suitable
plants is particularly limited in India. In India, commercial application of
Phytoremediation of soil Heavy metal or Organic compounds is in its earliest
phase. Fast growing plants with high biomass and good metal uptake ability
Phytoremediation 6.13

are needed. In most of the contaminated sites, hardy and tolerant weed
species exist and phytoremediation through these and other non-edible
species can restrict the contaminant from being introduced into the food
web. However, several methods of plant disposal have been described, but
data regarding these methods are scarce. Composting and compaction can be
treated as pre-treatment steps for volume reduction, but care should be taken
to collect leachate resulting from compaction. Between the two methods that
significantly reduce the contaminated biomass, incineration seems to be least
time-consuming and environmentally sound than direct burning or ashing.
 CHAPTER

7 Solid Waste Disposal and Management

Waste is unwanted or undesired material left over after the completion of

a process. In other words, it can also be stated that any substance or object,
which the holder discards or intend to discard, is also waste. Waste is a
continually growing problem at global and regional as well as at local levels.
A waste product is regarded as a pollutant when it damages the environment.
Often wastes and pollutants are intricately linked. In simple words, pollutants
are generally wastes but all wastes are not pollutants.
Wastes may be:
• Biological,
• Chemical, or
• Physical in nature, and may originate from the following activities:
— Manufacturing
— Agriculture and dairy
— Energy production
— Transport and
— House building and house keeping.
Basically, the waste can be categorised into (according to their Properties):
• Biodegradable Waste and
• Non-Biodegradable Waste.
— Biodegradable waste is a type of waste, typically originating from
Plant or Animal sources, which may be broken down by other
living organisms. With the proper treatment, Biodegradable waste
can be used for composting, animal feed, or converted into energy.
Biodegradable waste accounts for approximately 60% of municipal
waste; the most common types of biodegradable waste are food
waste, garden waste, paper and cardboard waste, and biodegradable
plastics.
7.2 Environmental Biotechnology

— Waste that cannot be broken down by other living organisms may

be called non-biodegradable. Examples are plastics, metal and glass.
Some dangerous chemicals and toxins are also non-biodegradable,
as are plastic grocery bags, Styrofoam (polystyrene), and other
similar materials, but will eventually break down over time.
Waste is broadly segregated into solid, liquid and gaseous waste
materials. Solid wastes arise from human and animal activities that are
normally discarded as useless or unwanted. In other words, solid wastes may
be defined as the organic and inorganic waste materials produced by various
activities of the society and which have lost their value to the first user. As the
result of rapid increase in production and consumption, urban society rejects
and generates solid material regularly which leads to considerable increase in
the volume of waste generated from several sources such as, domestic wastes,
commercial wastes, institutional wastes and industrial wastes of most diverse
categories.

7.1 TYPES OF SOLID WASTE

Solid waste can be classified into different types depending on their source:
• Municipal solid waste (Household waste), 
• Industrial solid waste (Hazardous waste) and 
• Biomedical waste (Hospital waste as infectious waste).

7.1.1 Municipal solid waste

The term Municipal Solid Waste (MSW) is normally assumed to include all of
the waste generated in a community, with the exception of waste generated
by municipal services, treatment plants, and industrial and agricultural
processes. In the urban context the term municipal solid wastes is of special
importance. The term refers to all wastes collected and controlled by the
municipality and comprises of most diverse categories of wastes. It comprises
of wastes from several different sources such as, domestic wastes, commercial
wastes, institutional wastes and building materials wastes.
Following are the different types of wastes.
• Biodegradable waste: food and kitchen waste, green waste, paper (can
also be recycled).
• Recyclable material: paper, glass, bottles, cans, metals, certain plastics,
etc.
• Inert waste: construction and demolition waste, dirt, rocks, debris.
• Composite wastes: waste clothing, Tetra Packs, waste plastics such as
toys.
• Domestic hazardous waste (also called “household hazardous waste”)
and toxic waste: medication, e-waste, paints, chemicals, light bulbs,
Solid Waste Disposal and Management 7.3

fluorescent tubes, spray cans, fertilizer and pesticide containers,

batteries, shoe polish.
In 1947, cities and towns in India generated an estimated 6 million
tonnes of solid waste in 2011; it was about 90 million tonnes. More than 25%
of the municipal solid waste is not collected at all; 70% of the Indian cities
lack adequate capacity to transport it and there are no sanitary landfills to
dispose of the waste. Over the last few years, the consumer market has grown
rapidly leading to products being packed in cans, aluminium foils, plastics,
and other such non-biodegradable items that cause incalculable harm to the
environment.
The type of litter we generate and the approximate time
it takes to degenerate
Type of litter Approximate time it takes
to degenerate the litter
Organic waste such as vegetable and fruit a week or two.
peels, leftover foodstuff, etc.
Paper 10–30 days
Cotton cloth 2–5 months
Wood 10–15 years
Woolen items 1 year
Tin, aluminium, and other metal items such as 100–500 years
cans
Plastic bags one million years
Glass bottles Undetermined

7.1.2 Industrial solid waste

Industrial waste is defined as waste generated by manufacturing or industrial
processes. The types of industrial waste generated include cafeteria garbage,
dirt and gravel, masonry and concrete, scrap metals, trash, oil, solvents,
chemicals, weed grass and trees, wood and scrap lumber, and similar
wastes. Industrial solid waste—which may be solid, liquid or gases held in
containers—is divided into hazardous and non-hazardous waste.
• Hazardous waste may result from manufacturing or other industrial
processes. Certain commercial products such as cleansing fluids, paints
or pesticides discarded by commercial establishments or individuals,
can also be defined as hazardous waste.
• Non-hazardous industrial wastes are those that do not meet the EPA’s
definition of hazardous waste—and are not municipal waste.
Industrial waste has been a problem since the industrial revolution.
Industrial waste may be toxic, ignitable, corrosive or reactive. If improperly
7.4 Environmental Biotechnology

managed, this waste can pose dangerous health and environmental

consequences.
The other major generators of industrial solid wastes are the thermal power
plants, producing coal ash; the integrated Iron and Steel mills, producing blast
furnace slag and steel melting slag; non-ferrous industries like aluminum, zinc
and copper, producing red mud and tailings; sugar industries, generating
press mud, pulp; and paper industries, producing lime and fertilizer; and
allied industries, producing gypsum.

7.1.3 Biomedical Waste

Biomedical waste, also known as infectious waste or medical waste, is defined
as solid waste generated during the diagnosis, testing, treatment, research or
production of biological products for humans or animals. Biomedical waste
includes syringes, live vaccines, laboratory samples, body parts, bodily fluids
and waste, sharp needles, cultures and lancets. The main sources of biomedical
waste are hospitals, medical clinics and laboratories. Because biomedical waste
can be detrimental to human health, the law requires such facilities to follow
procedures that protect the public from coming into contact with it. Agencies
that regulate different aspects of biomedical waste include Occupational
Safety and Health Administration (OSHA), Food and Drug Administration
(FDA) and Nuclear Regulatory Commission.
Biomedical wastes may be categorized as follows:
• Animal Waste: Animals carcasses, tissues and body parts, blood and
bodily fluids and infectious bedding.
• Biological Laboratory Waste: Cultures, stocks or specimens of
microorganisms, live or attenuated vaccines, human or animal cell
cultures and laboratory material that has come into contact with these
(solid and liquid).
• Human Anatomical Waste: any part of the human body, including tissues
and organs but excluding extracted teeth, hair, and nail clippings.
• Human Blood and Body Fluid Waste: Human fluid blood and blood
products, items saturated or dripping blood, body fluids contaminated
with blood and body fluids removed for diagnosis during surgery,
treatment or autopsy. This does not include urine or feces. Material
with minimal amounts of non-infectious blood (i.e., does not release
blood, if compressed) are not considered biomedical waste.
• Sharps: Needles, syringes with needles, lancets, scalpels, razor blades,
and precision knives. Contaminated broken glass, pipettes, test tubes,
microscope.
• Biohazardous waste: Waste, that is known or suspected to contain
infectious material or, which because of its physical or biological nature
may be harmful to humans, animals, plants or the environment.
Solid Waste Disposal and Management 7.5

• Infectious waste: Waste, which contains microorganisms in sufficient

quantity which could result in the multiplication and growth of those
microorganisms in a host.
• Pathological waste: Any waste which contains microorganisms capable
of causing disease.
Surveys carried out by various agencies show that the health care
establishments in India are not giving due attention to their waste management.
After the notification of the Biomedical Waste (Handling and Management)
Rules, 1998, these establishments are slowly streamlining the process of waste
segregation, collection, treatment, and disposal. Many of the larger hospitals
have either installed the treatment facilities or are in the process of doing so.

7.2 SOLID WASTE DISPOSAL AND MANAGEMENT:

Solid Waste Management (SWM) is the collection, transport, processing,
disposal, managing and monitoring of waste materials. The term usually
relates to materials produced by human activity, and the process is generally
undertaken to reduce their effect on health, the environment or aesthetics.
• Waste management is a distinct practice from resource recovery, which,
focuses on delaying the rate of consumption of natural resources.
• The management of wastes treats all materials as a single class, whether
solid, liquid, gaseous or radioactive substances, and tries to reduce the
harmful environmental impacts of each through different methods.
• Waste management practices differ for developed and developing
nations, for urban and rural areas, and for residential and industrial
producers.
• Management for non-hazardous waste—residential and institutional
waste in metropolitan areas—is usually the responsibility of local
government authorities, while management for non-hazardous
commercial and industrial waste, is usually the responsibility of the
generator.
Disposal is the discharge, deposit, injection, dumping, spilling, leaking,
or placing of any solid waste or hazardous waste into, or on any land, or
water, so that such solid waste or hazardous waste or any constituent thereof,
may enter the environment or be emitted into the air or discharged into
any waters, including ground waters. Within our modern scheme of waste
management, disposal is the last phase. As cities are growing in size with a
rise in the population, the amount of waste generated is increasing becoming
unmanageable.
• As waste management issues gain public awareness, concern has risen
about the appropriateness of various disposal methods. However,
in most places, disposal of waste is the most neglected area of Solid
Waste Management (SWM) services and the current practices are
grossly unscientific.
7.6 Environmental Biotechnology

• Almost all municipal authorities deposit solid waste at a dump yard

situated within or outside the city haphazardly and do not bother to
spread and cover the waste with inert material. These sites emanate
foul smell and become breeding grounds for flies, rodent, and pests.
Following are different methods for the disposal of waste – open dumps,
landfills, sanitary landfills, incineration plants, pyrolysis and recycling. One of
the important methods of waste treatment is composting.
Open dumps or Non-engineered disposal: Open dumps refer to
uncovered areas that are used to dump solid waste of all kinds. The waste is
untreated, uncovered, and not segregated. This is the most common method
of disposal in low-income countries, which have no control, or with only slight
or moderate controls. They tend to remain for longer time and environmental
degradation could be high, including mosquito, rodent and water pollution,
and degradation of the land. In some countries, open dumps are being phased
out.
Landfills: Historically, landfills have been the most common methods of
organized waste disposal, and remain so, in many places around the world.
• A landfill site is a site for the disposal of waste materials by burial and
is the oldest form of waste treatment.
• Landfills may include internal waste disposal sites (where a producer
of waste carries out their own waste disposal at the place of production)
as well as sites used by many producers.
• Many landfills are also used for waste management purposes, such
as the temporary storage, consolidation and transfer, or processing of
waste material (sorting, treatment, or recycling).
While new methods of hazardous waste disposal are being developed, it
appears that landfills will, at least for the time being, continue to be the most
favoured technique. In many countries, land is a readily available commodity
and often areas of non-productive or derelict land may be made available for
waste disposal. In many instances, land can be utilized in the near vicinity or on
the premises of industrial companies, thereby reducing transportation costs.
The potential also exists to reclaim certain areas for recreational purposes.
• Land filling is still the major disposal method in many countries. Yet,
in many instances, land filling sites are not properly chosen in terms of
geophysical soil properties, hydrogeology, topography and climate.
• On a proposed site, there is a need to carefully consider the potential
for ground or surface water contamination from pollution by leachate
migration or surface run-off from the site.
• Nonetheless, even when a site appears to have the right geophysical
properties, its selection, and use are not an absolute guarantee that
contamination of groundwater can be avoided.
Solid Waste Disposal and Management 7.7

• Hence, continuous surveillance of the site and its surroundings must be

maintained to check that the disposal of hazardous wastes can continue
without posing a threat to the environment and to the general public.
• To reduce this threat, landfill sites have been lined, for example,
with plastic materials, in order to prevent leaching into groundwater
supplies.
Sanitary landfill: Sanitary landfill is a fully engineered disposal option,
which avoids harmful effects of uncontrolled dumping by spreading,
compacting and covering the wasteland that has been carefully engineered
before use.
The four minimum requirements for setting up a sanitary landfill are:
• full or partial hydrological isolation,
• formal engineering preparation,
• permanent control and
• planned waste placement and covering.
Land filling relies on containment rather than treatment (for control)
of wastes. Appropriate liners for protection of the groundwater, leachate
collection and treatment, monitoring wells and, appropriate final cover design
are integral components of an environmentally sound sanitary landfill.
Incineration (or) thermal treatment: Incineration is a disposal method
in which solid organic wastes are subjected to combustion so as to convert
them into residue and gaseous products. This method is useful for disposal of
residue of both solid waste management and solid residue from wastewater
management.
• This process reduces the volumes of solid waste to 20 to 30 percent of
the original volume. Incineration and other high temperature waste
treatment systems are sometimes described as “thermal treatment”.
• Incinerators convert waste materials into heat, gas, steam and ash.
• Incineration is carried out both on a small scale by individuals and on a
large scale by industry. It is used to dispose of solid, liquid and gaseous
waste.
• It is recognized as a practical method of disposing of certain hazardous
waste materials (such as biological medical waste). Incineration is a
controversial method of waste disposal, due to issues such as emission
of gaseous pollutants.
• Incineration is common in countries such as Japan where land is more
scarce, as these facilities generally do not require as much area as
landfills.
• Waste-to-Energy (WtE) or Energy-from-Waste (EfW) are broad terms
for facilities that burn waste in a furnace or boiler to generate heat,
steam or electricity.
7.8 Environmental Biotechnology

• Combustion in an incinerator is not always perfect and there have been

concerns about pollutants in gaseous emissions from incinerator stacks.
Particular concern has focused on some very persistent organics such
as dioxins, furans, PAHs which may be created and may have serious
environmental consequences.
• Biomedical waste can be disposed of through incineration or
decontamination by heating with steam under pressure in an autoclave.
Trash chutes must not be used for the transfer or disposal of biomedical
waste.
Pyrolysis/Gasification, Plasma Pyrolysis Vitrification (PPV)/Plasma Arc
Process: Pyrolysis is a form of treatment that chemically decomposes organic
materials by heat in the absence of oxygen. Pyrolysis, typically, occurs under
pressure and at operating temperatures above 430°C.
• Pyrolysis gasification processes are established for homogenous
organic matter like wood, pulp, etc., while plasma pyrolysis vitrification
is a relatively new technology for disposal of particularly hazardous
wastes, radioactive wastes, etc.
• Toxic materials get encapsulated in vitreous mass, which is relatively
much safer to handle than incinerator/gasifier ash. These are now being
offered as an attractive option for disposal of MSW also.
• In all these processes, besides net energy recovery, proper destruction
of the waste is also ensured. These processes, therefore, have an edge
over incineration.
• This process produces fuel gas/fuel oil, which replace fossil fuels and
compared to incineration, atmospheric pollution can be controlled
at the plant level. NO and SO gas emissions do not occur in normal
operations due to the lack of oxygen in the system.
• It is a capital and energy intensive process and net energy recovery
may suffer in case of wastes with excessive moisture and inert content.
• High viscosity of Pyrolysis oil maybe problematic for its transportation
and burning. Concentration of toxic/hazardous matter in gasifier ash
needs care in handling and disposal.
No commercial plant has come up in India or elsewhere for the disposal of
Municipal Solid Waste (MSW). It is an emerging technology for MSW, yet to be
successfully demonstrated for large-scale application.
Recycling and reuse: Recycling involves the collection of used and
discarded materials, processing these materials, and making them into new
products. It reduces the amount of waste that is thrown into the community
dustbins thereby making the environment cleaner and the air more fresh to
breathe. Hence, recycling refers to the collection and reuse of waste materials
such as empty beverage containers. The materials from which the items are
made can be reprocessed into new products. Material for recycling may be
Solid Waste Disposal and Management 7.9

collected separately from general waste using dedicated bins and collection
vehicles, or sorted directly from mixed waste streams. Known as kerb-side
recycling, it requires the owner of the waste to separate it into various different
bins (typically wheelie bins) prior to its collection.

The Schematic diagram below depicts recycling of municipal wastes

The most common consumer products recycled include aluminum such
as beverage cans, copper such as wire, steel food and aerosol cans, old steel
furnishings or equipment, polyethylene and PET bottles, glass bottles and jars,
paperboard cartons, newspapers, magazines and light paper, and corrugated
fiberboard boxes.
• Different plastics (polymers) like PVC, LDPE, PP, and PS (see resin
identification code) are also recyclable. These items are usually
composed of a single type of material, making them relatively easy to
recycle into new products. The recycling of complex products (such
as computers and electronic equipment) is more difficult, due to the
additional dismantling and separation required.
• The type of material accepted for recycling varies by city and country.
Each city and country have different recycling programs in place that
can handle the various types of recyclable materials. However, variation
in acceptance is reflected in the resale value of the material, once it is
reprocessed.
Surveys carried out by Government and non-government agencies in the
country have all recognized the importance of recycling wastes. However, the
methodology for safe recycling of waste has not been standardized. Studies
have revealed that 7-15% of the waste is recycled. If recycling is done in a proper
manner, it will solve the problems of waste or garbage. At the community
level, a large number of NGOs (Non-Governmental Organizations) and
private sector enterprises have taken an initiative in segregation and recycling
of waste.
Biomedical waste can be managed properly by ensuring proper segregation
at the source, the use of accurate packaging (leak resistant, puncture resistant
and not susceptible to degradation by cleaning agents in case the packaging
is reused), appropriate color coding, proper in-house movement of waste
7.10 Environmental Biotechnology

(minimizing employee exposure to biomedical waste in a workplace),

designating waste storage areas and ensuring safe disposal.

Waste to energy or Energy from waste (Energy recovery)

The energy content of waste products can be harnessed directly by using them
as a direct combustion fuel, or indirectly, by processing them into another type
of fuel.
• Thermal treatment ranges from using waste as a fuel source for cooking
or heating and the use of the gas fuel, to fuel for boilers, to generate
steam and electricity in a turbine.
• Pyrolysis and gasification are two related forms of thermal treatment
where waste materials are heated to high temperatures with limited
oxygen availability. The process usually occurs in a sealed vessel under
high pressure.
• Pyrolysis of solid waste converts the material into solid, liquid and gas
products. The liquid and gas can be burnt to produce energy or refined
into other chemical products (chemical refinery).
• The solid residue (char) can be further refined into products such as
activated carbon. Gasification and advanced Plasma arc gasification are
used to convert organic materials directly into a synthetic gas (syngas)
composed of carbon monoxide and hydrogen.
• The gas is then burnt to produce electricity and steam. An alternative
to pyrolysis is high temperature and pressure supercritical water
decomposition
Even though the technology of waste to energy (WTE) projects has
been proven worldwide, its viability and sustainability is yet to be to be
demonstrated and established in India. The main factors that determine the
techno-economic viability of WTE projects are quantum of investment, scale
of operation, availability of quality waste, statutory requirements and project
risks.

7.4 BIOLOGICAL REPROCESSING OF SOLID WASTE DISPOSAL

Solid waste materials that are organic in nature, such as plant material, food
scraps, and paper products, can be recycled using biological composting and
digestion processes to decompose the organic matter. The resulting organic
material is then recycled as mulch or compost for agricultural or landscaping
purposes. In addition, waste gas from the process (such as methane) can be
captured and used for generating electricity and heat (CHP/cogeneration),
maximizing efficiencies. The intention of biological processing in waste
management is to control and accelerate the natural process of decomposition
of organic matter.
• There is a large variety of composting and digestion methods and
technologies varying in complexity from simple home compost heaps,
Solid Waste Disposal and Management 7.11

to small town scale batch digesters, industrial-scale enclosed-vessel

digestion of mixed domestic waste.
• Methods of biological decomposition are differentiated as being aerobic
or anaerobic methods, though hybrids of the two methods also exist.
• Anaerobic digestion of the organic fraction of MSW has been found to
be in a number of Life cycle analysis studies to be more environmentally
effective, than landfill, incineration or pyrolysis.
• The resulting biogas (methane) though must be used for cogeneration
(electricity and heat, preferably, on or close to the site of production)
and can be used with a little upgrading in gas combustion engines or
turbines.
• With further upgrading to synthetic natural gas, it can be injected
into the natural gas network or further refined to hydrogen for use
in stationary cogeneration fuel cells. Its use in fuel cells eliminates the
pollution from products of combustion.

7.4.1 Composting
Composting is a technology known in India since times immemorial.
Composting is the decomposition of organic matter by microorganism in
warm, moist, aerobic and anaerobic environment. Farmers have been using
compost made out of cow dung and other agro-waste.
• The compost made out of urban heterogeneous waste is found to be
of higher nutrient value as compared to the compost made out of cow
dung and agro-waste.
• Composting of MSW is, therefore, the most simple and cost-effective
technology for treating the organic fraction of MSW.
• Full-scale commercially viable composting technology is already
demonstrated in India and is in use in several cities and towns. Its
application to farm land, tea gardens, fruit orchards or its use as soil
conditioner in parks, gardens, agricultural lands, etc., is however,
limited on account of poor marketing.
• Main advantages of composting include improvement in soil texture
and augmenting of micronutrient deficiencies.
• It also increases moisture-holding capacity of the soil and helps in
maintaining soil health. Moreover, it is an age-old established concept
for recycling nutrients to the soil.
• It is simple and straightforward to adopt, for source separated MSW.
It does not require large capital investment, compared to other waste
treatment options. The technology is scale-neutral.
• Composting is suitable for organic biodegradable fraction of MSW, yard
(or garden) waste / waste containing high proportion of lignocelluloses
7.12 Environmental Biotechnology

materials, which do not readily degrade under anaerobic conditions,

waste from slaughterhouse and dairy waste.
• This method, however, is not very suitable for wastes that may be too
wet and, during heavy rains, open compost plants have to be stopped.
• Land required for open compost plants is relatively large. Also, issues of
methane emission, odor, and flies from badly-managed open compost
plants, remain. At the operational level, if waste segregation at source
is not properly carried out, there is possibility of toxic material entering
the stream of MSW.
• It is essential that compost produced be safe for application.
Standardization of compost quality is, therefore, necessary.

A pilot system diagram for composting

Biotechnological processes for soil and land treatment: Enhanced
bioremediation is a process in which indigenous or inoculated microorganisms
(e.g., fungi, bacteria, and other microbes) degrade (metabolize) organic
contaminants found in soil and/or ground water, converting them to innocuous
end products.
• Nutrients, oxygen, or other amendments may be used to enhance
bioremediation and contaminant desorption from subsurface materials.
• In the presence of sufficient oxygen (aerobic conditions), and other
nutrient elements, microorganisms will ultimately convert many
organic contaminants to carbon dioxide, water, and microbial cell mass.
• In the absence of oxygen (anaerobic conditions), the organic
contaminants will be ultimately metabolized to methane, limited
amounts of carbon dioxide, and trace amounts of hydrogen gas.
Under sulfate-reduction conditions, sulfate is converted to sulfide or
elemental sulfur, and under nitrate-reduction conditions, dinitrogen
gas is ultimately produced.
• Sometimes contaminants may be degraded to intermediate or final
products that may be less, equally, or more hazardous than the original
contaminant. For example, TCE is anaerobically biodegrades to the
Solid Waste Disposal and Management 7.13

persistent and more toxic vinyl chloride. To avoid such problems, most
bioremediation projects are conducted in situ. Vinyl chloride can easily
be broken down further, if aerobic conditions are created.
• Enhanced bioremediation of soil, typically involves the percolation
or injection of groundwater or uncontaminated water mixed with
nutrients and saturated with dissolved oxygen. Sometimes, acclimated
microorganisms (bioaugmentation) and/or another oxygen source
such as hydrogen peroxide, are also added. An infiltration gallery or
spray irrigation is typically used for shallow contaminated soils, and
injection wells are used for deeper contaminated soils.
• Although successful in situ bioremediation has been demonstrated in
cold weather climate, low temperature slows the remediation process.
For contaminated sites with low soil temperature, heat blankets may be
used to cover the soil surface to increase the soil temperature and the
degradation rate.
Enhanced bioremediation may be classified as a long-term technology
which may take several years for cleanup of a plume.

7.5 BIOTECHNOLOGICAL METHODS OF SOLID WASTE

(AGRICULTURE, DOMESTIC AND INDUSTRIAL)
DEGRADATION
In the present techno-economic era, the energy and environmental crises
developed due to huge amount of cellulosic materials are disposed as “waste.”
Municipal solid waste is composed of 40–50% cellulose, 9–12% hemicellulose,
and 10—15% lignin on a dry weight basis. Annually, Asia alone generates
4.4 billion tons of solid wastes, and municipal solid waste comprises
790 million tons, of which about 48 million tons are generated in India. By
the year 2047, municipal solid waste generation in India is expected to reach
300 million tons and land requirement for disposal of this waste would be
169.6 km2. Unscientific disposal causes an adverse impact on all components
of the environment and human health. Microorganism performs their
metabolic processes rapidly and with remarkable specificity under ambient
conditions, catalyzed by their diverse enzyme-mediated reactions. An enzyme
alternative to harsh chemical technologies has led to intensive exploration of
natural microbial biodiversity to discover enzymes. There is a wide spectrum
of microorganisms which can produce the variety of enzymes like cellulase
under appropriate conditions.
Cellulases are a consortium of free enzymes comprised of endoglucanases,
exoglucanases, and cellobiases are found in many of the 57 glycosyl hydrolase
families. Several studies were carried out to produce cellulolytic enzymes
in organic waste degradation process by several microorganism, including
fungi such as Trichoderma sp., Penicillium sp., and Aspergillus sp., respectively.
Many fungi capable of degrading cellulose synthesize large quantities of
7.14 Environmental Biotechnology

extracellular cellulases that are more efficient in depolymerising the cellulose

substrate. Most commonly studied cellulolytic organisms include fungal
species: Trichoderma, Humicola, Penicillium, and Aspergillus.
Many cellulases produced by bacteria appear to be bound to the cell
wall and are unable to hydrolyze native lignocellulose preparations to
any significant extent. A wide variety of gram-positive and gram-negative
species are reported to produce cellulose, including Clostridium thermocellum,
Streptomyces spp., Ruminococcus spp., Pseudomonas spp., Cellulomonas spp.,
Bacillus spp., Serratia, Proteus, Staphylococcus spp., and Bacillus subtilis. Various
biological studies have been carried out to identify the major microbiological
agents responsible for biodegradation. Today, environmental policies and
regulation progress lead to the development of biodegradation processes to
turn organic wastes into a valuable resource by potential microbes because
only few strains are capable of secreting a complex of cellulase enzymes, which
could have practical application in the enzymatic hydrolysis of cellulose as
well as biodegradation of organic municipal solid waste.
Probably, the best and the most economical approach to the problem
of—solid waste disposal, involve microbial degradation of solid wastes. Bio-
degradable wastes can be conveniently handled in this way. Recalcitrant
and refractory solids like plastics, polymers and many plasticizers create
problems. These materials are synthetic substances, and as such, are often too
new to the biological system. The microbial system simply lacks the enzymatic
machinery needed for their decomposition.
Recent strides taken in the field of biotechnology has come to be of
great help in this direction. Genetically engineered microbes have been
produced which can decompose a number of organic compounds which were
considered to be nondegradable earlier. It was only an Indian born American
Scientist Chakrabarty A.N., who has patented for the first time a genetically
engineered strain of bacteria, Pseudomonas, which decomposes a number of
complex and toxic hydrocarbons. Efforts are also being made to produce bio-
degradable plastic and polymers through genetically engineered microbes.
Biologically produced plastic and polymers shall create much less problem as
microbial system shall decompose them quickly and effectively into simpler
constituents.

7.6 IMPORTANCE, HEALTH IMPACTS AND AWARENESS OF

WASTE MANAGEMENT
Importance of waste reduction: In the affluent countries, the main motivations
for waste reduction are frequently related to the high cost and scarcity of sites for
landfills, and the environmental degradation caused by toxic materials in the
deposited wastes. The same considerations apply to large metropolitan areas
in developing countries that are surrounded by other populous jurisdictions.
The places that currently do not have significant disposal pressures can still
Solid Waste Disposal and Management 7.15

benefit from encouraging waste reduction. Their solid waste departments,

already overburdened, cannot afford to spend more money and effort on the
greater quantities of wastes that will inevitably be produced as consumption
levels rise and urban wastes change. Solid waste managers in developing
countries tend to pay little attention to the topic of reducing non-organic
wastes because the wastes they collect are between 50 to 90% organics, dirt
and ashes. These municipal wastes, however, are amenable to composting or
digestion, provided they contain very low levels of synthetic materials. Solid
waste departments thus have an interest in promoting diversion of synthetic
recyclables from the waste stream.
Each household generates garbage or waste, day in and day out. Items
that are no longer needed or do not have any further use, fall in the category of
waste and we tend to throw them away. There are different types of solid waste
depending on their source. In today’s polluted world, learning the correct
methods of handling the waste generated has become essential. Segregation is
an important method of handling municipal solid waste. Segregation at source
can be understood clearly by schematic representation. One of the important
methods of managing and treating wastes is composting.
As the cities are growing in size and in problems such as the generation
of plastic waste, various municipal waste treatment and disposal methods
are now being used to try and resolve these problems. One common sight in
all cities is the rag picker who plays an important role in the segregation of
this waste. Garbage generated in households can be recycled and reused to
prevent creation of waste at source and reducing amount of waste thrown into
the community dustbins.

7.7 HEALTH IMPACTS OF SOLID WASTE

Modernization and progress has had its share of disadvantages and one of the
main aspects of concern is the pollution it is causing to the earth—be it land,
air, and water. With increase in the global population and the rising demand
for food and other essentials, there has been a rise in the amount of waste
being generated daily by each household. This waste is ultimately thrown
into municipal waste collection centers from where it is collected by the area
municipalities to be further thrown into the landfills and dumps. However,
either due to resource crunch or inefficient infrastructure, not all of this waste
gets collected and transported to the final dumpsites. If, at this stage, the
management and disposal is improperly done, it can cause serious impacts on
health and problems to the surrounding environment.
Waste that is not properly managed, especially excreta and other liquid
and solid waste from households and the community, are a serious health
hazard and lead to the spread of infectious diseases. Unattended waste lying
around attracts flies, rats, and other creatures that, in turn, spread disease.
Normally, it is the wet waste that decomposes and releases a bad odor. This
7.16 Environmental Biotechnology

leads to unhygienic conditions, and thereby, to a rise in the health problems.

The plague outbreak in Surat is a good example of a city suffering due to
the callous attitude of the local body in maintaining cleanliness in the city.
Plastic waste is another cause for ill health. Thus, excessive solid waste that is
generated should be controlled by taking certain preventive measures.
• The group, at risk, from the unscientific disposal of solid waste
include—the population in areas where there is no proper waste
disposal method, especially the pre-school children; waste workers;
and workers in facilities producing toxic and infectious material. Other
high-risk group include, population living close to a waste dump and
those, whose water supply has become contaminated either due to
waste dumping or leakage from landfill sites. Uncollected solid waste
also increases risk of injury, and infection.
• In particular,  organic domestic waste  poses a serious threat, since they
ferment, creating conditions favorable to the survival and growth
of microbial pathogens. Direct handling of solid waste can result in
various types of infectious and chronic diseases with the waste workers
and the rag pickers being the most vulnerable.
• Exposure to hazardous waste  can affect human health, children being
more vulnerable to these pollutants. In fact, direct exposure can lead to
diseases through chemical exposure, as the release of chemical waste
into the environment leads to chemical poisoning. Many studies have
been carried out in various parts of the world to establish a connection
between health and hazardous waste.
• Waste from agriculture and industries  can also cause serious health
risks. Other than this, co-disposal of industrial hazardous waste
with municipal waste can expose people to chemical and radioactive
hazards. Uncollected solid waste can also obstruct storm water run-
off, resulting in the forming of stagnant water bodies that become the
breeding ground for disease. Waste dumped near a water source also
causes contamination of the water body or the ground water source.
Direct dumping of untreated waste in rivers, seas, and lakes results
in the accumulation of toxic substances in the food chain through the
plants and animals that feed on it.
• Disposal of hospital and other medical waste  requires special attention
since this can create major health hazards. This waste generated from
the hospitals, health care centres, medical laboratories, and research
centers such as discarded syringe needles, bandages, swabs, plasters,
and other types of infectious waste are often disposed with the regular
non-infectious waste.
• Waste treatment and disposal sites  can also create health hazards for
the neighborhood. Improperly operated incineration plants cause air
pollution and improperly managed and designed landfills attract all
types of insects and rodents that spread disease. Ideally, these sites
Solid Waste Disposal and Management 7.17

should be located at a safe distance from all human settlement. Landfill

sites should be well-lined and walled to ensure that there is no leakage
into the nearby ground water sources.
• Recycling, too, carries health risks if proper precautions are not taken.
Workers working with waste containing chemical and metals may
experience toxic exposure. Disposal of health-care wastes require
special attention since it can create major health hazards, such as
Hepatitis B and C, through wounds caused by discarded syringes. Rag
pickers and others, who are involved in scavenging in the waste dumps
for items that can be recycled, may sustain injuries and come into direct
contact with these infectious items.
Diseases: Certain chemicals, if released untreated, e.g. cyanides, mercury,
and polychlorinated biphenyls, are highly toxic and exposure can lead to
disease or death. Some studies have detected excesses of cancer in residents
exposed to hazardous waste. Many studies have been carried out in various
parts of the world to establish a connection between health and hazardous
waste.
The role of plastics: The unhygienic use and disposal of plastics and its
effects on human health has become a matter of concern. Colored plastics are
harmful as their pigment contains heavy metals that are highly toxic.
Some of the harmful metals found in plastics are copper, lead, chromium,
cobalt, selenium, and cadmium. In most industrialized countries, colour
plastics have been legally banned. In India, the Government of Himachal
Pradesh has banned the use of plastics and so has Ladakh district. Other states
should emulate their example.
Preventive measures: Proper methods of waste disposal have to be
undertaken to ensure that it does not affect the environment around the area
or causes health hazards to the people living there. At the household-level
proper segregation of waste has to be done and it should be ensured that all
organic matter is kept aside for composting, which is undoubtedly the best
method for the correct disposal of this segment of the waste. In fact, the organic
part of the waste that is generated decomposes more easily, attracts insects and
causes disease. Organic waste can be composted and then used as a fertilizer.

7.8 KEY CONCEPTS IN MUNICIPAL WASTE REDUCTION

Waste reduction: All means of reducing the amounts of waste that must be
collected and disposed of by solid waste authorities. It ranges from legislation
and agreements at the national level for packaging and product redesign to
local programs to prevent recyclables and compostable organics from entering
final waste streams.
Source reduction: Any procedure to reduce wastes at the point of
generation, in contrast to sorting out recyclable components after they have
been mixed together for collection.
7.18 Environmental Biotechnology

Source separation: Keeping different categories of recyclables and

organics separate at source, i.e., at the point of generation, to facilitate reuse,
recycling, and composting.
Waste recovery, materials recovery, or waste diversion: Obtaining
materials/organics (by source separation or sorting out from mixed wastes)
that can be reused or recycled.
Reuse: Reusing a product for the same or a different purpose.
Recycling: The process of transforming materials into secondary resources
for manufacturing new products is called recycling.
Redemption center: Waste trading enterprise that buys recyclable
materials and sells to brokers. Sometimes also called “buy-back centre”.
Producer responsibility: Producers of products or services accept a
degree of responsibility for the wastes that result from the products/services
they market, by reducing materials used in production, making repairable/
recyclable goods, and/or reducing packaging.
Promoting waste reduction and materials recovery at the national and
local levels: Action for waste reduction can take place at both national and
local levels. At the national level, the main routes to waste reduction are:
• redesign of products or packaging;
• promotion of consumer awareness; and
• promotion of producer responsibility for post-consumer wastes (this
applies mostly to industrialized countries).
At the local level, the main means of reducing waste are:
• diversion of materials from the waste stream through source separation
and trading;
• recovery of materials from mixed waste;
• pressure on national or regional governments for legislation on
redesigning packaging or products; and
• support of composting, either centralized or small-scale.

7.9 WASTEWATER
Water is crucial for all aspects of life, the defining feature of our planet. Ninety
seven and a half per cent of all water is found in the oceans; of the remaining
freshwater only one per cent is accessible for extraction and use. Functioning
and healthy aquatic ecosystems provide us with a dazzling array of benefits
– food, medicines, recreational amenity, shoreline protection, processing our
waste, and sequestering carbon. At the beginning of the 21st century, the
world faces a water crisis, both of quantity and quality, caused by continuous
population growth, industrialization, food production practices, increased
living standards and poor water use strategies. Wastewater management or the
lack of, has a direct impact on the biological diversity of aquatic ecosystems,
Solid Waste Disposal and Management 7.19

disrupting the fundamental integrity of our life support systems, on which

a wide range of sectors from urban development to food production and
industry depend. It is essential that wastewater management is considered as
part of integrated, ecosystem-based management that operates across sectors
and borders, freshwater and marine.

What do we mean by wastewater?

Wastewater can mean different things to different people with a large
number of definitions in use. But in broad perspective wastewater may be
defined as “a combination of one or more of: domestic effluent consisting
of black water (excreta, urine and fecal sludge) and grey water (kitchen and
bathing wastewater); water from commercial establishments and institutions,
including hospitals; industrial effluent, storm water and other urban run-off;
agricultural, horticultural and aquaculture effluent, either dissolved or as
suspended matter.

7.9.1 Wastewater treatment and Management

Wastewater treatment plants can be divided into two major types:
1. Biological and
2. Physical/Chemical.
• Biological plants are more commonly used to treat domestic, or
combined domestic and industrial, wastewater from a municipality.
They use basically the same processes that would occur naturally in
the receiving water, but give them a place to happen under controlled
conditions, so that the cleansing reactions are completed before the
water is discharged into the environment.
• Physical/chemical plants are more often used to treat industrial
wastewaters directly, because they often contain pollutants which
cannot be removed efficiently by microorganisms— although industries
that deal with biodegradable materials, such as food processing,
dairies, breweries, and even paper, plastics and petrochemicals, may
use biological treatment. And biological plants generally use some
physical and chemical processes, too.
A physical process usually treats suspended, rather than dissolved
pollutants. It may be a passive process, such as simply allowing suspended
pollutants to settle out or float to the top naturally—depending on whether they
are more or less dense than water, Or, the process may be aided mechanically,
such as by gently stirring the water to cause more small particles to bump into
each other and stick together, forming larger particles which will settle or rise
faster—a process known as flocculation.
• Chemical flocculants may also be added to produce larger particles. To
aid flotation processes, dissolved air under pressure may be added to
cause the formation of tiny bubbles which will attach to particles.
7.20 Environmental Biotechnology

Filtration through a medium such, as sand as a final treatment stage, can

result in a very clear water. Ultrafiltration, nanofiltration, and reverse osmosis
are processes which force water through membranes and can remove colloidal
material (very fine, electrically charged particles, which will not settle) and
even some dissolved matter. Absorption (adsorption, technically) on activated
charcoal is a physical process which can remove dissolved chemicals. Air or
steam stripping can be used to remove pollutants that are gases or low-boiling
liquids from water, and the vapors which are removed in this way, are also
often passed through beds of activated charcoal, to prevent air pollution.
These last processes are used mostly in industrial treatment plants, though
activated charcoal is common in municipal plants, as well, for odor control.
Some examples of chemical treatment processes, in an industrial setting,
would be:
• converting a dissolved metal into a solid, settleable form by precipitation
with an alkaline material like sodium or calcium hydroxide. Dissolved
iron or aluminum salts or organic coagulant aids like polyelectrolytes can
be added to help flocculate and settle (or float) the precipitated metal.
• converting highly toxic cyanides used in mining and metal finishing
industries into harmless carbon dioxide and nitrogen by oxidizing them
with chlorine
• destroying organic chemicals by oxidizing them using ozone or hydrogen
peroxide, either alone or in combination with catalysts (chemicals which
speed up reactions) and/or ultraviolet light.
In municipal treatment plants, chemical treatment in the form of aluminum
or iron salts is often used for removal of phosphorus by precipitation. Chlorine
or ozone (or ultraviolet light) may be used for disinfection, that is, killing
harmful microorganisms before the final discharge of the wastewater. Sulfur
dioxide or sulfite solutions can be used to neutralize (reduce) excess chlorine,
which is toxic to aquatic life. Chemical coagulants are also used extensively in
sludge treatment to thicken the solids and promote the removal of water.
A typical treatment plant consists of a train of individual unit processes
set up in a series, with the output (effluent) of one process becoming the input
(influent) of the next process. The first stages will usually be made up of
physical processes that take out easily removable pollutants. After this, the
remaining pollutants are generally treated further by biological or chemical
processes. These may—
• convert dissolved or colloidal impurities into a solid or gaseous form,
so that they can be removed physically, or
• convert them into dissolved materials which remain in the water, but
are not considered as undesirable as the original pollutants.
The solids (residuals or sludges) which result from these processes form a
side stream which also has to be treated for disposal.
Solid Waste Disposal and Management 7.21

A common set of processes that might be found at a municipal treatment

plant would be:
• Preliminary treatment to remove large or hard solids that might clog or
damage other equipment. These might include grinders (comminuters),
bar screens, and grit channels.
— The first chops up rags and trash;
— the second simply catches large objects, which can be raked off;
— the third allows heavier materials, like sand and stones, to settle out,
so that they will not cause abrasive wear on downstream equipment.
Grit channels also remove larger food particles (i.e., garbage).
• Primary settling basins, where the water flows slowly for up to a few
hours, to allow organic suspended matter to settle out or float to the
surface. Most of this material has a density not much different from
that of water, so it needs to be given enough time to separate. Settling
tanks can be rectangular or circular.
In either type, the tank needs to be designed with some type of scrapers
at the bottom to collect the settled sludge and direct it to a pit from which it
can be pumped for further treatment— and skimmers at the surface, to collect
the material that floats to the top (which is given the rather inglorious name
of “scum”.)
— Secondary treatment, usually biological, tries to remove the remaining
dissolved or colloidal organic matter. Generally, the biodegradation of
the pollutants is allowed to take place in a location where plenty of
air can be supplied to the microorganisms. This promotes formation
of the less offensive, oxidized products. Engineers try to design the
capacity of the treatment units so that enough of the impurities will be
removed to prevent significant oxygen demand in the receiving water
after discharge.
There are two major types of biological treatment processes: attached
growth and suspended growth.
In an attached growth process, the microorganisms grow on a surface,
such as rock or plastic. Examples are:
(1) open trickling filters, where the water is distributed over rocks and
trickles down to underdrains, with air being supplied through vent
pipes,
(2) enclosed biotowers, which are similar, but more likely to use shaped,
plastic media instead of rocks, and
(3) so-called rotating biological contacters, or RBCs, which consist of large,
partially submerged discs which rotate continuously, so that the
microorganisms growing on the disc’s surface are repeatedly exposed
alternately to the wastewater and to the air.
7.22 Environmental Biotechnology

The most common type of suspended growth process is the so-called

activated sludge system. This type of system consists of two parts, an aeration
tank and a settling tank, or clarifier. The aeration tank contains a “sludge” which
is what could be best described as a “mixed microbial culture”, containing
mostly bacteria, as well as protozoa, fungi, algae, etc. This sludge is constantly
mixed and aerated either by compressed air bubblers located along the bottom,
or by mechanical aerators on the surface. The wastewater to be treated enters
the tank and mixes with the culture, which uses the organic compounds for
growth — producing more microorganisms — and for respiration, which results
mostly in the formation of carbon dioxide and water. The process can also be
set up to provide biological removal of the nutrients nitrogen and phosphorus.
After sufficient aeration time to reach the required level of treatment, the
sludge is carried by the flow into the settling tank, or clarifier, which is often
of the circular design (An important condition for the success of this process is
the formation of a type of culture which will flocculate naturally, producing a
settling sludge and a reasonably clear upper, or supernatant layer. If the sludge
does not behave this way, a lot of solids will be remain in the water leaving the
clarifier, and the quality of the effluent wastewater will be poor). The sludge
collected at the bottom of the clarifier is then recycled to the aeration tank to
consume more organic material. The term “activated” sludge is used, because
by the time the sludge is returned to the aeration tank, the microorganisms
have been in an environment depleted of “food” for some time, and are in a
“hungry”, or activated condition, eager to get busy biodegrading some more
wastes. Since the amount of microorganisms, or biomass, increases as a result
of this process, some must be removed on a regular basis for further treatment
and disposal, adding to the solids produced in primary treatment.

Activated sludge process

Variations
Sequencing Batch Reactor (SBR):The type of activated sludge system,
described above, is a continuous flow process. There is a variation in which the
entire activated sludge process take place in a single tank, but at different times.
Solid Waste Disposal and Management 7.23

Steps include filling, aerating, settling, drawing off supernatant, etc. A system
like this can provide more flexibility and control over the treatment, including
nutrient removal, and is amenable to computer control.
Membrane Bioreactor (MBR): In this more recent innovation, treated
water is pumped out of the aeration tank through banks of microfiltration
membranes. Clarifiers are not needed. The sludge concentration can be higher
than in a conventional system, which allows treatment in a smaller volume;
and the sludge’s ability to flocculate well is no longer a consideration. Low
effluent solids concentrations can be achieved, which can help in phosphorus
removal and disinfection (see below).

Removal of heavy metals from waste streams

Nutrient removal: Nutrient removal refers to the treatment of the
wastewater to take out nitrogen or phosphorus, which can cause unnecessary
growth of algae or weeds in the receiving water.
• Nitrogen is found in domestic wastewater mostly in the form of
ammonia and organic nitrogen.
• These can be converted to nitrate by bacteria, if the plant is designed
to provide enough oxygen and a long enough “sludge age” to develop
these slow-growing types of organisms.
• The nitrate which is produced, may be discharged; it is still usable as a
plant nutrient, but it is much less toxic than ammonia.
• If more complete removal of nitrogen is required, a biological process
can be set up which reduces the nitrate to nitrogen gas (and some
nitrous oxide).
• There are also physical/chemical processes which can remove
nitrogen, especially ammonia; they are not as economical for domestic
wastewater, but might be suited for an industrial location where no
other biological processes are in use (These methods include alkaline
air stripping, ion exchange, and “breakpoint” chlorination).
Phosphorous removal: Phosphorous removal is most commonly done
by chemical precipitation with iron or aluminum compounds, such as ferric
chloride or alum (aluminum sulfate).
7.24 Environmental Biotechnology

• The solids which are produced can be settled along with other sludges,
depending upon where the treatment process takes place. (“Lime”,
or calcium hydroxide, also works, but makes the water very alkaline,
which has to be corrected, and produces more sludge.).
• There is also a biological process for phosphorus removal, which
depends on designing an activated sludge system in such a way as
to promote the development of certain types of bacteria which have
the ability to accumulate excess phosphorus within their cells. These
methods mainly convert dissolved phosphorus into particulate form.
• For treatment plants which are required to discharge only very low
concentrations of total phosphorus, it is common to have a sand (or
other type of) filter as a final stage, to remove most of the suspended
solids which may contain phosphorus.
Disinfection: Disinfection, usually the final process before discharge, is
the destruction of harmful (pathogenic) microorganisms, i.e., disease-causing
germs. The object is not to kill every living microorganism in the water, which
would be sterilization, but to reduce the number of harmful ones to levels
appropriate for the intended use of the receiving water.
The most commonly used disinfectant is chlorine, which can be supplied
in the form of a liquefied gas which has to be dissolved in water, or in the
form of an alkaline solution called sodium hypochlorite, which is the same
compound as common household, chlorine bleach.
Chlorine is quite effective against most bacteria, but a rather high dose is
needed to kill viruses, protozoa, and other forms of pathogen. Chlorine has
several problems associated with its use, among them being:
(1) It reacts with organic matter to form toxic and carcinogenic chlorinated
organics, such as chloroform,
(2) Chlorine is very toxic to aquatic organisms in the receiving water— the
USEPA recommends no more than 0.011 parts per million (mg/L) and
(3) It is hazardous to store and handle.
Hypochlorite is safer, but still produces problems 1 and 2. Problem 2 can
be dealt with by adding sulfur dioxide (liquefied gas) or sodium sulfite or
bisulfite (solutions) to neutralize the chlorine. The products are nearly harmless
chloride and sulfate ions. This may also help somewhat with problem 1.
A more powerful disinfectant is ozone, an unstable form of oxygen
containing three atoms per molecule, rather than the two found in the ordinary
oxygen gas which makes up about 21% of the atmosphere. Ozone is too
unstable to store, and has to be made as it is used. It is produced by passing
an electrical discharge through air, which is then bubbled through the water.
While chlorine can be dosed at a high enough concentration so that some of it
remains in the water for a considerable time, ozone is consumed very rapidly
and leaves no residual. It may also produce some chemical byproducts, but
probably not as harmful as those produced by chlorine.
Solid Waste Disposal and Management 7.25

The other commonly used method of disinfection is ultraviolet light.

The water is passed through banks of cylindrical, quartz-jacketed fluorescent
bulbs. Anything which can absorb the light, such as fouling or scale formation
on the bulbs’ surfaces, or suspended matter in the water, can interfere with
the effectiveness of the disinfection. Some dissolved materials, such as iron
and some organic compounds, can also absorb some of the light. Ultraviolet
disinfection is becoming more popular because of the increasing complications
associated with the use of chlorine.

7.9.2 Water Purification By Waterweeds And Membrane Filters

Water Purification: Water purification is the process of removing undesirable
chemicals, biological contaminants, suspended solids and gases from
contaminated water. The goal is to produce water fit for a specific purpose.
Most water is purified for human consumption (drinking water) but water
purification may also be designed for a variety of other purposes, including
meeting the requirements of medical, pharmacology, chemical and industrial
applications. In general, the methods used include physical processes such as
filtration and sedimentation, biological processes such as slow sand filters or
activated sludge, chemical processes such as flocculation and chlorination and
the use of electromagnetic radiation such as ultraviolet light.
• The purification process of water may reduce the concentration of
particulate matter including suspended particles, parasites, bacteria,
algae, viruses, fungi; and a range of dissolved and particulate material
derived from the surfaces that water may have made contact with after
falling as rain.
• The standards for drinking water quality are typically set by
governments or by international standards. These standards will
typically set minimum and maximum concentrations of contaminants
for the use that is to be made of the water.
• It is not possible to tell whether water is of an appropriate quality by
visual examination. Simple procedures such as boiling or the use of a
household activated carbon filter are not sufficient for treating all the
possible contaminants that may be present in water from an unknown
source. Even natural spring water—considered safe for all practical
purposes in the 19th century—must now be tested before determining
what kind of treatment, if any, is needed. Chemical analysis, while
expensive, is the only way to obtain the information necessary for
deciding on the appropriate method of purification.
According to a 2007 World Health Organization report, 1.1 billion people
lack access to an improved drinking water supply, 88% of the 4 billion annual
cases of diarrheal disease are attributed to unsafe water and inadequate
sanitation and hygiene, and 1.8 million people die from diarrheal diseases each
year. The WHO estimates that 94% of these diarrheal cases are preventable
7.26 Environmental Biotechnology

through modifications to the environment, including access to safe water.

Simple techniques for treating water at home, such as chlorination, filters, and
solar disinfection, and storing it in safe containers could save a huge number
of lives each year. Reducing deaths from waterborne diseases is a major public
health goal in developing countries.
Purification of water by water weeds: Water weeds or the aquatic
plants, often treated as a problem for development of lakes actually help in
rejuvenation of water bodies by absorbing sewage and heavy metals that affect
most of the lakes. The water weeds cover the lake surface reducing foul smell,
manufacture biomass, purify the water body and absorb all heavy metals
including sulphur, phosphate, nitrate, etc. 
Growing water weeds especially Hyacinth which has more absorbing
capacity for sometime, harvesting it and replanting it again will rejuvenate the
water. But, the growth of the weeds needs to be monitored well as they grow
at a very rapid pace, and once they are harvested, they have to be disposed
scientifically to ensure that they have no adverse effect on the environment.
The harvested weeds can be dumped into a compost pit for being converted
into manures.
Purification of water by Membrane filters: Membrane technology has
become a dignified separation technology over the past decennia. The main
force of membrane technology is the fact that it works without the addition
of chemicals, with a relatively low energy use and easy and well-arranged
process conductions.
Membrane technology is a generic term for a number of different, very
characteristic separation processes.
These processes are of the same kind, because in each of them a membrane
is used. Membranes are used more and more often for the creation of process
water from groundwater, surface water or wastewater. Membranes are now
competitive for conventional techniques. The membrane separation process is
based on the presence of semi-permeable membranes.
Principle: The principle is quite simple: the membrane acts as a very
specific filter that will let water flow through, while it catches suspended
solids and other substances.
There are various methods to enable substances to penetrate a membrane.
Examples of these methods are the applications of high pressure, the
maintenance of a concentration gradient on both sides of the membrane and
the introduction of an electric potential.
Membranes occupy through a selective separation wall. Certain substances
can pass through the membrane, while other substances are caught.
There are two factors that determine the affectivity of a membrane
filtration process—selectivity and productivity.
Solid Waste Disposal and Management 7.27

• Selectivity is expressed as a parameter called retention or separation

factor.
• Productivity is expressed as a parameter called flux. Selectivity and
productivity are membrane-dependent.

Schematic representation of the water purification by membrane filters

Membrane filtration can be divided up between micro and ultra filtration,
on the one hand and nano filtration and Reverse Osmosis (RO or hyper
filtration), on the other hand.
When membrane filtration is used for the removal of larger particles,
microfiltration and ultrafiltration are applied. Because of the open character
of the membranes, the productivity is high while the pressure differences are
low.
When salts need to be removed from water, nano filtration and Reverse
Osmosis are applied. Nano filtration and RO membranes do not work
according to the principle of pores; separation takes place by diffusion through
the membrane. The pressure that is required to perform nano filtration and
Reverse Osmosis is much higher than the pressure required for micro and
ultrafiltration, while productivity is much lower.

Steps involved in membrane filter technology

7.28 Environmental Biotechnology

Membrane filtration has a number of benefits over the existing water

purification techniques:
• It is a process that can take place while temperatures are low. This is
mainly important because it enables the treatment of heat-sensitive
matter. That is why these applications are widely used for food
production.
• It is a process with low-energy cost. Most of the energy that is required
is used to pump liquids through the membrane. The total amount of
energy that is used is minor, compared to alternative techniques, such
as evaporation.
• The process can easily be expanded.
However, no filtration can remove substances that are actually dissolved
in the water such as phosphorus, nitrates and heavy metal ions.

7.9.3 Indicator Organisms

The following microbiological parameters are particularly important from the
health point of view:

Indicator Organisms
Coliforms and Faecal Coliforms: The Coliform group of bacteria comprises
mainly species of the genera Citrobacter, Enterobacter, Escherichia and Klebsiella
and includes Faecal Coliforms, of which Escherichia coli is the predominant
species. Several of the Coliforms are able to grow outside of the intestine,
especially in hot climates, hence their enumeration is unsuitable as a parameter
for monitoring wastewater reuse systems. The Faecal Coliform test may also
include some non-faecal organisms which can grow at 44°C, so the E. coli count
is the most satisfactory indicator parameter for wastewater use in agriculture.
Faecal Streptococci: This group of organisms includes species mainly
associated with animals (Streptococcus bovis and S. equinus), other species with
a wider distribution (e.g. S. faecalis and S. faecium, which occur both in man
and in other animals) as well as two biotypes (S. faecalis var liquefaciens and
a typical S. faecalis, that hydrolyzes starch) which appear to be ubiquitous,
occurring in both polluted and non-polluted environments. The enumeration
of Faecal Streptococci in effluents is a simple routine procedure but has the
following limitations: the possible presence of the non-faecal biotypes as part
of the natural microflora on crops may detract from their utility in assessing
the bacterial quality of wastewater irrigated crops; and the poorer survival
of Faecal Streptococci at high than at low temperatures. Further studies are
still warranted on the use of Faecal Streptococci as an indicator in tropical
conditions and especially, to compare survival with that of Salmonellae.
Clostridium perfringens: This bacterium is an exclusively faecal spore-
forming anaerobe normally used to detect intermittent or previous pollution
Solid Waste Disposal and Management 7.29

of water, due to the prolonged survival of its spores. Although this extended
survival is usually considered to be a disadvantage for normal purposes,
it may prove to be very useful in wastewater reuse studies, as Clostridium
perfringens may be found to have survival characteristics similar to those of
viruses or even helminth eggs.
Pathogens: The following pathogenic parameters can only be considered,
if suitable laboratory facilities and suitably trained staff are available:
Salmonella spp. Several species of Salmonellae may be present in raw
sewage from an urban community in a tropical developing country, including
S. typhi (causative agent for typhoid) and many others. It is estimated that a
count of 7000 Salmonellae/liter is typical in a tropical urban sewage with similar
numbers of Shigellae, and perhaps 1000 Vibrio cholera/liter. Both Shigella spp.
and V. cholera are more rapidly killed in the environment so; if removal of
Salmonellae can be achieved, then the majority of other bacterial pathogens will
also have been removed.
Enteroviruses. May give rise to severe diseases, such as Poliomyelitis and
Meningitis, or to a range of minor illnesses such as respiratory infections.
Although there is no strong epidemiological evidence for the spread of these
diseases via sewage irrigation systems, there is some risk and, it is desirable
to know to what extent viruses are removed by existing and new treatment
processes, especially under tropical conditions. Virus counts can only be
undertaken in a dedicated laboratory, as the cell culture techniques required
are very susceptible to bacterial and fungal contamination.
Rotaviruses. These viruses are known to cause gastro-intestinal problems
and, though usually present in lower numbers than enteroviruses in sewage,
they are known to be more persistent, so it is necessary to establish their
survival characteristics relative to enteroviruses and relative to the indicator
organisms in wastewaters. It has been claimed that the removal of viruses
in wastewater treatment occurs in parallel simultaneously or insimultaneity
would such better with the removal of suspended solids, as most virus
particles are solids-associated. Hence, the measurement of suspended solids
in treated effluents should be carried out as a matter of routine.
Intestinal Nematodes: It is known that nematode infections, in particular
from the roundworm Ascaris lumbricoides, can be spread by effluent reuse
practices. The eggs of A. lumbricoides are fairly large (45-70 mm × 35-50 mm)
and several techniques for enumeration of nematodes have been developed.

7.9.4 Reclaim of Treated Wastewater

Reclaimed water or recycled water, is former wastewater (sewage) that is treated
to remove solids and certain impurities, and used in sustainable landscaping
irrigation or to recharge groundwater aquifers. The purpose of these processes
is sustainability and water conservation, rather than discharging the treated
water to surface waters such as rivers and oceans. Cycled repeatedly through
7.30 Environmental Biotechnology

the planetary hydrosphere, all water on Earth is recycled water. But, typically
when we hear the term “recycled water” or “reclaimed water”, it means
wastewater that is sent from our home or business through a pipeline system
to a treatment facility where is treated to a level consistent with its intended
use. It is then routed directly to a recycled water system for uses such as
irrigation or industrial cooling.
• The recycling and recharging is often done by using the treated
wastewater for designated municipal sustainable gardening irrigation
applications. In most locations, it is intended to only be used for non-
potable uses, such as irrigation, dust control, and fire suppression.
• Reclaimed water is highly engineered for safety and reliability so that
the quality of reclaimed water is more predictable than many existing
surface and groundwater sources. Reclaimed water is considered safe
when appropriately used.
• Reclaimed water planned for use in recharging aquifers or augmenting
surface water receives adequate and reliable treatment before mixing
with naturally occurring water and undergoing natural restoration
processes. Some of this water eventually becomes part of drinking
water supplies.

7.9.5 Uses and benefits of reclaimed water

• The cost of reclaimed water exceeds that of potable water in many
regions of the world, where a fresh water supply is plentiful. However,
reclaimed water is usually sold to citizens at a cheaper rate to encourage
its use. As fresh water supplies become limited from distribution costs,
increased population demands, or climate change reducing sources,
the cost ratios will also evolve.
• Using reclaimed water for non-potable uses, saves potable water for
drinking, since less potable water will be used for non-potable uses.
• It sometimes contains higher levels of nutrients such as nitrogen,
phosphorus and oxygen which may somewhat help fertilize garden
and agricultural plants, when used for irrigation.
• The usage of water reclamation decreases the pollution sent to sensitive
environments. It can also enhance wetlands, which benefits the wildlife
depending on that ecosystem.
In most locations, reclaimed water is not directly mixed with potable
(drinking) water for several reasons:
• Utilities providing reclaimed water for non-potable uses do not treat
the water to drinking water standards.
• Varying amounts of pathogens, pharmaceutical chemicals (e.g.,
hormones from female hormonal contraception) and other trace
chemicals are able to pass through the treatment and filtering process,
Solid Waste Disposal and Management 7.31

potentially causing danger to humans. Modern technologies such as

reverse osmosis may help to somewhat overcome this problem. An
experiment by the University of New South Wales reportedly showed
a reverse osmosis system removed ethinylestradiol and paracetamol
from the wastewater, even at 1000 times the expected concentration.
• Drinking water standards were developed for natural ground water,
and are not appropriate for identifying contaminants in reclaimed
water. In addition to pathogens, and organic and endocrine disrupting
chemicals, a large number of compounds may be present in reclaimed
water. They cannot all be tested for, and there is a paucity of toxicity
information on many of the compounds.
Because of this, state regulatory agencies do not allow reclaimed water
to be used for drinking, bathing, or filling swimming pools. They also warn
those, who use reclaimed water for irrigation, to place a sign on their property,
warning people not to drink from the irrigation system, and to not use it
directly on fruits or vegetables.
 CHAPTER

8 Biological Methods of Pest Management

Biological methods of pest management in agriculture is a method of

controlling pests (including insects, mites, weeds and plant diseases) that
relies on predation, parasitism, herbivory, or other natural mechanisms. The
use of biological control is a fundamental tactic for pest suppression within an
effective Integrated Pest Management (IPM) program, and biological control
refers to the use of natural enemies against a pest population to reduce the
pest’s density and damage to a level lower than would occur in their absence.
IPM is an ecologically-based pest management strategy that forms a part
of the overall crop production system. Ideally, it incorporates all appropriate
methods from many scientific disciplines into a systematic approach to
minimize pest damage. IPM control tactics include a variety of approaches
including cultural control, resistant plant varieties, chemical control, and
biological control.
• Biological control as a management tool dates back over 1,000 years
when ancient Chinese citrus growers used ants to control caterpillar
larvae infesting their trees. It is one of the safest methods of control
since it is not toxic, pathogenic or injurious to humans.
• Biological control has the advantage of being self-perpetuating once
established and usually does not harm non-target organisms found in
the environment. In addition, it is not polluting or as disruptive to the
environment as chemical pesticides, nor does it leave residues on food,
a concern to many people today.
• However, the use of biological control does require detailed knowledge
of the pest’s biology and population dynamics, as well as the natural
enemies associated with the pest and their impact.
• Control is usually not complete with this IPM method since a residual
population of the pest is often necessary for the natural enemies to
remain in the environment, so some non-economic population levels
of pests must be acceptable or tolerated.
8.2 Environmental Biotechnology

• Biological control also fits well in combination with other IPM strategies.
There are many factors (crop, pest complex, and environment) that can
influence the success of beneficial organisms in reducing pest densities
to manageable levels. In many situations, the biological control method
will need to be utilized in concert with other tactics.
• Selecting the least disruptive management tactic is recommended by
IPM and should help conserve natural enemies.
• The use of biological control to manage pests is divided into three types
of approaches.
o
Importation refers to the search for better natural enemies to
introduce and permanently establish. The need for importation
biological control occurs when a pest is accidentally introduced into
an area and its natural enemies are left behind. An attempt is made
to locate these enemies and introduce them to reestablish the control
that often existed in the native range of the pest. This may be from
another country or another region of the same country.
o
Augmentation is an attempt to reduce a pest’s population to non-
economic levels by temporarily increasing natural enemy numbers
in an area through periodic releases. The natural enemies then
identifies and attack the pest. In some cropping systems, technology
has been developed to rear natural enemies artificially; so these
releases can be made economically. A number of commercial
companies have been created to produce a wide variety of natural
enemies, both predators and parasites.
o The third approach, conservation, is concerned with protecting the
natural enemies that are already present in an area. In conservation,
an attempt is made to manipulate the environment or the farming
practices to protect the natural enemies or provide needed resources
(e.g. alternate prey or food for adults) for them to survive and
build up populations to levels where they can manage the pest
and prevent it from causing economic damage to crops. Naturally-
occurring or indigenous natural enemies prevent many plant-
feeding insects from achieving pest status. Conservation of these
natural enemies allows them to operate, near their full potential.
Conserving natural enemies requires the use of farming practices
that are less disruptive to natural enemy populations. Insecticide
use destroys the target pest as well as many natural enemies that are
present. Reduced or carefully-timed insecticide treatments lower
the negative impact on beneficial organisms. Effective conservation
of natural enemies depends on: understanding the agro ecosystem;
use of selective pesticides; use of the least disruptive formulation of
the chemical; application of the insecticide only when necessary and
based on reasonable economic injury levels of the pest; and pesticide
Biological Methods of Pest Management 8.3

application at the time or place that is the least injurious to natural

enemies.
Natural enemies of insect pests fall basically into three types:
• Parasites (also called parasitoids) adults are free-living; the immature
stage lives on or inside a host and kills the host before the host completes
its development. Parasites lay one or more eggs on the outside of the
host body or they insert the eggs inside their host. The immature
parasite feeds on the host and requires only a single individual prey to
complete its development. Free-living adults may feed on nectar from
flowering plants or obtain nutrients by piercing the body of host insects
and withdrawing fluids (host-feeding). Parasites attack a particular
stage of the host, but all host stages are attacked by various parasites.
Parasites are often small, easily overlooked and can be difficult to
distinguish from other small non-parasitic flies and wasps. Parasites
are not harmful to humans and tend to attack and parasitize one, or at
most a few, closely related species of pest insects. Parasites are usually
members of the order Hymenoptera and a few are members of the order
Diptera. Parasites are often considered more effective natural enemies
than predators because many have a narrower host range, require
only one host to complete development, have an excellent ability to
locate and kill their host and, can respond rapidly to increases in host
populations.
• Predators include birds, fish, amphibians, reptiles, small mammals,
and arthropods. Arthropods (insects, mites and spiders) are the
most important predators in pest management and include lady
beetles, ground beetles, syrphid flies, green lacewings, assassin bugs,
predaceous bugs, minute pirate bugs, predatory mites, and spiders.
Predators are usually larger than the prey which they capture and kill.
They may use camouflage to “sit and wait” for prey or may be active
hunters. Predators usually deposit their eggs near their prey, so the
immature ones can immediately find their host and begin feeding.
Immature stages are mobile, usually consume more than one prey
during their development, are often generalist feeders (more than one
species of host is attacked), and usually, both the adults and immature
population feed on the prey insect.
• Diseases also occur among insects. Insect diseases are caused by
fungi, viruses, bacteria, protozoans, and other microorganisms. Insect-
parasitic nematodes are also included in this group of natural enemies.
Insect-parasitic nematodes are small worms that attack and kill insects
that live in moist habitats. A few species are currently being sold
commercially for insect control. Insect pathogens, including nematodes,
are an important component in suppressing pest species. Some insect
pathogens and nematodes are commercially available and can be
8.4 Environmental Biotechnology

manipulated to achieve biological control of specific pests. Both diseases

and nematodes, like parasites, tend to be specific to certain species
or groups of pests; they do not harm non-target organisms, such as
beneficial insects, animals, humans, or plants. They can quickly spread
through an insect population causing rapid mortality in a short period
of time, and can be important in the natural control of pest populations.
This phenomenon, called an epizootic, occurs when the insect pest
population level is high or environmental conditions are especially
suitable for the pathogen or disease-causing organism, enabling the
disease organism to spread from insect to insect very quickly. In high-
value crops, the pest population usually cannot be allowed to reach
a level where an epizootic can occur. However, epizootics can be an
important natural control of pests of forests, rangeland, and certain
types of field crops.
Insect viral pathogens vary in how they attack and kill their host. Most
insect viruses need to be ingested to successfully infect their host, though
some can be transferred from the parent insect to the offspring through the
egg. Symptoms usually occur within a few days after the virus is ingested.
The infected insect will appear sluggish, feeding will stop, and the cuticle will
have a pale discoloration and will often hang from its legs.

Caterpillar killed by viral infection

The infected insect will die one to two days after the symptoms appear.
The decomposing cadaver will burst, liberating the viral particles into the
environment. Important groups of viruses that attack insects are the—
• nuclear polyhedrosis viruses (NPV),
• cytoplasmic polyhedrosis viruses (CPV) and
• granulosis viruses (GV).
Biological Methods of Pest Management 8.5

Viruses usually attack the caterpillar stage, such as the Helicoverpa NPV
that invades the corn earworm larva or the codling moth GV that infects the
codling moth larva.
Baculoviruses is the most popular choice for microbial control as they are
distinct from any type of virus recorded from vertebrates. They have been
used regularly for pest control since the 1950s, particularly in forestry, where
they have been highly effective at controlling sawflies. Baculoviruses can
infect many species, mostly caterpillars and sawflies, but also some species
of beetle and flies. Baculoviruses infect their hosts through ingestion. Virus
particles invade the cells of the gut before colonizing the rest of the body.
Infection reduces mobility and feeding and insects are killed in five to eight
days. Mass production of baculoviruses can be done only in insects, but this is
economically viable for larger hosts such as caterpillars, and formulation and
application are straightforward.
The bacteria most important in insect pest management are in the genus
Bacillus. Species in this genus form spores that are toxic to the insect when
ingested. Symptoms of infected insects include, a loss of appetite, sluggishness,
discharge from the mouth and anus, discoloration and liquefaction and
putrefaction of the body tissues.

Caterpillar killed by a bacterial infection

Bacillus thuringiensis (commonly called Bt) is the most widely-used
bacterium for insect pest control. Different strains of Bt are specific against
caterpillars, mosquito larvae and some beetles and their larvae. Bacillus popillae
and B. lentimorbus cause “milky disease” of white grubs. “Milky disease” refers
to the white discoloration of the insect blood. The spores of B. popillae and B.
lentimorbus survive in the soil and are ingested by the grubs as they feed on
8.6 Environmental Biotechnology

roots of grasses. Bacillus thuringiensis, B. popillae, and B. lentimorbus have been

formulated into microbial insecticides by several companies for application to
crops in an augmentative manner.

Spores and bipyramidal crystals of Bacillus thuringiensis

• The major advantage of the Bacillus thuringiensis (Bt) toxin is that it is
harmful to only a few species of insects, while it is essentially harmless
to other animals and humans. These biological pesticides also degrade
rapidly in the environment. Thus, the use of such biological pesticides
appears to be a significantly more environmentally safe solution to pest
control than the classical (synthetic chemical) pesticides.
• Features of Bt biopesticides limit their use in insect control. In contrast
to contact insecticides, Bt insecticides must be ingested by the target
insect. The timing of Bt sprays is critical to attaining economic levels
of insect control. Usually Bt is applied when early instar larvae are
present, as older larvae are more tolerant. Bt sprays persist only a few
days on the leaf surface.
• The chemistry of the leaf surface, proteinases and sunlight contribute
to the degradation of CRY proteins. It is rare for a Bt insecticide to have
greater efficacy than the best available chemical control. Hence, Bt
adoption suffers at the hand of more efficacious chemical insecticides.
• Bt biopesticides have inherent advantages in certain pest control
applications. They are used as a resistance management tools in insect
control. Due to their distinct mode-of-action, they are alternated or
combined with chemical pesticides.
• Bt is especially suited for specialty or ‘high value crops.’ The tightening
of registration procedures for new chemical pesticides has led many of
the larger crop protection companies to take the decision not to register
products for use on specialty crops.
• Increased usage of Bt biopesticides will occur as organic markets
expand and consumer demand for eco-friendly pest control alternatives
in home gardens and treatments for high-value alternative crops.
Biological Methods of Pest Management 8.7

Most recently, genes that produce Bt toxins have been genetically

engineered into crop plants (corn, cotton, tobacco and potato) for season-long
protection against larval pests and some adult insects. Larval and adult pests
ingest the toxin after they have fed on the foliage and subsequently die.
A beneficial soil bacterium, Saccharopolyspora spinosa, produces a natural
metabolite, Spinosad, when cultured under aerobic fermentation conditions.
Spinosad has been formulated as a microbial insecticide. Insects become
poisoned with Spinosad when they ingest treated foliage or come into contact
with the microbial sprays of the metabolite. Sickened insects stop feeding,
become limp and are unable to move, and may appear to have weak tremors.
Spinosad is effective against a wide spectrum of insect pests, including
armyworms, European corn borer, diamondback moth, leaf miners, and
Colorado potato beetle.
Examples of Bacterial Insecticides:
Pathogen: Bacillus thuringiensis var. kurstaki (Bt)
Host range: Caterpillars (larvae of moths and butterflies)
Uses and comments: Effective for foliage-feeding caterpillars (and
Indian meal moth in stored grain). Deactivated rapidly in sunlight; apply
in the evening or on overcast days and direct some spray to lower surfaces
of leaves. Does not cycle extensively in the environment. Available as liquid
concentrates, wettable powders, and ready-to-use dusts and granules. Active
only if ingested.
Pathogen: Bacillus thuringiensis var, israelensis (Bti)
Host range: Larvae of Aedes and Psorophora mosquitoes, black flies, and
fungus gnats
Uses and comments: Effective against larvae only. Active only if ingested.
Culex and Anopheles mosquitoes are not controlled at normal application
rates. Activity is reduced in highly turbid or polluted water. It does not cycle
extensively in the environment. Applications generally made over wide areas
by mosquito and black-fly abatement districts. Not all Bti products are labeled
for use against fungus gnats.
Pathogen: Bacillus thuringiensis var. san diego
Host range: Larvae of Colorado potato beetle, elm leaf beetle adults
Uses and comments: Effective against Colorado potato beetle larvae
and the elm leaf beetle. Like other bacteria, it must be ingested. It is subject
to breakdown in ultraviolet light and does not cycle extensively in the
environment.
Pathogen: Bacillus thuringiensis var. tenebrionis
Host range: Larvae of Colorado potato beetle
8.8 Environmental Biotechnology

Uses and comments: Very similar to Bacillus thuringiensis var. san diego.
Foil contains a bacterial conjugate that produces toxins that act against these
same beetles and caterpillars.
Pathogen: Bacillus thuringiensis var. aizawa
Host range : Wax moth Caterpillars
Uses and comments : Used only for the control of wax moth infestations
in honeybee hives.
Pathogen: Bacillus popilliae and Bacillus lentimorbus
Host range : Larvae (grubs) of Japanese beetle
Uses and comments : The main Illinois lawn grub (the annual white grub,
Cyclocephala sp.) is not susceptible to milky spore disease. The disease is very
effective against Japanese beetle grubs and cycles effectively for years in the
soil.
Pathogen: Bacillus sphaericus
Host range : Larvae of Culex, Psorophora, and Culiseta mosquitoes, larvae
of some Aedes spp.
Uses and comments : Active only if ingested. Under development for
use against Culex, Psorophora, and Culiseta species; also effective against Aedes
vexans. Remains effective in stagnant or turbid water. Commercial formulations
will not cycle to infect subsequent generations.
Insect pathogenic fungi produces spores that germinate when they come
in contact with the insect cuticle during favorable conditions. Germinating
spores penetrate the insect cuticle and invade the body cavity. Hyphae rapidly
grow, filling the body cavity with a fungal mass, killing the insect. The fungus
also may produce a toxin. Hyphae penetrate outward through the softer parts
of the insect and under favorable moisture conditions, produce spores that
ripen and are released into the environment to complete the life cycle.
• Insects that are attacked by fungi often retain their shape, but usually
become hardened or “mummy-like” and appear “fuzzy” from the
fungal growth. There are many genera of fungi that attack insects.
• The most important ones are Metarhizium, Beauveria, Entomophthora and
Zoopthora. Metarhizium anisopliae and B. bassiana attack a wide range
of insects, such as grasshoppers, true bugs, aphids, caterpillars, and
beetles.
• Entomophthora muscae attacks many types of adult flies including the
seed corn maggot and hover fly. Zoophthora radicans attacks the potato
leafhopper and many aphids, and Z. phytonomi infects the alfalfa
weevil.
Fungi used as insecticides include the following:
• Beauveria bassiana: This common soil fungus has a broad host range
that includes many beetles. It infects both larvae and adults of many
Biological Methods of Pest Management 8.9

species. Understanding the interactions between Beauveria bassiana

and other soil microorganisms may be the key to successful use of this
fungus.
• Nomuraea rileyi: In soybeans, naturally occurring epidemic infections
of Nomuraea rileyi cause dramatic reductions in populations of foliage-
feeding caterpillars. Research, directed at predicting disease outbreaks
caused by this fungus, may help in determining the need for application
of insecticides.
• Verticillium lecanii: This fungus has been used in greenhouses to control
aphids and whiteflies.
• Lagenidium giganteum: This aquatic fungus is highly infectious to
larvae of several mosquito genera. It cycles effectively in the aquatic
environment (spores produced in infected larvae persist and infect
larvae of subsequent generations), even when mosquito density is low.
Its effectiveness is limited by high temperatures.
• Hirsutella thompsonii: Hirsutella thompsonii is a pathogen of the citrus
rust mite.

8.1 ADVANTAGES OF MICROBIAL INSECTICIDES

Individual products differ in important ways, but the following list of beneficial
characteristics applies to most microbial insecticides.
• The organisms used in microbial insecticides are essentially non-toxic
and non-pathogenic to wildlife, humans, and other organisms not
closely related to the target pest.
• The toxic action of most microbial insecticides is specific to a single
group or species of insects and this specificity means that most
microbial insecticides do not directly affect beneficial insects (including
predators or parasites of pests) in treated areas. If necessary, most
microbial insecticides can be used in conjunction with synthetic
chemical insecticides, because in most cases, the microbial product is
not deactivated or damaged by residues of conventional insecticides.
• Because their residues present no hazards to humans or other animals,
microbial insecticides can be applied even when a crop is almost ready
for harvest.
• In some cases, the pathogenic microorganisms can become established
in a pest population or its habitat and provide control during subsequent
pest generations or seasons.
Microorganisms that are pathogenic to insects provide a wealth of
biological material that can be exploited by humans to control insect pests.
Innovative applications of a few such entomopathogens are found throughout
the world, but widespread commercial production of microbial insecticides
awaits further studies of the biology, ecology, and pathogenicity of the agents.
8.10 Environmental Biotechnology

Genetic engineering techniques may be used to increase the virulence of

these microorganisms, as well as, to make them more tolerant of physical and
chemical conditions and perhaps, to broaden their host ranges. The use of
microbial insecticides could decrease our dependence on chemical pesticides.

8.2 DISADVANTAGES OF MICROBIAL INSECTICIDES

The limitations or disadvantages listed below do not prevent the successful
use of microbial insecticides.
• Understanding how these limitations affect specific microorganisms,
will help users to choose effective products and take necessary steps to
achieve successful results.
• Because a single microbial insecticide may be toxic to only a specific
species or group of insects, each application may control only a portion
of the pests present in a field, garden, or lawn. If other types of pests
are present in the treated area, they will survive and may continue
to cause damage. Conventional insecticides are subject to similar
limitations because, they too, are not equally effective against all pests.
Nonetheless, the negative aspect of selectivity is often more noticeable
for microbials.
• Heat, desiccation (drying out), or exposure to ultraviolet radiation
reduces the effectiveness of several types of microbial insecticides.
Consequently, proper timing and application procedures are especially
important for some products.
• Special formulation and storage procedures are necessary for some
microbial pesticides. Although these procedures may complicate the
production and distribution of certain products, storage requirements
do not seriously limit the handling of microbial insecticides that are
widely available. (Store all pesticides, including microbial insecticides,
according to label directions.)
• Because several microbial insecticides are pest-specific, the potential
market for these products may be limited. Their development,
registration, and production costs cannot be spread over a wide
range of pest-control sales. Consequently, some products are not
widely available or are relatively expensive (several insect viruses, for
example).

8.3 INSECT GROWTH REGULATORS

Several features of Insect Growth Regulators (IGRs) make them attractive as
alternatives to broad-spectrum insecticides. Because they are more selective,
they are less harmful to the environment and more compatible with pest
management systems that include biological controls.
Compared with conventional pesticides, insect growth regulators are:
Biological Methods of Pest Management 8.11

• More selective
• Less harmful to the environment
• More compatible with biological controls
• Less likely to be lost because of resistance
Insects have demonstrated a propensity to develop resistance to
insecticides. Broad-spectrum insecticides that are used routinely will
eventually be lost because of resistance. Intelligent use of IGRs should reduce
the likelihood of resistance-developing. IGRs show good potential on pears
because their selectivity preserves the natural enemies that can help control
pear psylla. Because of its ability to rapidly develop resistance to insecticides,
it is important that psylla be controlled by an integrated system, incorporating
several control factor. The selectivity of IGRs is due to the different way they
act on insects, compared with most conventional insecticides. Virtually, all
chemicals used to control insects fall into one of three categories: neurotoxins,
growth regulators and behavior modifiers.
Neurotoxins: Most chemicals used to control insects are neurotoxins
which interfere with normal nerve function. Organophosphate insecticides
were derived from nerve gases that were first exploited for military purposes.
Other insecticides were discovered by testing chemicals to find those that
killed pests quickly. About the only thing that kills quickly is a neurotoxin;
so chemicals that acted on neurotransmissions were sought and developed
as insecticides. In the early discovery and development of insecticides, efforts
were focused on chemistry rather than biology. Because all animals share,
basically the same neurochemical systems, neurotoxins are toxic to all animals.
Insect growth regulators: The origin of IGRs was entirely different. Their
discovery was based on knowledge of how insects grow, develop, function
and behave. They have been discovered in two ways.
• One way was to expose an insect to IGRs and observe abnormalities in
how it develops, functions or behaves. Chemicals that produce desired
effects were developed.
• Another was to find out what processes in the insects’ development
involve hormones and to use those hormones as models to synthesize
chemical analogs that will interfere with normal insect growth and
development. Because IGRs act on systems unique to insects, or shared
with close relatives, they are less likely to affect other organisms.
Behavior modifiers: Behavior-affecting chemicals, such as pheromones,
are discovered in the same way as IGRs but tend to be even more specific.
Pheromones aid the sexes of a single species to find each other so that effort is
not wasted chasing mates of a different species.
How insect growth regulators work? : Insects wear their skeletons on
the outside. The skeletons are called exoskeletons. As the insect grows, a
new exoskeleton must be formed inside the old exoskeleton and the old one
8.12 Environmental Biotechnology

shed. The new one then swells to a larger size and hardens. The process is
called molting. The changes from larval to adult form, a process called
metamorphosis, also take place during molting. Hormones control the phases
of molting by acting on the epidermis, which is part of the exoskeleton.
Chitin synthesis inhibitors: These prevent the formation of chitin,
a carbohydrate that is an important structural component of the insect’s
exoskeleton. When treated with one of these compounds, the insect grows
normally until, the time to molt. When the insect molts, the exoskeleton is not
properly formed and it dies. Death may be quick, but in some insects it may
take several days. As well as disrupting molting, chitin synthesis inhibitors
can kill eggs by disrupting the normal development of the embryo.
Role of Juvenile hormones and its analogues for pest management:
Juvenile hormone, also called Neotenin, is a hormone in insects, secreted by
endocrine glands near the brain (corpora allata), that controls the retention
of juvenile characters in larval stages. The hormone affects the process of
molting, the periodic shedding of the outer skeleton during development, and
in adults, it is necessary for normal egg production in females.

Juvenile hormone is produced in the corpora allata of insects

• Juvenile Hormones (JHs) are a group of acyclic sesquiterpenoids that
regulate many aspects of insect physiology. JHs regulate development,
reproduction, diapause, and polyphenisms.
• Juvenile Hormone Analogs (JHAs) represent a class of insecticides that
were designed specifically to disrupt endocrine-regulated processes
relatively unique to insects.
Synthetic analogues of the juvenile hormone are used as an insecticide,
preventing the larvae from developing into adult insects. Since the early 1970s,
numerous analogs of JH have been tested for insecticidal activity
JH itself is expensive to synthesize and is unstable in light. At high levels
of JH, larva can still molt, but the result will only be a bigger larva, not an
adult. Thus, the insect’s reproductive cycle is broken.
• One JH analogue, methoprene, is approved by the WHO for use in
drinking water cisterns to control mosquito larvae. Methoprene is a
growth regulator and prevents the immature insect from developing
Biological Methods of Pest Management 8.13

normally. Juvenile hormone has several functions within an insect. If

the hormone is present when the larva molts the insect will remain a
larva. If the hormone is absent, the larva will become a pupa and begin
to develop into an adult insect. Since methoprene mimics juvenile
hormone, the larva is prevented from developing into an adult.
• Pyriproxyfen, 4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether,
is a juvenile hormone analog with relatively low mammalian toxicity
that was first registered in Japan in 1991 for controlling public health
pests. It has been used for controlling a variety of insect pests including
mosquitoes and other flies, whiteflies, scale insects and aphids,
cockroaches, lepidopteron, ants, fleas, locusts and grasshoppers, and
thrips.

Chemical structures of the juvenile hormones, terpenoidal (methoprene),

and nonterpenoidal (fenoxycarb, phyproxyfen and diofenolan)
juvenile harmone analogs
Juvenile hormone analogs and mimics: When applied to an insect, these
abnormal sources of juvenilizing agent can have striking consequences.
• For example, if the normal course of events calls for a molt to the pupal
stage, an abnormally high level of juvenilizing agent will produce
another larval stage or produce larval-pupal intermediates.
Juvenoid insect growth regulators (IGRs) can also act on eggs. They can
cause sterilization, disrupt behavior and, disrupt diapause—the process that
triggers dormancy before the onset of winter. In theory, all insect systems
influenced by juvenile hormone are potential targets for a juvenoid IGR.
8.14 Environmental Biotechnology

The early juvenoid IGRs were true analogs of juvenile hormone and were
unstable when exposed to ultraviolet light. This seriously limited their use
in plant protection. Another group of juvenoid IGRs, called juvenile mimics,
was discovered. Entomologist found that extracts of many plant tissues have
juvenilizing effects, but they have different chemical structures from juvenile
hormones and are much more stable. They have been used as models to
synthesize some highly effective and stable juvenile hormone mimics which
have potential to control tree fruit pests.
Anti-juvenile hormone agents: Anti-juvenile hormone agents cancel the
effect of juvenile hormone by blocking juvenile hormone production.
• For example, an early instar, treated with an anti-juvenile hormone
agent, molts prematurely into a non-functional adult. A disadvantage
of these chemicals is that, they are so selective that, they may not be
economic for a manufacturer to develop.

8.4  USE OF PHEROMONES FOR PEST MANAGEMENT

With increasing public concern about the use of toxic pesticides to control
insects and other pestiferous organisms, resource managers are turning
toward other techniques of integrated pest management. Some of these
techniques are common-sense approaches, such as completing sanitation or
clean-up activities before the season when the damaging stages of an insect
pest are present.
Other tools are more “hi-tech”, such as the use of odors called
semiochemicals, and in particular, pheromones, to manipulate the behavior of
insect pests. With these non-toxic and biodegradable chemicals, insects can
be lured into traps or foiled into wasting energy that they normally need
for locating food and mates. Semiochemicals are chemical signals that are
produced by a plant or animal and are detected by a second plant or animal
and cause a response in the second organism. Many species depend on these
chemical signals for survival.
Pheromones: Pheromones are a class of semiochemicals that insects and
other animals release to communicate with other individuals of the same
species. The key to all of these behavioral chemicals is that they leave the body
of the first organism, pass through the air (or water) and reach the second
organism, where they are detected by the receiver.
• In insects, these pheromones are detected by the antennae on the head.
The signals can be effective in attracting faraway mates, and in some
cases, can be very persistent, remaining in place and active for days.
• Long-lasting pheromones allow marking of territorial boundaries or
food sources. Other signals are very short-lived, and are intended
to provide an immediate message, such as a short-term warning of
danger or a brief period of reproductive readiness.
Biological Methods of Pest Management 8.15

• Pheromones can be of many different chemical types, to serve differ-

ent functions. As such, pheromones can range from small hydrophobic
molecules to water-soluble peptides.
• Over the last 40 years, scientists have identified pheromones from over
1,500 different species of insects. Pheromones have also been isolated
from many higher animals such mammals and reptiles. Human phero-
mones remain elusive.
• Scientists have found certain chemical effects associated with the hu-
man reproductive cycle, but have not identified any powerful attrac-
tants for humans, so far.
• With insects, though, pheromones have found wide application in the
fields of agriculture, forestry, and urban pest management, and there
are companies that specialize in the discovery, manufacturing, and
sales of pheromone-related products.

8.5  PHEROMONES: INSECT PEST MANAGEMENT

There are three main uses of pheromones in the integrated pest management
of insects.
• The most important application is in monitoring a population of insects
to determine if they are present or absent in an area or to determine
if enough insects are present to warrant a costly treatment. This
monitoring function is the keystone of integrated pest management.
Monitoring is used extensively in urban pest control of cockroaches,
in the management of stored grain pests in warehouses or distribution
centers, and to track the nationwide spread of certain major pests
such as the gypsy moth, Medfly, and the Japanese beetle. With major
increases in worldwide trade, exotic pests are being brought into ports
of entry in cargo containers and packaging materials (ship dunnage).
Sometimes, containers from ships are transferred uninspected to semi-
trailers and trucked far inland. When the containers are opened and
packaging materials are removed, the exotic insect pests are able to
disperse without the usual level of scrutiny provided at ports of entry.
Pheromone traps are currently in use to monitor the movement of such
exotic insect pests into most major North American ports of entry.
• A second major use of pheromones is to mass trap insects to remove
large numbers of insects from the breeding and feeding population.
Massive reductions in the population density of pest insects, ultimately,
help to protect resources such as food or fiber for human use. Mass
trapping has been explored with pine bark beetles and has resulted
in millions of insects attracted specifically into traps and away from
trees. Relatives of bark beetles, called ambrosia beetles, have been
mass-trapped from log sorting and timber processing areas throughout
British Columbia. These trapping operations have reduced damage to
8.16 Environmental Biotechnology

the wood in raw logs and newly cut boards. Mass trapping has also
been used successfully against the codling moth, a serious pest of
apples and pears. Another common example of mass trapping involves
yellow jackets, which can become bothersome at the end of the summer
season. However, mass trapping of yellow jackets in colorful yellow-
green traps is carried out with a food attractant, rather than pheromone
bait.
• A third major application of pheromones is in the disruption of
mating in populations of insects. This has been most effectively used
with agriculturally-important moth pests. In this scenario, synthetic
pheromone is dispersed into crops and the false odor plumes attract
males away from females that are waiting to mate. This causes a
reduction of mating, and thus, reduces the population density of the
pests. In some cases, the effect has been so great that the pests have
been locally eradicated.
In summary, pheromones are species-specific chemicals that affect insect
behavior, but are not toxic to insects. They are active (e.g. attractive) in
extremely low doses (one millionth of an ounce) and are used to bait traps or
confuse a mating population of insects. Pheromones can play an important
role in integrated pest management for structural, landscape, agricultural, or
forest pest problems.

8.6  BIOLOGICAL CONTROL OF WEEDS

Biological control is a long-term solution which is most effective as part of
an integrated weed management approach. Plants that have become weeds
in Australia are rarely invasive and troublesome in their home country. This
is often because populations in the home country are regulated by a variety
of natural enemies such as insects and pathogens (disease-causing organisms
like fungi and bacteria) that attack the seeds, leaves, stems and roots of the
plant. If plants are introduced to a new country without these natural enemies,
their populations may grow unchecked to the point where they become so
prevalent that they are regarded as weeds.
The biological control approach makes use of the invasive plant’s
naturally-occurring enemies, to help reduce  the invasive plant’s impact on
agriculture and the environment. It simply aims to reunite weeds with their
natural enemies and achieve sustainable weed control. These natural enemies
of weeds are often referred to as biological control agents. It is critical that
the biological control agents do not become pests themselves. Considerable
host-specificity testing is done prior to the release of biological control agents
to ensure they will not pose a threat to non-target species such as native and
agricultural plants. Not all weeds are suitable for biological control.
A biological control agent is generally only used when the cost of
conventional control methods such as herbicides, mechanical control or fire is
Biological Methods of Pest Management 8.17

so great, both in dollar terms and impact on the environment, that there is little
option than to pursue the biological control avenue.

8.6.1  The process

A weed becomes a problem in the introduced range because its population
density fluctuates around an equilibrium that is above a threshold at which
the weed begins to affect the economic or ecological sustainability of the
ecosystem.

The process of biological control of weeds

Best case scenario of the course of events in classical biological control
programs targeting weeds invading habitats such as rangeland, pasture, or
natural ecosystem.
 Following their introduction and establishment, populations of biological
control agents build up to very high levels due to the abundance of their
host plant. Eventually, their attack on the plant causes a decline in the weed
population. This, in turn, leads to a decline in the numbers of biological
control agents until an equilibrium is reached between the amount of damage
caused by an agent and regeneration by the weed. In a successful biological
control program, this new equilibrium is below the damage threshold that the
ecosystem can tolerate.
The advantages of biological control of weeds are:
• It is inexpensive.
• It poses little threat to non-target organisms.
• Once established, biological control agents are self-perpetuating and
can spread on their own.
• Little additional effort is required once a biological control organism
is established, while other control methods require action or inputs,
periodically.
• The environmental impact is generally low.
8.18 Environmental Biotechnology

The main drawbacks to biological control of weeds are:

• There is always some risk and concern with introducing an exotic
organism into the environment. The main concern is a host shift of a
biological control agent that results in the agent feeding on a desirable
plant.
• Biological control agents are not available for all target pests. Some of
the target weeds are closely related to plants that are desirable, so the
risk of introducing an agent is too great.
• Research time and money is needed to locate biological agents and
screen them for host range before the agents are released. It generally
takes years of research and testing, before agents are released.
• Biological control takes place slowly, in most cases. Localized weed
problems may not be eliminated quickly enough to satisfy public
opinion.
• Biological control relies on populations of the weed and the agent to
maintain the system. Thus, weeds are not completely eliminated. Also,
there is often highs and lows in population levels of the weed and the
agent both in time and space. Thus, there are some situations where
biological control does not seem to work well.
If there are advantages and disadvantages, how is the decision to use
biological control determined?
The decision is made on a case-by-case basis. The potential impact of the
weed, the alternative control measures available, the risk to the environment,
and the consequences of doing nothing are all considered. The scientific
information and the social values, all influence the decision. Once the organism
is released, there may be little good alternative to reverse the decision, so this
decision is taken seriously. This is further complicated by the fact that social
values change through time and, that the scientific information available will
also change as new information becomes available. There are a number of
regulations in place that impact the release of organisms.
Biocontrol and the environment:
• Biological control is attractive because it has negligible environmental
impact. Most biocontrol organisms are very selective to their target
pests thus posing little danger of damage to non-target species.
• They do not cause contamination of groundwater or leave residues
behind.
• Biocontrol also has its limitations. It may not consistently provide a
high level of pest control because environmental conditions may not
always be suitable for the growth and reproduction of the biocontrol
organism.
• Therefore, biological control is well adapted for use within an
integrated pest management program where other cultural practices
will complement and boost the level of control.
Biological Methods of Pest Management 8.19

• Since biological control uses living organisms, the user must be

educated on how to handle the product and under what circumstances
it will work best.

8.7  CHROMOSOMAL MANIPULATION

Methods to manage invasive species range from ignoring them and hoping,
they will go away through to a variety of options for physical removal, biocides
and biological control. For well-established and widely distributed pests,
the only realistic options currently available are augmentative and classical
biological control, and sterile male release programs, both of which have
significant constraints on their application. As a result, most invasive pests
remain uncontrolled except at small scales and for short periods. In the 1960s,
entomologists speculated that genetic techniques could be a powerful means
of controlling pest populations, based on the observation that meiotic drive
(a genetic sex ratio distorter) had apparently driven some insect populations
to extinction. Practical development of such techniques lied fallow, however,
until recent developments in recombinant genetics stimulated renewed
interest in the field. Currently, at least three recombinant methods for pest
control are being tested in the laboratory. They are:
• repressible male sterility,
• virally vectored immuno contraception, and
• female-biased sex ratio distortion.
Another widely publicized study has speculated that the escape of even
one carrier of a “Trojan gene” (a construct, that pleiotropically enhances mating
advantage, while otherwise, reducing fitness) could cause species extinction,
an issue of considerable concern with regards to accidental escapement of
genetically modified (GM) organisms, but also, one, that offers options for pest
control, if properly managed. A number of recent studies have modeled the
potential for pest control of methods that have been proposed, incorporating
varying degrees of ecological reality. All conclude that pest control using
genetic methods is feasible, at least under the conditions specified in the
models. Three broadly different approaches for using recombinant genetics to
control pests have been investigated:
• genetically engineered viruses that, when incorporated into a bait, act
as a species-specific toxin or sterilizer;
• self-disseminating engineered viral diseases; and
• Non-disseminating (i.e., sexually transmitted) “autocidal” genes.
In the last, include as a special case, chromosomal modifications, designed
to achieve the same outcomes, as the recombinant approaches
Release of Insects carrying a Dominant Lethal: A method using
recombinant DNA technology to create genetically modified insects called
8.20 Environmental Biotechnology

RIDL (Release of Insects carrying a Dominant Lethal) is under development

by a company called Oxford Insect Technologies (Oxitec), UK.
• The method works by introducing a repressible “Dominant Lethal”
gene into the insects. This gene kills the insects but it can be repressed
by an external additive, which allows the insects to be reared in
manufacturing facilities.
• This external additive is commonly administered orally, and so can be
an additive to the insect food. The insects can also be given genetic
markers, such as DsREDfluorescence, that make monitoring the
progress of eradication easier, preferably under the field conditions.
• There are potentially several types of RIDL, but the more advanced
forms have a female-specific dominant lethal gene. This avoids the need
for a separate sex separation step, as the repressor can be withdrawn
from the final stage of rearing, leaving only males.
• These males are then released in large numbers into the affected region.
The released males are not sterile, but any female offspring their mates
produce will have the dominant lethal gene expressed, and so will die.
The number of females in the wild population will therefore decline,
causing the overall population to decline.
• Using RIDL means that the males will not have to be sterilized by
radiation before release, making the males healthier when they need to
compete with the wild males for mates.
• Progress towards applying this technique to mosquitos has been made
by researchers at Imperial College London who created the world’s
first transgenic malaria mosquito.
• A similar technique is the daughterless carp, a genetically modified
organism produced in Australia by the CSIRO in the hope of eradicating
the introduced carp from the Murray River system.
• As stated earlier, biotechnological approaches based on genetically
modified organism (transgenic organisms) are still under development.
However, since no legal framework exists to authorize the release of
such organisms in the nature, sterilization by irradiation remains the
most used technique.

8.8  STERILE MALE TECHNOLOGY

Screwworm was the first pest successfully eliminated from an area through
the sterile male technique by the use of an area-wide approach. The sterile
insect technique is a method of ‘biological control, whereby overwhelming
numbers of sterile insects are released’. The released insects are normally male,
as it is the female that causes the damage, usually by laying eggs in the crop,
or, in the case of mosquitoes, taking a blood-meal from humans. The sterile
males compete with the wild males for female insects. If a female mates with a
sterile male then it will have no offspring, thus reducing the next generation’s
Biological Methods of Pest Management 8.21

population. Repeated release of insects can eventually wipe out a population,

though it is often more useful to consider controlling the population rather
than eradicating it.
• The technique has successfully been used to eradicate the Screw-worm
fly (Cochliomyia hominivorax) in areas of North America. There have
also been many successes in controlling species of fruit flies, most
particularly, the Medfly (Ceratitis capitata), and the Mexican fruit fly
(Anastrepha ludens).
• Insects are mostly sterilized with radiation, which might weaken the
newly sterilized insects, if doses are not correctly applied, making
them less able to compete with wild males. However, other sterilization
techniques are under development which would not affect the insects’
ability to compete for a mate.
• The technique was pioneered in the 1950s by American entomologists
Dr. Raymond C. Bushland and Dr. Edward F. Knipling. For their
achievement, they jointly received the 1992 World Food Prize.
Drawbacks
• As with insecticide treatment, repeated treatment is sometimes
required to suppress the population before the use of sterile insects.
• Sex separation could be difficult for some species, though this can be
easily performed on Medfly and screwworm, for example.
• Radiation treatment in some cases affects the health of males, so
sterilized insects in such cases are at a disadvantage when competing
for females.
• The technique is species-specific. For instance, there are 22 species of
Tsetse fly in Africa, and the technique must be implemented separately
for each.
• Standard operating procedures of mass rearing and irradiation do not
leave room for mistakes. Since the Fifties, when SIT was first used as
a means for pest control, several failures have occurred in different
places around the world where non-sterilized artificially produced
insects were released before the problem was spotted.
• Application to large areas should be long-lasting, otherwise, migration
of wild insects from outside the control area could repopulate.
• The major drawback to this technique is that the cost of producing
such a large number of sterile insects is often prohibitive in poorer
countries.

8.9  ENVIRONMENTAL EPIDEMIOLOGICAL SURVEYS AS INDICES

OF HEALTH HAZARDS FOR ENVIRONMENTAL POLLUTION
Epidemiology is the study of the distribution and determinants of disease
frequency in human populations and the application of this study to control
health problems.
8.22 Environmental Biotechnology

• The term study includes both, surveillance, whose purpose is to monitor

aspects of disease occurrence and, spread—that are related to effective
control, and epidemiologic research, whose goal is to collect valid and
precise information about the causes, preventions, and treatments for
disease.
• The term ‘disease’ refers to a broad array of health-related states and
events including diseases, injuries, disabilities, and death.
Epidemiologic research encompasses several types of study designs,
including experimental studies and observational studies such as cohort
and case-control studies. Each type of epidemiologic study design simply
represents a different way of harvesting information. The selection of one
design over another depends on the particular research question, concerns
about validity and efficiency, and practical and ethical considerations. For
example, experimental studies, also known as trials, investigate the role of
some factor or agent in the prevention or treatment of a disease. In this type
of study, the investigator assigns individuals to two or more groups that
either receive or do not receive the preventive or therapeutic agent. Because
experimental studies closely resemble controlled laboratory investigations,
they are thought to produce the most scientifically rigorous data of all the
designs.
However, because experimental studies are often infeasible because of
difficulties in enrolling participants, high costs, and thorny ethical issues,
most epidemiologic research is conducted using observational studies.
Observational studies are considered “natural” experiments because the
investigator lets nature take its course. Observational studies take advantage
of the fact that people are exposed to noxious and/or healthy substances
through their personal habits, occupation, place of residence, and so on. The
studies provide information on exposures that occur in natural settings, and
they are not limited to preventions and treatments.
Furthermore, they do not suffer from the ethical and feasibility issues
of experimental studies. For example, although it is unethical to conduct an
experimental study of the impact of drinking alcohol on the developing fetus
by assigning newly-pregnant women to either a drinking or non-drinking
group, it is perfectly ethical to conduct an observational study by comparing
women who choose to drink during pregnancy with those, who decide not to
do so.
The two principal types of observational studies are cohort and case-
control studies. A classical cohort study examines one or more health effects
of exposure to a single agent. Subjects are defined according to their exposure
status and followed over time to determine the incidence of health outcomes.
In contrast, a classical case-control study examines a single disease in relation
to exposure to one or more agents. Cases who have the disease of interest
and controls, who are a sample of the population that produced the cases,
Biological Methods of Pest Management 8.23

are defined and enrolled. The purpose of the control group is to provide
information on the exposure distribution in the population that gave rise to
the cases. Investigators obtain and compare exposure histories of cases as well
as controls.
Additional observational study designs include cross-sectional studies and
ecologic studies. A cross-sectional study examines the relationship between a
disease and an exposure among individuals in a defined population at a point
in time. Thus, it takes a snapshot of a population and measures the exposure
prevalence in relation to the disease prevalence. An ecologic study evaluates
an association using the population rather than the individual as the unit of
analysis. The rates of disease are examined in relation to factors described
on the population level. Both the cross-sectional and ecologic designs have
important limitations that make them less scientifically rigorous than cohort
and case-control studies.
An overview of these study designs is provided in the following table:
Main Types of Epidemiologic Studies
Type of study Characteristics
Experimental Studies preventions and treatments for diseases;
investigator actively manipulates which groups receive
the agent under study.
Observational Studies causes, preventions, and treatments for diseases:
investigator passively observes, as nature takes its
course.
Cohort Typically examines multiple health effects of an exposure:
subjects are defined according to their exposure levels
and followed for disease occurrence.
Case-control Typically examines multiple exposures in relation to a
disease; subjects are defined as cases and controls, and
exposure histories and compared.
Cross-sectional Examines relationship between exposure and disease
prevalence in a defined population at a single point in
time.
Ecological Examines relationship between exposure and disease
with population-level rather than individual-level data.
The goal of all these studies is to determine the relationship between
an exposure and a disease with validity and precision using a minimum of
resources. Validity is defined as the lack of bias and confounding. Bias is an
error committed by the investigator in the design or conduct of a study that
leads to a false association between the exposure and disease. Confounding,
on the other hand, is not the fault of the investigator, but rather, reflects the
8.24 Environmental Biotechnology

fact that epidemiologic research is conducted among free-living humans with

unevenly distributed characteristics.
As a result, epidemiological studies that try to determine the relationship
between an exposure and disease are susceptible to the disturbing influences
of extraneous factors, known as confounders. Precision is the lack of random
error, which leads to a false association between the exposure and disease just
by “chance,” an uncontrollable force that seems to have no assignable cause.
Several factors help epidemiologists determine the most appropriate study
design for evaluating a particular association, including the hypothesis being
tested, state of knowledge, the frequency of the exposure and the disease, and
the expected strength of the association between the two.

8.10 TOXICITY
Toxicity is the degree to which a substance can damage a living or non-living
organism. Toxicity can refer to the effect on a whole organism, such as an
animal, bacterium, or plant, as well as the effect on a substructure of the
organism, such as a cell (cytotoxicity) or an organ (organ toxicity), such as the
liver (hepatotoxicity). By extension, the word may be metaphorically used to
describe toxic effects on larger and more complex groups, such as the family
unit or society at large.

Symbol of Toxicity
A central concept of toxicology is that effects are dose-dependent; even
water can lead to water intoxication when taken in too many doses, whereas,
for even a very toxic substance such as snake venom, there is a dose below
which there is no detectable toxic effect. Toxicity is species-specific, lending
cross-species analysis, problematic. Newer paradigms and metrics are
evolving to bypass animal-testing, while maintaining the concept of toxicity
endpoints.
• Toxicology: Toxicology is the study of the nature, effects, detection, and
mitigation of poisons and the treatment or prevention of poisoning.
• Toxin: A toxin is a toxic substance.
Biological Methods of Pest Management 8.25

8.10.1  Types of toxicity

There are generally three types of toxic entities; chemical, biological, and
physical:
• Chemical toxicants include inorganic substances such as lead, mercury,
asbestos, hydrofluoric acid, and chlorine gas, organic compounds such
as methyl alcohol, most medications, and poisons from living things.
• Biological toxicants include bacteria and viruses that can induce
disease in living organisms. Biological toxicity can be difficult to
measure because the “threshold dose” may be a single organism.
Theoretically, one virus, bacterium or worm can reproduce to cause
a serious infection. However, in a host with an intact immune system
the inherent toxicity of the organism is balanced by the host’s ability to
fight back; the effective toxicity is then a combination of both parts of
the relationship. A similar situation is also present with other types of
toxic agents.
• Physical toxicants are substances that, due to their physical nature,
interfere with biological processes. Examples include coal dust and
asbestos fibers, both of which can ultimately be fatal if inhaled.

8.10.2  Factors influencing toxicity

Toxicity of a substance can be affected by many different factors, such as the
pathway of administration (whether the toxin is applied to the skin, ingested,
inhaled, injected), the time of exposure (a brief encounter or long-term), the
number of exposures (a single dose or multiple doses, over time), the physical
form of the toxin (solid, liquid, gas), the genetic makeup of an individual,
an individual’s overall health, and many others. Several of the terms used to
describe these factors have been included here.
• Acute exposure: A single exposure to a toxic substance which may
result in severe biological harm or death; acute exposures are usually
characterized as lasting no longer than a day.
• Chronic exposure: Continuous exposure to a toxin over an extended
period of time, often measured in months or years; it can cause
irreversible side effects.

8.11  MEASURING TOXICITY

(Use of LC50 and LD50 of organisms for evaluation of toxicity)
Toxicity can be measured by its effects on the target (organism, organ, tissue
or cell). Because individuals typically have different levels of response to the
same dose of a toxin, a population-level measure of toxicity is often used which
relates the probabilities of an outcome for a given individual in a population.
LC50: Lethal Concentration 50. An LC50 value is the concentration of a
material in air that will kill 50% of the test subjects (animals, typically mice
8.26 Environmental Biotechnology

or rats) when administered as a single exposure (typically 1 or 4 hours). This

value gives you an idea of the relative toxicity of the material.
This value applies to vapors, dusts, mists and gases. Solids and liquids use
the closely related LD50 value (50% lethal dose).
LD50: Lethal Dose 50. It’s the amount of a solid or liquid material that it
takes to kill 50% of test animals (for example, mice or rats) in one dose.
Toxicologists can use many kinds of animals, but most often, testing is
done with rats and mice. It is usually expressed as the amount of chemical
administered (e.g., milligrams) per 100 grams (for smaller animals) or per
kilogram (for bigger test subjects) of the body weight of the test animal.
The LD50 can be found for any route of entry or administration, but dermal
(applied to the skin) and oral (given by mouth) administration methods are
the most common.
Common procedures to do LD50/LC50 tests:
• In nearly all cases, LD50 tests are performed using a pure form of the
chemical. Mixtures are rarely studied.
• The chemical may be given to the animals by mouth (oral); by applying
on the skin (dermal); by injection at sites such as the blood veins (i.v.-
intravenous), muscles (i.m. - intramuscular) or into the abdominal
cavity (i.p. - intraperitoneal).
• The LD50 value obtained at the end of the experiment is identified as the
LD50 (oral), LD50 (skin), LD50 (i.v.), etc., as appropriate.
• Researchers can do the test with any animal species but they use rats
or mice most often. Other species include dogs, hamsters, cats, guinea-
pigs, rabbits, and monkeys.
• In each case, the LD50 value is expressed as the weight of chemical
administered per kilogram body weight of the animal and it states the
test animal used and route of exposure or administration; e.g., LD50
(oral, rat) - 5 mg/kg, LD50 (skin, rabbit) - 5 g/kg. So, the example “LD50
(oral, rat) 5 mg/kg” means that 5 milligrams of that chemical for every
1 kilogram body weight of the rat, when administered in one dose by
mouth, causes the death of 50% of the test group.
• If the lethal effects from breathing a compound are to be tested, the
chemical (usually a gas or vapor) is first mixed in a known concentration
in a special air chamber where the test animals will be placed.
• This concentration is usually quoted as parts per million (ppm)
or milligrams per cubic meter (mg/m3). In these experiments, the
concentration that kills 50% of the animals is called an LC50 (Lethal
Concentration 50) rather than an LD50.
• When an LC50 value is reported, it should also state the kind of test
animal studied and the duration of the exposure, e.g., LC50 (rat) - 1000
ppm/ 4 hr or LC50 (mouse) - 5mg/m3/ 2hr.
Biological Methods of Pest Management 8.27

8.11.1  Toxic
Toxic is defined by OSHA (Occupational Safety and Health Administration)
as, a chemical which falls in any of these three categories:
• A chemical that has a median lethal dose (LD50) of more than 50
milligrams per kilogram but not more than 500 milligrams per kilogram
of body weight when administered orally to albino rats weighing
between 200 and 300 grams each.
• A chemical that has a median lethal dose (LD50) of more than 200
milligrams per kilogram but not more than 1,000 milligrams per
kilogram of body weight when administered by continuous contact for
24 hours (or less, if death occurs within 24 hours) with the bare skin of
albino rabbits weighing between two and three kilograms each.
• A chemical that has a median lethal concentration (LC50) in air of more
than 200 parts per million but not more than 2,000 parts per million
by volume of gas or vapor, or more than two milligrams per liter but
not more than 20 milligrams per liter of mist, fume, or dust, when
administered by continuous inhalation for one hour (or less, if death
occurs within one hour) to albino rats weighing between 200 and 300
grams each.

8.11.2  Highly toxic

Highly toxic is defined by OSHA (Occupational Safety and Health
Administration) as:
• A chemical that has a median lethal dose (LD50) of 50 milligrams or less
per kilogram of body weight when administered orally to albino rats
weighing between 200 and 300 grams each.
• A chemical that has a median lethal dose (LD50) of 200 milligrams or
less per kilogram of body weight when administered by continuous
contact for 24 hours (or less, if death occurs within 24 hours) with the
bare skin of albino rabbits weighing between two and three kilograms
each.
• A chemical that has a median lethal concentration (LC50) in air of 200
parts per million by volume or less of gas or vapor, or 2 milligrams per
liter or less of mist, fume, or dust, when administered by continuous
inhalation for one hour (or less, if death occurs within one hour) to
albino rats weighing between 200 and 300 grams each.
 CHAPTER

9 Algae-Biotechnology

Algae, belonging to the kingdom Protista, are simple photosynthetic

organisms. Based on the pigment and food reserve, algae are classified into
different types, namely, blue green algae (BGA), green algae, red algae and
brown algae.
• Algae are simple, autotrophic organisms that can synthesize their
own food by means of photosynthesis. The taxonomy of algae is very
confusing. Previously, algae were classified under the kingdom Plantae,
as they possess chlorophyll for photosynthesis. However, algae are
mostly aquatic and lack true roots, stem and leaves, which are not so
in plants. Hence, in the modern classification, they are excluded from
Plantae and categorized under Protists. The size of algae may range
from few micrometers to several meters. For example, the freshwater
alga Micromonas is about 1 micrometer, whereas the giant marine kelp
can grow to about 60 meters in length. The branch of science that deals
with the study of algae is called Phycology. Those who specialize in the
study of algae are known as phycologists.
Types of Algae: Algae are classified based on the type of pigments and
food reserves present in the particular species. The difference in the pigments
play a major role in determining the habitat distribution of the particular
algal species. Regarding the distribution of algae, they can adapt in diverse
environmental conditions. Majority of algae are found in aquatic habitats,
either in freshwater or marine water. Some of the species are found in extreme
environment like snow and ice, whereas some are adapted in hot springs.
Blue Green Algae (BGA): Blue green algae (BGA), also referred to as
Cyanobacteria, are the simplest forms of algae. Examples of BGA are Nostoc and
Calothrix. As the name suggests, they are blue green in color, ranging from
single-celled organization to colonial forms. BGA contain chlorophyll a, b
and phycobilins. They are prokaryotic in cellular organization, that resembles
9.2 Environmental Biotechnology

bacteria. BGA are considered to be an intermediate between bacteria and

plants. Hence, the name cyanobacteria is assigned to these algal species. Since
BGA lack specialized organelles, they photosynthesize directly through the
cytoplasm.
Green Algae: Green algae, belonging to the phylum Chlorophyta, contain
chlorophyll a, b, carotenoids and xanthophylls. The main food reserve of green
algae is starch. Some examples of green algae are Ulva, Codium and Caulerpa.
As of now, about 7000 species of green algae are identified. They may be either
unicellular or multicellular. Most of them are freshwater algae, while a few
species are found in the marine water.
Red Algae: Red algae, belonging to Rhodophyta, contain chlorophyll a,
d, carotenoids, xanthophylls and phycobilins. The food reserve of red algae is
floridean starch. The examples of red algae are Chondrus and Gelidiella species.
Majority of red algae are marine species. More than 6500 species of red algae
have been identified, out of which about 200 are freshwater species. The red
pigment, phycobilin helps in harvesting light at a greater depth, hence some
members of red algae are found in such a depth in the ocean floor, where no
other photosynthetic organisms can adapt.
Brown Algae: Brown algae, belonging to the class Paeophyceae,
contain chlorophyll a, c and fucoxanthin pigment. Due to the green color,
chlorophyll and brown pigment, fucoxanthin, the members belonging
to phaeophyta exhibit a typical greenish-brown coloration. The food reserve of
brown algae are complex carbohydrate polymers, called laminarin. Laminaria 
and  Macrocystis are the examples of brown algae. Similar to red algae,
majority of these algal groups are adapted in marine water. Brown algae are
the most complex algae, in which some species are adapted at certain depths
in the seas and oceans. The giant kelps, found in the ocean floor are brown
algae belonging to the order Laminarales. Kelps are the only algae with tissue
differentiation.
Algal species are very sensitive to the changes in the environmental
conditions. Hence, they are used as biological indicators to determine any
modification in the environment. For example, simple freshwater algae
like Euglena and Chlorella are used to indicate the extent of water pollution.
Products Obtained from Algae (Industrial uses of algae) : Humans use
algae as food, for production of useful compounds, as biofilters to remove
nutrients and other pollutants from wastewaters, to assay water quality, as
indicators of environmental change, in space technology, and as laboratory
research systems. Algae are commercially cultivated for Pharmaceuticals,
Nutraceuticals, Cosmetics and Aquaculture purpose.
Agar: Agar, a substance made from algae, is gelatinous in nature. It is an
ingredient in many different Japanese desserts. The Japanese red bean jelly,
called Mizuyôkan, is made from agar, and is a very popular delicacy. Agar is
Algae-Biotechnology 9.3

commercially produced with the help of Gelidium amansii—a species of red

algae. Agar is used very commonly as a laxative, and has also been used as a
vegetarian substitute for gelatin. Sometimes, it is also used as a soup thickener.
In Southeast Asia, agar is commonly used in jellies, ice creams and desserts.
It is also used as an industrial clarifying agent for brewing and paper sizing
fabrics.
Alginic Acid: Another very useful form of algae is alginic acid. Alginic
acid is a viscous, gum-like substance, derived from algae. It is used as an
additive in dehydrated products. It is also a very important ingredient in the
manufacturing of papers and textiles. As it possess most of the properties
of gum, it is also used in the water-proofing and fire-proofing industry. It is
especially helpful in making fabrics that are fire and water-resistant. In the
pharmaceutical industry, it is used in the manufacture of Gaviscon, Asilone,
Bisodol, etc. It is used as a mold-making material in life casting, prosthetics
and dentistry. Like most of the forms or products of algae, alginic acid is used
extensively in the food processing industry as an ingredient of soups and
jellies.
Carrageenan: The carrageenan is also one of the substances that has been
derived from the red algae, found near the Irish coastline. Like many algae
products, it is used as an ingredient in food products such as ice creams,
milkshakes, and sauces, to increase the viscosity of the delicacy. In many parts
of Europe, local beer and alcoholic drink manufacturers use carrageenan as
a protein remover. Manufacturers of processed and canned meats use it as
a substitute for fats. The presence of carrageenan also increases the water
retaining capacity of meat products. Apart from that, carrageenan has a wide
range of uses. It is prominently used in shampoos, toothpaste, diet sodas, pet
food and soy milk.
Fuels from Algae: The energy crisis that hit the world in the recent
decades has triggered off the race for invention of effective as well as cheap
biofuels. Algae oleum is one of the 3rd generation biofuel that has been derived
from algae. The concept of algae culture or algae farming has been derived as
a result, and many forms of algae fuels like cooking oil, biodiesel, bioethanol,
biogasoline, etc. are in the process of development.
Algae is one of the best example of putting eco-friendly resources to use,
as none of the products derived from algae are considered to be pollutants.
On the upside, many products of algae can be used to curb pollution. Algae
can also be used to treat sewage, and is an excellent alternative for chemical
fertilizers. It can also be used to curb and arrest the toxic chemicals that are
present in water bodies. Algae is also the ideal substitute for chemical dyes and
pigments. In many industries, algae bioreactors are used to curb the emission
of carbon and carbon compounds. Humans use algae as food, for production
of useful compounds, as biofilters to remove nutrients and other pollutants
from wastewaters, to assay water quality, as indicators of environmental
9.4 Environmental Biotechnology

change, in space technology, and as laboratory research systems. Algae is

commercially cultivated for Pharmaceuticals, Nutraceuticals, Cosmetics and
Aquaculture purpose.

Food supplement
• It is a complete protein with essential amino acids (unlike most plant
foods) that are involved in major metabolic processes such as energy
and enzyme production.
• It contains high amounts of simple and complex carbohydrates which
provide the body with a source of additional fuel. In particular, the
sulfated complex carbohydrates are thought to enhance the immune
system’s regulatory response.
• It contains an extensive fatty acid profile, including Omega 3 and
Omega 6. These essential fatty acids also play a key role in the
production of energy.
• It has an abundance of vitamins, minerals, and trace elements in
naturally-occurring synergistic design.
Stabilizing agent: Chondrus crispus (probably confused with Mastocarpus
stellatus, common name: Irish moss), is also used as “carrageen”. It is an
excellent stabiliser in milk products—it reacts with the milk protein caesin
other products include: petfoods, toothpaste, icecreams and lotions, etc.
Alginates in creams and lotions are absorbable through the skin.
Fertilizer: Algae are used by humans in many ways. They are used as
fertilizers, soil conditioners and are a source of livestock feed. Because many
species are aquatic and microscopic, they are cultured in clear tanks or ponds
and either harvested or used to treat effluents pumped through the ponds

Role of Algae in Pollution control

• Algae are used in Wastewater Treatment facilities, reducing the need
for greater amounts of toxic chemicals than are already used.
• Algae can be used to capture fertilizers in runoff from farms. When
subsequently harvested, the enriched algae itself can be used as
fertilizer.
• Algae Bioreactors are used by some power plants to reduce CO2
emissions. The CO2 can be pumped into a pond, or some kind of tank,
on which the algae feed. Alternatively, the Bioreactor can be installed
directly on top of a smokestack.

9.1  ALGAE AS A SOURCE OF FOOD AND FEED

Algae are primitive eukaryotic organisms that do not have true stems, roots
and leaves. They are primary producers in aquatic ecosystems and marine
organisms. Most of these are edible and have been consumed in different forms
Algae-Biotechnology 9.5

since ancient times. The foods we consume, often dictate our physical and
mental health. A healthy diet will always help fight illness-causing infections,
whereas, a poor diet will make us more susceptible to illness. 

9.1.1  Microalgae nutritional composition

As for higher plants, the nutritional composition of microalgae is made up
mainly of proteins, carbohydrates, lipids and trace nutrients, including
vitamins, antioxidants, and trace elements. Their content varies with different
species, even strains and growing conditions, including nutrient supplies,
temperature, sunlight, etc.
Following Table illustrates general composition of some animal feeds and
foods, and microalgae species (% of dry matter of maximum values achieved in each
commodity).
General composition of some animal feeds and foods, and microalgae
Commodity Crude Protein Carbohydrates Lipids
Meat 43 1 34
Fish 55 — 38
Egg 49 3 45
Milk 26 38 28
]Rice 8 77 2
Soyabean 37 30 20
Corn 10 85 4
Wheat 14 84 2
Fish-meal 60–72 — 6–10
Anaboena cylindrical 43–56 25–30 4–7
Chlamydomonos rheinhordff 48 17 21
Chlorella vulgoris 51–58 12–17 14–22
Chlorella pyrenoidoisa 57 26 2
Dunatlela salina 57 32 6
Euglena gracitis 39–61 14–18 14–20
Porphyridium cruentum 28–39 40–57 9–14
Scenedesmus obliquus 50–56 10–17 12–14
Arthrospira maxima 60–71 13–16 6–7
Spirutina platensis 46–63 8–14 4–9
Spirogyro sp. 6–20 33–64 11–21
Synechococcus sp. 73 15 11
Tetraselmis suecica 41
Isochrysis golbana 39
Dunolietta tertiolecta 54
Chlorella stigmatophora 39
9.6 Environmental Biotechnology

It can be noted that there is a large variation in compositions among

conventional feeds / foods and microalgae. Indeed, depending on growth
conditions, microalgae even the same strain—can exhibit large variations in
composition, with protein, carbohydrate and lipid contents, each ranging
from about 15 to over 50% of dry weight. This plasticity in composition is an
important attribute in their use as animal feeds.
About six decades ago, the mass production of certain protein-rich
microalgae was considered as a possibility to close the predicted “food
gap”. Although this “gap” failed to materialize, as a result of the “green
revolution”, catalyzed in large part by the enormous amounts of fertilizers
produced by the Harber Bosch process, the early interest in microalgae
resulted in many comprehensive nutritional studies in both animals and
humans. These nutritional studies continued during the 60s and 70s, due in
part to the following interest in microalgae for space applications, and the
early commercial production of two algae species, Chlorella and Spirulina.
However, by the 1970s, the scale-up of commercial production to reduce
production costs was not achieved, and the field of large-scale production is
delaying this potential.
Essential Amino Acids: These early and extensive nutritional studies of
microalgae as foods and feeds demonstrated that algal proteins are of high
quality, comparable to conventional vegetable proteins in terms of their content
of essential amino acids, which mainly determine the nutritional quality of a
protein source. The amino acid profile of various algae are compiled in the
following table and compared with some basic conventional food items and
a reference pattern of a well-balanced protein, recommended by WHO/FAO.
List of Amino acids present in conventional foods and some microalgae
Leu Val Arg Lys He Phe Thr Met Try His
WHO/FAO 7 5 — 5.5 4 6.0 — 3.50 1 —
Egg 8,8 7,2 6,2 5,3 6,6 5,8 5 3,2 1,7 2,4
Meat 7,8 5,3 6,6 8,2 5,1 4,2 4,5 2,4 — 3,2
Milk 9,2 5,7 3,3 7,8 4,3 5,6 4,5 2,5 — 2,6
Soyabean 7,7 5,3 7,4 6,4 5,3 5 4 1,3 1,4 2,6
Fish-meal 4,48 2,77 3,82 4,72 2,66 4,35 2,31 2,31 0,57 1,45
Chlorella 8,8 5,5 6,4 8,4 3,8 5 4,8 2,2 2,1 2
vulgaris
Dunatella 11 5,8 7,3 7 4,2 5,8 5,4 2,3 0,7 1,8
bordawil
Scenedesmus 7,3 6 7,1 5,6 3,6 4,8 5,1 1,5 0,3 2,1
obliquus
Arthrospira 8 6,5 6,5 4,6 6 4,9 4,6 1,4 1,4 1,8
maxima
Spirutinep 9,8 7,1 7,3 4,8 6,7 5,3 6,2 2,5 0,3 2,2
latensis
Tetraselmis 9,3 5,6 7,6 9,8 4,1 5,9 5,3 1,5 — 2,5
suecica
Algae-Biotechnology 9.7

Leu Val Arg Lys He Phe Thr Met Try His

Isochrysis 10,5 6 8,7 12,3 4,9 6,1 6,1 0,7 — 2,5
galbena
Dunaliela 10,7 5,3 7,2 13,6 4,2 6,6 2,6 0,8 — 2,5
Chlorella 9,3 5,7 8,6 13,4 3,8 5,5 4,9 1,4 — 2,3
Most of the early work in determining which amino acids could be classed
as indispensable for humans and animals was carried out with rats fed on
purified diets. The following ten indispensable amino acids are required for
growth in the rat: Arginine, Histidine Isoleucine, Leucine, Lysine, Methionine,
Phenylalanine, Threonine, Tryptophan and Valine. Some animals differ
slightly in these requirements (the pig can synthesize arginine, for example),
but the content of these essential amino acids determines in large measure the
nutritional value of these feeds.
Vitamins and Trace Elements: A similar comparison can be made for
vitamins, demonstrating that microalgae contain high levels of essential
vitamins, similar to the best food sources such as baker’s yeast and liver and
are vastly superior to all commodity feeds (such as soybeans, corn, fishmeal,
etc.) which have little, if any, vitamin content.
List of vitamins and trace elements present in conventional
foods and some algae
Vit Vit Vit Vit Vit Vit Vit Nicoti- Blotin Folic Pantho-
A B1 B2 B6 B12 C E nate Acid tenic
Acid
RDI (mg/d) 1.7 1.5 2.0 2.5 0.005 50.0 30.0 18.0 — 0.6 8.0
Liver 60.0 3.0 29.0 9.0 0.65 310.0 10.0 136.0 1.0 2.9 73.0
Spinach 130.0 0.9 1.8 1.8 — 470.0 — 5.5 0.007 0.7 2.8
Baker’s yeast trace 9.1 16.5 21.0 — trace 112.0 4.0 5.0 53.0
Spiruling 840.0 44.0 39.0 3.0 9.0 80 120.0 — 0.3 0.4 13.0
platenis
Chlorella 480.0 10.0 36.0 23.0 — — — 240.0 0.15 — 20.0
pyrenoldolsa
Scenedesmus 554.0 11.5 29.0 — 1.1 396.0 — 108.0 — — 46.0
quadricauda

The nutritionally necessary trace elements, of which, there are about two
dozen, have similarly high levels in microalgae to those of vitamins, compared
to most feed sources. However, data on trace elements is more limited, contents
are even more variable between and even within species, and composition
more plastic, depending on growing conditions, than is the case even for the
major constituents, essential amino acids or vitamins. Indeed, microalgae with
desired high concentrations of trace elements could be produced on demand
by adjusting the trace elements in their growth medium.
In the case of both vitamins and trace elements, but also for essential amino
acids, the bioavailability of these trace nutrients is often more important and
decisive in terms of feed quality than just their bulk constituents. Bioavailability
9.8 Environmental Biotechnology

studies are more difficult than just simple analytical measurements, but,
overall, bioavailability of all these components is good to high for various
microalgae biomass sources studied; in most cases; the microalgae already
being produced commercially (e.g. Chlorella and Spirulina).
Health Benefits Associated with Algae: Over the years, research has
proven many health benefits associated with algae. It is commonly used as
a dietary supplement to improve general health and wellness. Some of the
important health benefits of algae include:
• It is known to have a high concentration of beta-carotene that helps fight
some types of cancer and cardiovascular diseases.
• Full of antioxidants, algae restricts the growth of free radicals and toxins.
Antioxidants also aid in production of necessary enzymes needed to
keep the body’s function smooth.
• As it is organic in nature and full of enzymes, it is easily digestible and
light on the stomach. It is known to facilitate smooth bowel movements,
thereby preventing abdominal muscle associated diseases.
• Algae is considered a complete source of protein, as it contains amino
acids, minerals and many vitamins, essential for the growth of hair, skin
and nails.
• Algae contains immune boosting and stimulating properties such as amino
acids, minerals, proteins and vitamins. These help fight infections,
detoxify the immune system, and aid in building the body’s resistance
to infections.
• There are only two sources of GLA found; mother’s milk and spirulina,
a blue-green algae. GLA is essential for the development and growth of
babies. Deficiency in nutrition reduces GLA in the mother’s milk, which
results in poor baby health.
• Algae is known to treat diabetes, anemia, liver disease, ulcers, allergies,
radiation and chemical poisoning. Its concentrated sugar stabilizes blood
sugar levels in people with high or low blood sugar.
• Algae  ensures a healthy nutrient level for people who diet, as well as
people who detoxify their bodies regularly. The amino acids present in
algae are known to influence neurotransmitters in the brain that control
appetite.
• A balanced diet consists 80% alkaline food and 20% acidic food. An
acidic body is vulnerable to diseases. Algae is considered as a natural
source of alkaline food.
Other benefits of Algae:
• It contains calcium and magnesium, which are essential for strong
bones.
• Has high levels of iron, useful for anemic people and pregnant or
lactating women.
Algae-Biotechnology 9.9

• It helps to reduce anxiety and treat sleep disorders.

• It is popular in weight reduction diets.
• Algae contains the zeaxanthin nutrient, which is good for the eyes.
• Regular consumption of algae aids in enhancing memory and increasing
concentration and focus.
Consuming algae as a regular health supplement helps improve the
overall development and growth of the body, as it contains almost all nutrients,
minerals, vitamins, proteins, amino acids and nucleic acids, chlorophyll, fiber,
etc. There are many types of algae available, like Spirulina and Chlorella. It is
mostly available as powders, capsules, tablets, etc. While algae health benefits
are enormous, it is best to consult a medical practitioner on the type and the
amount one needs to consume to derive maximum health benefits.

9.2  MASS CULTIVATION OF COMMERCIALLY VALUBLE MARINE

MICROALGAE FOR AGAR AGAR, ALGINATES
Microalgae were one of the first organisms to come into existence in the
Earth’s ocean more than 3 billion years ago, when the Earth’s environment
formed. They are also called phytoplankton. These unicellular organisms have
chlorophyll and produce oxygen (O2) by immobilizing carbon dioxide (CO2)
in the atmosphere through photosynthesis. There are about 100,000 different
types of microalgae living not only in the oceans but also in fresh water (lakes,
ponds, and rivers). Marine microalgae, the largest primary biomass, have been
attracting attention as resources for new metabolites and biotechnologically
useful genes. The diversified marine environment harbors a large variety of
microalgae.
Industrial culture technique: Along with rising expectations for
microalgae, it has become necessary to develop technology to industrially
and efficiently produce the required amounts of microalgae. In particular, it
is essential to establish culture techniques ranging from small-to large-scale
culturing. It is also necessary to enhance productivity based on individual
culture methods and accumulate know-how with regard to ensuring quality.
In the case of producing relatively small amounts along with rising
expectations for microalgae, it has become necessary to develop technology
to industrially and efficiently produce the required amounts of microalgae. In
particular, it is essential to establish culture techniques ranging from small-
to large-scale culturing. It is also necessary to enhance productivity based
on individual culture methods and accumulate know–how with regard to
ensuring quality. In the case of producing relatively small amounts of medicine,
food and feed, which require purity and safety, an enclosed culture system
(Enclosed System) is used. In the case of biofuel and biomass, large-scale-yet
low-cost, production methods are required. Currently, as a large-scale culture
method, culturing in open spaces such as ponds (Open Pond System) has been
employed.
9.10 Environmental Biotechnology

At present, such methods are a long way from being efficient. In particular,
the development of a culture method to industrially produce low-priced
products in large quantities is still at the study stage. A study is now under
way to develop an enclosed culture system that utilizes light more effectively
than open culture methods and is suitable for low-priced, mass culture.
Macroalgae have long been used for the production of phycocolloids such
as alginates, carrageenans or agars. These polymers are either located in cell
walls or within the cells serving as storage materials. A characteristic of marine
algae is the abundance of sulphated polysaccharides in their cell walls.
AGAL-AGAL: Agars are 1,3-b-1,4-b-galactans from cell walls of red algae
that are substituted with sulphate groups. Like the carrageenans, the agals
are extracted with hot water. The genera Gelidium and Gracilaria supply most
of the raw material for agar production. Gelidium used for commercial agal
production is harvested from the wild, whereas Gracilaria species have also
been cultivated in Chile, China and Indonesia, in protected bays in the ocean,
on line ropes or nets, or in ponds on land. Like carrageenans, agals are used as
stabilisers for emulsions and suspensions and as gelling agents. About 90% of
the agal produced was for food applications and the remaining 10% were used
for bacteriological and other biotechnological uses.
The production process of agal-agal from red algae contains the following
parts:
• Drying of algae: Once collected, the algae are dried in the sun.
• Alkalinization of algae: Once dried, they are placed in a container with
very hot water in which an alkali product, such as caustic soda, has
been poured.
• Washing and bleaching with cold water: Then, the algae are washed
with cold water to remove all impurities. Sulfuric acid is added to
dealkalinize them and sodium hypochlorite for bleaching them .
Finally, they are cleaned with cold water.
• Extraction by cooking: The product is extracted by subjecting the
washed and bleached algae to a process of cooking for two hours.
• Filtering: The filtering aims to separate the product from other waste
such as rocks, shells, dirt, etc. This is accomplished by passing the
water with the algae thorough different filtering tanks.
• Gelling: A process intended to bring the product to the texture of a gel.
To do this, the algae are cooled by different processes from 80°C to
25°C.
• Pressing and drying: After this, the product is compacted by a press
and dried with hot air.
• Grinding, sieving and packaging: Finally, the algae are ground and
sieved before being packed.
Algae-Biotechnology 9.11

The properties of agar agar are numerous. It is used primarily in the

following areas:
• Food Industry: The food industry uses it as a food additive with the
code E-406. It is a natural thickener, non-toxic, with no flavor, not
degradable by acid or proteolytic enzymes, and with no many calories.
The thickening ability of agar agar comes from its power to make the
liquid more viscous so that it produces, what is called, a gelling effect,
that is to say, it makes a liquid to become a gel.
Therefore, it is very suitable for the manufacture of canned fish and
meat because it compacts them and provides them a better texture.
Moreover, this product forms a layer that protects these foods from
contact with the metal walls of the container. By doing this, it prevents
canned food to become rusted (cooked meat, ham, shoulder, sausage,
canned chicken, etc.)
The beverage industry (beer, wine, etc.) uses it to purify and clarify the
liquids because it settles the impurities to the bottom. Other packaged
products such as fruit juices, sauces, soups, yogurt, curds, ice cream,
cakes ... also contain agar-agar as a thickener or stabilizer.
It is the natural substitute for animal gelatin obtained from bones and
skin. In this regard, agar agar is highly valued within the vegetarian
people who use it in many recipes.
• Metalworking: Used to improve the coating of metal objects made out
of lead or zinc or to encourage the stretching of certain metals.
• Paper industry: Its use provides waterproofing properties to paper.
• Leather industry: Used to promote leather tanning.
• Textile industry: It gives strength and stiffness to clothes and facilitates
printing.
• Pharmaceuticals: The pharmaceutical industry includes it in the
composition of many drugs and excipients, to improve taste, color,
texture, etc. It is also used to form the cover of some preparations such
as suppositories, pessaries or gels. It is one of the basic ingredients in
the preparation of drugs for constipation.
• Natural medicine: In natural medicine, is mainly used as a laxative and
in slimming cures.
• Cosmetics industry: It becomes a part of the manufacture of skin
creams.
• Microbiological research: agar agar is one of the main culture media
for microbiological research. It presents a greater capacity than
any animal gelatine to resist microorganisms and remain stable at a
suitable temperatures for incubating them. On the other hand, it can
be solidified or become, easily a gel, allowing the microorganisms to be
mixed in it easily.
9.12 Environmental Biotechnology

• Traditionally, agar agar is produced in Asia, mainly China, Korea,

Japan and Indonesia. Although it has been used since ancient times in
the East, it is documented for the first time in the seventeenth century
in Japan. In Europe it was not used until the nineteenth century.
• At present, there are many countries that produce it. These include, for
example, United States, Korea, Spain, France, Morocco, Chile, South
Africa and Portugal. Among all the varieties, the Atlantic agar agar
stands out. This is obtained form the alga Gelidium sesquipedale. Spanish
production of this type of alga in Galicia is very well known.
• Most of agar agar is produced for human consumption, and only about
10% is used for other applications.
Most of the agar agar is derived from the following two kinds of algae:
Gelidium: It inhabits rocky places of eulitoral and sublitoral zone. Eulitoral
area (= interdital) is that part of the coast that lies between tides, so it is devoid
of water at low tide and is covered with it when the tide rises. The sublitoral
zone extends from eulitoral area and is characterized by being permanently
covered by water. In general, the species of this genus do not like excessive sun
exposure and prefer moderate temperatures between 15 and 20°C.
Formerly, there were plenty of productive areas of these algae in Japan,
but industrial pollution has extinguished most populations in this area. The
main collection areas are currently on the north coast of Spain, the east coast
of Portugal, the west coast of Morocco and the southwest coast of France. To a
lesser proportion, they are also found in Indonesia, on the southwest coast of
Mexico and the southeastern coast of Korea.
Gracilaria: They inhabit the eulitoral areas of sandy soils. They need
higher temperatures, although there are species adapted to colder waters, and
more easily resist external aggressions, such as freshwater input or fertilizer.
The species that prefer warmer areas live in coastal areas of Indonesia
and southern China. Some of these species are grown in ponds and estuaries.
Other places with lower production are the west coast of South Africa and the
coast of Namibia. Species adapted to cold areas can be found in places such as
southern Chile and eastern Canada.
Other genres used extensively in some regions are Gelidiella which is
the most commonly used in India, Pterocladia in Russia and Ahnfeltia in New
Zealand and the Azores.
Collection and cultivation of agal agal: Although most of the agal agal
is obtained from collecting it naturally in coastal areas, it is increasingly
spreading in cultivation. Among the species grown, Gracilaria is the most used
and the only one that, in most cases, can be considered economically viable.
There are three main methods for the cultivation of Gracilaria:
• Open cultivation in estuaries or bays: Sandy soils are required. On
these types of soils, algae are placed for them to take root and develop
new algae.
Algae-Biotechnology 9.13

• On ropes or nets: They form a sort of row with filaments or nylon

or polypropylene ropes to which algae are attached so that they can
thrive. Each line is held on stakes that are inserted and fastened to the
bottom of the water. Sometimes they are fixed on nets that, in turn, are
placed into the sea ground with bamboo poles.
• On ponds or pools: In this case, a pond is sown so that the algae
become entrenched to the bottom. Since they are not anchored as in the
previous cases, we need to do so in not very windy ponds.
Each pond should be about 60 or 70 cm deep and must contain a whole
surface of one or half hectare. Salinity and temperature must be controlled,
something which is done by means of a water supply from the outside, so that
each pool or pond should be connected with a salt water source and another
source of fresh water.
To get a proper salinity, water should be added or removed. Since it
requires a constant temperature of about 15 to 30°C, more water should be
added when the outdoor temperature is higher so that the sun does not heat
the water from the bottom too much. On the contrary, some water should be
removed when the temperature is lower to allow that sunlight to access the
deeper level of water and heat it.
Alginates: Alginates are polymers from the cell walls of a wide variety
of species of the brown algae, particularly species of Laminaria, Macrocystis
and Ascophyllum. They are polymers composed of D-mannuronic acid and
L-guluronic acid monomers. The alginates are extracted from the cell walls
using hot alkali (sodium carbonate). Alginates are commonly used in the food
and pharmaceutical industries as stabilizers for emulsions and suspensions,
e.g. ice cream, jam, cream, custard, creams, lotions, tooth paste, as coating
for pills. They are also used in the production of paint, construction material,
glue and paper, oil, photo and textile industry. Brown seaweeds for alginate
production are harvested from the wild, and not cultivated, for this purpose.
Although these seaweeds are cultivated to produce food in China, their
cultivation to provide raw material for industrial uses would be too expensive.
Production: Biotechnological and Traditional: There has been significant
progress in the understanding of alginate biosynthesis over the last few
years. The fact that the alginate molecule enzymatically undergoes a post-
polymerization modification with respect to chemical composition and
sequence opens up the possibility for in vitro modification and tailoring of
commercially available alginates.
Isolation from Natural sources/fermentative production: All commercial
alginates today are produced from marine brown agar. Alginates with more
extreme composition can be isolated from the bacterium Azotobacter vinelandii,
which in contrast to Pseudomonas species, produces polymer containing
G-blocks. Production by fermentation, therefore, is technically feasible at the
moment.
9.14 Environmental Biotechnology

Molecular Genetics and in vitro Modification: Alginate with a high

content of guluronic acid can be prepared from special algal tissues by
chemical fractionation or by in vitro using mannuronam C-5 epimerases from
A. vinelandii. These epimerases, which convert M and G in the polymer chain,
recently have allowed for the production of highly programmed alginates
with respect to chemical composition and sequence.
Commercial application of alginates: The uses of alginates are based on
three main properties.
• The first property of alginates is their ability to thicken the solution
when dissolved in water (more technically, described as their ability to
increase the viscosity of aqueous solutions).
• The second is their ability to form gels; gels form, when a calcium salt
is added to a solution of sodium alginate in water. The gel forms by
chemical reaction, the calcium displaces the sodium from the alginate,
holds the long alginate molecules together and a gel is the result.
No heat is required and the gels do not melt, when heated. This is in
contrast to the agar gels where the water must be heated to about 80°C
to dissolve the agar and the gel forms, when cooled below about 40°C.
• The third property of alginates is the ability to form films of sodium or
calcium alginate and fibers of calcium alginates.
Alginate molecules are long chains that contain two different acidic
components, abbreviated here for simplicity to M and G. The way in which
these M and G units are arranged in the chain and the overall ratio, M/G, of
the two units in a chain, can vary from one species of seaweed to another.
In other words, all “alginates” are not necessarily the same. So some algae
may produce an alginate that gives a high viscosity when dissolved in water,
others may yield a low viscosity alginate. The conditions of the extraction
procedure can also affect viscosity lowering it, if conditions are too severe. All
of this results in sellers, normally—offering a range of alginates with differing
viscosities.
Similarly, the strength of the gel formed by the addition of calcium salts can
vary from one alginate to another. Generally, alginates with a higher content
of G will give a stronger gel; such alginates are said to have a low M/G ratio.
Some examples: Macrocystis can gives a medium-viscosity alginate, or a
high viscosity with a careful extraction procedure (lower temperature for the
extraction). Sargassum, usually, gives a low viscosity product. Laminaria digitata
gives a soft to medium strength gel, while Laminaria hyperborea and Durvillaea
give strong gels. These are some of the reasons why alginate producers like
to have a variety of seaweed sources, to match the alginate to the needs of
particular applications.
Textile printing: In textile printing, alginates were used as thickeners
for the paste containing the dye. These pastes may be applied to the fabric
by either screen—or roller printing equipment. Alginates became important
Algae-Biotechnology 9.15

thickeners with the advent of reactive dyes. These combine chemically with
cellulose in the fabric. Many of the usual thickeners, such as starch, react with
the reactive dyes, and this leads to lower colour yields and sometimes by-
products, that are not easily washed out.
Alginates do not react with the dyes, they easily wash out of the finished
textile and are the best thickeners for reactive dyes. Alginates are more
expensive than starch and recently starch manufacturers have made efforts
to produce modified starches that do not react with the reactive dyes, so it
is becoming a more competitive market. This use of alginate represents a
large market, but it is affected by economic recessions when there is often a
fall in demand for clothing and textiles. The types of alginate required vary
from medium-to-high viscosity with older screen printing equipment, to
low viscosity, if modern, high speed, roller printing is used. Textile printing
accounts for about 50 per cent of the global alginate market.
Food: The thickening property of alginate is useful in sauces and in syrups
and toppings for ice cream. By thickening pie fillings with alginate, softening
of the pastry by liquid from the filling is reduced. Addition of alginate can
make icings non-sticky and allow the baked goods to be covered with plastic
wrap. Water-in-oil emulsions such as mayonnaise and salad dressings are
less likely to separate into their original oil and water phases if thickened
with alginate. Sodium alginate is not useful when the emulsion is acidic,
because insoluble alginic acid forms; for these applications propylene glycol
alginate (PGA) is used, since this is stable in mild acid conditions. Alginate
improves the texture, body and sheen of yoghurt, but PGA is also used in
the stabilization of milk proteins under acidic conditions, as found in some
yoghurts. Some fruit drinks have fruit pulp added and it is preferable to keep
this in suspension; addition of sodium alginate, or PGA in acidic conditions,
can prevent sedimentation of the pulp. In chocolate milk, the cocoa can be kept
in suspension by an alginate/phosphate mixture, although in this application,
it faces strong competition from carrageenan. Small amounts of alginate can
thicken and stabilize whipped cream.
Alginates have some applications that are not related to either their
viscosity or gel properties. They act as stabilizers in ice cream; addition of
alginate reduces the formation of ice crystals during freezing, giving a smooth
product. This is especially important when ice cream softens between the
supermarket and the home freezer; without alginate or similar stabilizer, the
refrozen ice cream develops large ice crystals, giving it an undesirable crunchy
mouth feel. Alginate also reduces the rate at which the ice cream will melt.
Beer drinkers prefer some foam on the top of a newly-poured glass, and poor
foam leads to a subjective judgment that the beer is poor quality. Addition of
a very low concentration of propylene glycol alginate will provide a stable,
longer-lasting beer foam. A variety of agents are used in the clarification of
wine and removal of unwanted coloring—wine fining—but in more difficult
cases, it has been found that the addition of sodium alginate can be effective.
9.16 Environmental Biotechnology

The gelling properties of alginate were used in the first production of

artificial cherries in 1946. A flavored, colored solution of sodium alginate
was allowed to fall, in large drops, into a solution of a calcium salt. Calcium
alginate immediately formed as a skin on the outside of the drop and when the
drop was allowed to sit in the solution, the calcium gradually penetrated the
drop converting it all into a gel that hardened with further standing. Because
the cherry-flavored gels did not melt, they became very popular in bakery
products. Fruit substitutes can now be made by automated and continuous
processes that are based on similar principles. Either the calcium can be
applied externally, as above, or internally. In the latter case, a calcium salt that
does not dissolve is added to the fruit puree, together with a weak acid; the
weak acid slowly attacks the calcium salt and releases water-soluble calcium
that then reacts with the alginate and forms the gel.
Edible dessert jellies can be formed from alginate-calcium mixtures, often
promoted as instant jellies or desserts because they are formed simply by
mixing the powders with water or milk, no heat being required. Because they
do not melt, alginate jellies have a different, firmer mouth feel when compared
to gelatin jellies, which can be made to soften and melt at body temperature.
Mixtures of calcium salts and sodium alginate can be made to set to a gel at
different rates, depending on the rate at which the calcium salt dissolves. Gel
formation can also be delayed even after everything is mixed together; this is
done using a gel-retarder that reacts with the calcium before the alginate does,
so, no calcium is available to the alginate until all the retarder is used. In this
way, gel formation can be delayed for several minutes if desired, such as when
other ingredients need to be added and mixed before the gel starts to set.
Alginate gels are used in restructured or reformed food products. For
example, restructured meats can be made by taking meat pieces, binding them
together and shaping them to resemble usual cuts of meat, such as nuggets,
roasts, meat loaves, even steaks. The binder can be a powder of sodium alginate,
calcium carbonate, lactic acid and calcium lactate. When mixed with the raw
meat, they form a calcium alginate gel that binds the meat pieces together.
This is used for meats for human consumption, such as chicken nuggets; it has
become especially useful in making loaves of meat for fresh pet food; some
abattoir wastes are suitable as cheap ingredients. Up to 1 per cent alginate
is used. Similar principles are applied to making shrimp substitutes using
alginate, proteins such as soy protein concentrate, and flavors. The mixture is
extruded into a calcium chloride bath to form edible fibers which are chopped,
coated with sodium alginate and shaped in a mould. Restructured fish fillets
have been made using minced fish and a calcium alginate gel. Onion rings
are made from dried onion powder; pimento olive fillings are made using
pimento pulp. In 2001, a new line of olives launched in Spain were stuffed
with flavored pastes, such as garlic, herbs, hot pepper, lemon and cheese. Each
of these is made with green manzanilla olives and an alginate-based paste
containing the appropriate ingredient to provide the flavour.
Algae-Biotechnology 9.17

Calcium alginate films and coatings have been used to help preserve
frozen fish. The oils in oily fish such as herring and mackerel can become rancid
through oxidation even when quick frozen and stored at low temperatures. If
the fish is frozen in a calcium alginate jelly, the fish is protected from the air and
rancidity from oxidation is very limited. The jelly thaws with the fish, so they
are easily separated. If beef cuts are coated with calcium alginate films before
freezing, the meat juices released during thaw are reabsorbed into the meat
and the coating also helps to protect the meat from bacterial contamination. If
desired, the calcium alginate coating can be removed by redissolving it with
sodium polyphosphate.
Immobilized biocatalysts: Many commercial chemical syntheses and
conversions are best carried out using biocatalysts such as enzymes or active
whole cells. Examples include, the use of enzymes for the conversion of glucose
to fructose, the production of L-amino acids for use in foods, the synthesis
of new penicillin after hydrolysis of penicillin G, the use of whole cells for
the conversion of starch to ethanol (for beer brewing), and the continuous
production of yoghurt. To carry out these processes on a moderate to large
scale, the biocatalysts must be in a concentrated form and be recoverable from
the process for reuse.
This can be achieved by “immobilizing” the enzymes or cells by
entrapping them in a material that will still allow penetration by the substance
to be converted or changed. Originally, single enzymes were isolated and used
for a specific conversion, but now similar or better results can be obtained
using whole cells, and this is more economical. An added advantage of
immobilization is, that the cells last longer. Ordinary suspended cells may have
good activity for only 1-2 days, while immobilized cells can last for 30 days.
Beads made with calcium alginate were one of the first materials to be used
for immobilization. The whole cells are suspended in a solution of sodium
alginate and this is added dropwise to a calcium chloride solution. The beads
form in much the same way as described for artificial cherries. In use, they are
packed into a column and a solution of the substance to be converted is fed into
the top of the column and allowed to flow through the bed of beads containing
the immobilized biocatalyst in the cells. The conversion takes place and the
product comes out at the bottom. A simple example is to immobilize yeast
cells, flow a solution of sugar through the beads, and the sugar is converted
to alcohol.
Pharmaceutical and medical uses: If a fine jet of sodium alginate solution
is forced into a bath of a calcium chloride solution, calcium alginate is formed as
fibers. If low viscosity alginates are used, a strong solution can be used without
any viscosity problems and the calcium bath is not diluted as rapidly. The
fibres have very good strength when both wet and dry. As with most polymer
fibers formed by extrusion, stretching, while forming, increases the linearity
of the polymer chains and the strength of the fiber. Good quality stable fibers
9.18 Environmental Biotechnology

have been produced from mixed salts of sodium and calcium alginate, and
processed into non-woven fabric that is used in wound dressings. They have
very good wound healing and haemostatic properties and can be absorbed
by body fluids because the calcium in the fiber is exchanged for sodium from
the body fluid to give a soluble sodium alginate. This also makes it easy to
remove these dressings from large open wounds or burns since they do not
adhere to the wound. Removal can be assisted by applying saline solutions
to the dressing to ensure its conversion to soluble sodium alginate. Recently,
the consumer division of a multinational pharmaceutical company launched
a new line of adhesive bandages and gauze pads based on calcium alginate
fibers. They are being promoted as helping blood to clot faster—twice as fast
as their older, well established, product.
Alginic acid powder swells when wetted with water. This has led to its use
as a tablet disintegrant for some specialized applications. Alginic acid has also
been used in some dietary foods, such as biscuits; it swells in the stomach and,
if sufficiently taken, it gives a “full” feeling so the person is dissuaded from
further eating. The same property of swelling has been used in products such
as Gavisconä tablets, which are taken to relieve heartburn and acid indigestion.
The swollen alginic acid helps to keep the gastric contents in place and reduce
the likelihood of reflux irritating the lining of the oesophagus.
Alginate is used in the controlled release of medicinal drugs and other
chemicals. In some applications, the active ingredient is placed in a calcium
alginate bead and slowly released as the bead is exposed in the appropriate
environment. More recently, oral controlled-release systems involving alginate
microspheres, sometimes coated with chitosan to improve the mechanical
strength, have been tested as a way of delivering various drugs. Pronova
Biomedical AS, a leading supplier of ultra-pure alginates and chitosans for
controlled release and other medical materials applications, was acquired
by FMC Bioploymer in early 2002; FMC had previously acquired Pronova
Biopolymer, producer of food and technical grade alginates.

9.2.1  Other applications

Paper: The main use for alginate in the paper industry is in surface sizing.
Alginate added to the normal starch sizing gives a smooth continuous film
and a surface with less fluffing. The oil resistance of alginate films give a size
with better oil resistance and enhances grease-proof properties. An improved
gloss is obtained with high gloss inks. If papers or boards are to be waxed,
alginate in the size will keep the wax mainly at the surface. They give better
coating runability than other thickeners, especially in hot, on-machine coating
applications. Alginates are also excellent film–formers and improve ink
holdout and printability. The quantity of alginate used is usually 5-10 per cent
of the weight of starch in the size.
Alginate is also used in starch adhesives for making corrugated boards
because it stabilizes the viscosity of the adhesive and allows control of its rate
Algae-Biotechnology 9.19

of penetration. One per cent sodium alginate, based on the weight of starch
used, is usually sufficient.
Paper coating methods and equipment has developed significantly since
the late 1950s with the demand for a moderately priced coated paper for high
quality printing. Trailing blade coating equipment runs at 1,000 m/minute or
more, so the coating material, usually clay plus a synthetic latex binder, must
have consistent rheological properties under the conditions of coating. Up to
1 per cent alginate will prevent change in viscosity of the coating suspension
under the high shear conditions where it contacts the roller. The alginate
also helps to control water loss from the coating suspension into the paper,
between the point where the coating is applied and the point where the excess
is removed by the trailing blade. The viscosity of the coating suspension must
not be allowed to increase by loss of water into the paper because this leads
to uneven removal by the trailing blade and streaking of the coating. Medium
to high viscosity alginates are used, at a rate of 0.4-0.8 per cent of the clay
solids. Because of the solvent resistance of alginate films, the print quality of
the finished paper is improved.
Welding rods: Coatings are applied to welding rods or electrodes to act
as a flux and to control the conditions in the immediate vicinity of the weld,
such as temperature or oxygen and hydrogen availability. The dry ingredients
of the coating are mixed with sodium silicate (water glass) which gives some
of the plasticity necessary for extrusion of the coating onto the rod; it also acts
as the binder for the dried coating on the rod. However, the wet silicate has no
binding action and does not provide sufficient lubrication to allow effective
and smooth extrusion. An additional lubricant is needed, and a binder that
will hold the damp mass together before extrusion and maintain the shape of
the coating on the rod during drying and baking. Alginates are used to meet
these requirements. The quantities of alginates used are very dependent on
the type of welding rod being coated and the extrusion equipment being used.
Alginate manufacturers are the best source of information for using alginates
in welding rod applications.
Binders for fish feed: The worldwide growth in aquaculture has led to
the use of crude alginate as a binder in salmon and other fish feeds, especially,
moist feed made from fresh waste fish mixed with various dry components.
Alginate binding can lower consumption by up to 40 per cent and pollution of
culture ponds is sharply reduced.
Release agents: The poor adhesion of films of alginate to many surfaces,
together with their insolubility in non-aqueous solvents, have led to their use
as mould, release agents, originally for plaster moulds and later in the forming
of fiber glass plastics. Sodium alginate also makes a good coating for anti-tack
paper, which is used as a release agent in the manufacture of synthetic resin
decorative boards. Films of calcium alginate, formed in situ on a paper, have
been used to separate decorative laminates after they have been formed in a
hot-pressing system.
9.20 Environmental Biotechnology

Future prospects: The overall annual growth rate for alginates is 2-3
per cent, with textile printing applications accounting for about half of the
global market. However, the textile industry is flat at present, as it rides a trough
in the cycle of peaks and troughs, and it is 90 per cent based in Asia and the
Near East (Turkey). Pharmaceutical and medical uses are about 20 per cent by
value of the market and have stayed buoyant, with 2-4 per cent annual growth
rates, driven by ongoing developments in controlled release technologies and
the use of alginates in wound care applications. Food applications are worth
about 20 per cent of the market. That sector has been growing only slowly,
and recently has grown at only 1-2 per cent annually. The paper industry
takes about 5 per cent and the sector is very competitive, not increasing but
just holding its own. The alginate industry faces strong competition from
Chinese producers, whose prices do not reflect the real expense of cultivating
Laminaria japonica, even in China, yet they do not appear to import sufficient
wild seaweeds to offset those costs. The result is low profitability for most of
the industry, with the best opportunities lying in the high end of the market,
such as pharmaceutical and medical applications.

9.3  COMMERCIAL APPLICATION OF MICROALGAE AND THEIR

PRODUCTS
The use of microalgae by humans dates back thousands of years to the Chinese
who used Nostoc to survive famine. Other species of microalgae including, the
blue green algae species, Arthrospira (Spirulina) and Aphanizomenon, have also
been used by humans for thousands of years. Spirulina has been exploited
by ancient peoples in both Chad and Mexico as a source of food. Although,
microalgae have been used for food by humans for thousands of years,
microalgae culture is one of the modern biotechnologies. Unialgal cultures
were first achieved in 1890, with Chlorella vulgaris and, the use of this type of
cultures was used for the study of plant physiology in the early 1900. In 2004,
the global market for microalgal biomass was estimated to be 5,000 t of dry
matter per year and generated a turnover of US$ 1,250 million. Microalgae
contain about 50% carbon in their biomass and this carbon is obtained in
most cases from ‘‘atmospheric’’ carbon dioxide, and therefore, microalgae are
attracting interest as vehicles for carbon sequestration for industrial processes.
The use of algae for therapeutic purposes has a long history, but the search
for biologically-active substances from algae, especially, examination for
antibiotic activity began only in the 1950s. Much of that laboratory work up
until the 1980s, focused on macroalgae. Approximately, 15,000 natural marine
products have now been screened for biological activity and 45 marine derived
natural products have been tested to be used as medical drugs in preclinical
and clinical trials. Only 2 have been developed into registered drugs—one
from a marine snail and the other from a sea squirt, although, none, as yet, from
microalgae. It is reported that preclinical trials are currently being undertaken
on an anti-cancer drug, Curacin, derived from the blue-green algae, Lyngbya
majuscula. There have also been reports of the use of products derived from
algae being used to combat HIV infection.
Algae-Biotechnology 9.21

Microalgae are responsible for over 50% of primary photosynthetic

productivity on earth and are budding sunlight factories for a wide range
of potentially useful products, but are barely used. In the early 1950s, the
increase in world population lead to the search for new alternative food and
protein sources and algae, appeared at the time, a good. Although, algae have
and are still used for food, to some extent around the world, the large scale
production of algae to solve the world’s food calorie and protein shortage
has not materialized. The large-scale cultivation of microalgae and the use
of its biomass for the production of useful products were first considered
seriously in Germany during World War II. Commercial large–scale modern
algae culture started in the early 1960s in Japan with the culture of Chlorella.
In the early 1970s, culturing and harvesting of Spirulina began in Mexico. The
third major area of commercialisation of algae occurred, in Australia, with the
growth of Dunaliella salina for the production of b-carotene. Plants were then
subsequently built in the USA and Israel and production of blue-green algae
also commenced in India. Plants have recently been built in the USA and India
for the growth of Haematococcus pluvialis as a source of astaxanthin, approved
as food-coloring and also a powerful anti-oxidant.
A common feature of most of the algal species currently produced
commercially (i.e. Chlorella, Spirulina and Dunaliella) is that they grow in
highly selective environments which means that they can be grown in open
air cultures and still remain relatively free of contamination by other algae and
protozoa. Thus, Chlorella grows well in nutrient-rich media, Spirulina requires
a high pH and bicarbonate concentration and Dunaliella salina grows at very
high salinity. Those species of algae, which do not have environmental-selective
advantages, may need to be grown in closed systems. This includes most of
the marine algae grown as aquaculture feeds (e.g. Skeletonema, Chaetoceros,
Thalassiosira, Tetraselmis and Isochrysis) and the dinoflagellate, Crypthecodinium
cohnii, grown as a source of long-chain polyunsaturated fatty acids, as well as,
almost all other species being considered for commercial mass culture.

9.3.1  Current commercial uses of algae

‘Health’ foods: Chlorella is produced by more than 70 companies with the
largest producer, Taiwan Chlorella Manufacturing and Co, producing 400t
of dry algal biomass per year. Chlorella is sold as a health food or dietary
supplement. Several reports from Japan have described various potential
therapeutic effects of Chlorella, and although, these are encouraging findings,
the investigations were initiated by the Chlorella producing companies and
must be viewed as such. Suggested health benefits include, efficacy on gastric
ulcers, wounds and constipation, together with, preventive action against both
atherosclerosis and hyper-cholesterol and antitumor activity. The suggested
most important active substance is b-1,3-glucan which is believed to be an
active immune-stimulator, free radical scavenger and a reducer of blood lipids.
Unfortunately, the American Cancer Society concluded, ‘‘however, available
9.22 Environmental Biotechnology

scientific studies do not support its effectiveness for preventing or treating

cancer or any other disease in humans’’.
Spirulina (Arthrospira) is used in human nutrition because of it high
protein content and excellent nutrient value. It is also a valuable source of
the essential fatty acid, linolenic acid, that cannot be synthesised by humans.
A wide range of medical benefits for a broad range of conditions have been
claimed, but the full validations of many of these claims is still awaited. Many
companies are producing ‘‘nutraceuticals’’ (food supplements with claimed
nutritional and medicinal benefits) made from Spirulina. DIC claims to be the
biggest manufacture of Spirulina in China. Production of Spirulina in Hainan
by DIC was estimated at 300 t per annum. The largest plant is Earthrise Farms
in California, USA, covering over 444,000 m2, producing algal tablets and
powder, sold in over 20 countries, is owned by DIC in Japan. Cyanotech, in
Kona, Hawaii, produces a powder under the name Spirulina Pacifica. The
market for dried Spirulina was estimated to be US$ 40 million, in 2005.
Carotenoids: Algae contain carotenoids, yellow orange or red pigments,
that include b-carotene, a substance converted by the body to Vitamin A. There
are over 400 known carotenoids, but only a few are used commercially, the
two main compounds being b-carotene and astaxanthin. The most important
uses of carotenoids are as food colourants and as supplements for human and
animal feeds. The average concentration of carotenoids in most algae is only
0.1–2%, but Dunaliella, when grown under the right conditions of high salinity
and light intensity, will produce up to 14% b-carotene. Dunaliella is, therefore,
well suited to the commercial production of b-carotene and several industrial
production plants are in operation around the world including Australia,
Israel, USA and China. The major producer is Cognis Nutrition and Health,
whose farms cover 800 ha in Western Australia and produce b-carotene
extracts together with algal powder for human and animal use.
Until 1980, production of b-carotene was synthetic. Natural carotenoids,
although more expensive than synthetic, have the advantage of supplying the
natural isomers in their natural ratio and the natural isomers of b-carotene are
considered superior to the trans-synthetic form. In 1994, algal b-carotene was
sold in small quantities due to the cost and the majority of production was
synthetic, however, it was concluded that the increasing number of algal plants
producing b-carotene rich Dunaliella, at that time, may change the situation.
It has since been reported that b-carotene from Dunaliella, is a substantial
growing industry and commercial utilisation is economically viable.
Astaxanthin is another carotenoid that can be derived from algae and is
principally used in fish farming and as a dietary supplement or anti-oxidant.
The annual worldwide aquaculture market for this pigment in 2004 was
estimated to be US$ 200 million with an average price of US$ 2500/kg, but the
market is dominated by the synthetic form. Astaxanthin can be produced by
Haematococcus, a freshwater alga that normally grows in puddles, birdbaths
Algae-Biotechnology 9.23

and other shallow fresh water depressions. Haematococcus can contain up to

3% astaxanthin, but it requires a two-stage culture process which is not suited
to open pond cultivation. The first stage of the process is designed to optimize
algal biomass (green thin-walled flagellated stage with optimum growth at
a temperature 22–25°C) and the second stage (thick-walled resting stage)
under intense light and nutrient-poor conditions during which astaxanthin is
produced. Due to its high price, the astaxanthin produce from Haematococcus
cannot compete with synthetic forms. However, for some applications, natural
astaxanthin is preferred, for example, in carp, chicken and red sea bream
diets due to enhanced natural pigment deposition, regulatory requirements
and consumer demand for natural products. Commercial production is
being carried out in Hawaii, India and Israel, where Algatech sell a crushed
Haematococcus biomass on the pharmaceutical market. Cyanotech, in Hawaii,
claimed a market share of over 95% of the animal nutrition market for algae-
based astaxanthin products, but subsequently stopped selling into the animal
nutrition market in March, 2008.
Phycobiliproteins: In addition to chlorophyll and the lipophilic pigments,
such as the carotenoids, certain types of microalgae especially red algae, or
rhodophyta, contain phycocyanin and Phycoerythrin. These photosynthetic
accessory pigments, collectively known Phycobiliproteins, are deeply colored
(red or blue), water soluble, complex, proteinaceous compounds. These algae
pigments have the potential as natural colorants for food, cosmetics and
pharmaceuticals.
Dainippon Ink and Chemicals produces a blue food colorant from
Spirulina, called Lina blue, that is used in chewing gum, ice slush, sweets, soft
drinks, dairy products and wasabi. Phycobiliproteins can be commercially
produced from Spirulina and the red microalgae Porphyridium and Rhodella.
Phycobiliproteins are widely used in clinical or research immunology because
they have very powerful and highly sensitive fluorescent properties. In 1997,
the global market for Phycobiliproteins colorants was estimated at US$ 50
million and prices vary from US$ 3 to US$ 25/mg.
Fatty acids: Humans and animals lack the requisite enzymes to synthesize
polyunsaturated fatty acids (PUFAs) of more than 18 carbon atoms and they
must obtain them from food and are, therefore, often known as essential fatty
acids. A group of essential fatty acids which are attracting a lot of attention
currently are known as omega-3, a group of unsaturated fatty acids where a
carbon double bond is in the third position from the methyl or omega end.
Oily Fish and fish oils are well known sources of PUFAs, but issues have
been raised concerning possible accumulation of toxins in fish. Fish obtain
PUFAs from algae, with marine algae, such as diatoms, being a particularly
rich source and therefore, algae have been proposed as a Commercial source.
Docosahexaenoic acid (DHA) produced by heterotrophic (using plant or
animal materials as an energy source rather than light in photosynthesis
9.24 Environmental Biotechnology

(autotrophic)) culture of the dinoflagellate, Crypthecodinium cohnii, is the

only currently commercial produced fatty acid from algae. DHA (22:6) is a
22-carbon chain with six cis double bonds; the first double bond is located
at the third carbon from the omega end (an omega-3 fatty acid). It is used as
a supplement in infant formulas and as a dietary supplement. It is essential
for the proper functioning of our brains as adults, and for the development
of our nervous system and visual abilities during the first 6 months of life. In
addition, omega-3 fatty acids are part of a healthy diet that helps lower risk of
heart disease.
Although, infants that are breastfed should receive enough DHA if the
mother has an adequate intake of this fatty acid, many organizations have
suggested that DHA should be added to baby milk formula. The world
wholesale market for infant formula in 2005 was estimated to be about US$
10 billion per annum. Martek produce DHA using Crypthecodinium cohnii for
baby formula, and in 2003, production was 240 tons and it is now a company
with more than 525 employees and revenue of more than US$ 300 million.
Martek acquired, OmegaTech, another producer of a DHA, oil, known as DHA
gold an adult dietary supplement from Schizochytrium—Nutrinova, formerly
known as Hoechst until 1997, when it was taken over by Celanese, produced
an oilcontaining DHA from Ulkenia. In 2005, Lonza, based in Switzerland,
acquired Nutrinova’s DHA business and sells the DHA oil as a vegetarian
source of omega-3.
A process for producing high-purity Eicosapentaenoic acid, EPA, another
omega-3 fatty acid (20:5), from Phaeodactylum tricornutum, has been developed
by the University of Almeria in Spain. An economic analysis, on a potential
facility producing 430 kg 96% pure EPA per year, estimated the total cost of
production at US$ 4,602/kg, with 60% of the cost arising from the recovery
process and 40% from the biomass production. It is believed that the cost
needs to be reduced by 80% to be economically viable. The residual biomass
following the extraction of the EPA contains too much residual solvent to be
sold for animal feed, and therefore, must be incinerated. The annual worldwide
demand of EPA is 300 t. The production of EPA from Nannochloropsis and,
the diatom, Nitzschia, is reportedly under study. It is reported that studies
are being undertaken to produce omega-6 PUFAs; Linolenic acid (18:3) from
Arthrospira and Arachidonic acid (20:4) from Porphyridium.
Stable isotopic biochemicals: Microalgae are wellsuited as a source of
isotopically labelled compounds due to their ability to incorporate stable
isotopes from relatively inexpensive inorganic molecules into high value
isotopic organic chemicals. The market for these chemicals is in excess of US$ 13
million. Spectra Stable Isotopes, now part of Cambridge Isotope Laboratories,
sells marked amino acids at up to US$ 5900/g and marked nucleic acids at
US$ 28/mg.
Animal feed: Microalgae are an important food source and feed additive
in the commercial rearing of many aquatic animals. Over 30% of the current
Algae-Biotechnology 9.25

world algal production is sold for animal feed and over 50% of the world
production of Spirulina is used as feed supplements. In 1999, the production
of microalgae for use in aquaculture reached 1000 t. Approximately, a dozen
types are produced in relatively small quantities for the aquaculture industry.
Many studies have shown the suitability of algae as a potential animal feed
and as a replacement for conventional protein sources such as soybean
and fish-meal. Algae are the normal natural food for many animals used in
aquaculture and, it is not surprising that, they are considered the best food
source for aquaculture. Unfortunately, the trend is to avoid using live algae
due to their high cost and production difficulties. The cost of producing dry
algal biomass feed, in Australia, varies from US$ 80/kg to US$ 800/kg. Other
cost estimates have given costs between US$ 50/kg to US$ 150/kg with a peak
value of US$ 1000/kg. Yeast can be used as a replacement feed, but the omission
of algae from aquaculture may give less predictable performance and the total
replacement of algae in aquaculture diets are not yet considered sufficiently
advanced for widespread utilisation. In addition to direct feed, microalgae can
be used as a feed source for zooplankton which can, in turn, be used as a feed
for fish.
One problem that has been encountered is, with the notable exception
of Spirulina, poor digestibility due to the high content of cellulose cell wall
material. Ruminates, such as sheep and cattle, are capable of digesting cellulose
material and it is therefore, possible to feed algae direct to them, but this has
not gained much commercial favor yet. Poultry can be fed up to 5–10% algae
and, this can have a positive effect on the development of colour within the
skin and egg yolk due to the carotenoids. Higher concentrations of algae in the
feed can lead to adverse effects.
Human food: Although, microalgae are eaten as a food in China and Chad
and, had been considered as a solution to the world’s food shortage, their use
on a global scale appears limited to health food and food supplements.
The current state of microalgal production
Alga Annual Producer Applications and
production country products
Spindina 3,000 t dry wt China, Human and
India, USA, animal nutrition,
Myanmar, phycobiliproteins,
Japan cosmetics
Chlorella 2,000 t dry wt Taiwan, Human nutrition,
Germany, aquaculture, cosmetics
Japan
Davabella 1,200 t dry wt Australia, Human nutrition,
Israel, USA, cosmetics, b-carotene
China
9.26 Environmental Biotechnology

Apyani 500 t dry wt USA Human nutrition

Haemodo 300 t dry wt USA, India, Aquaculture,
Israel Astaxanthin
Crypthiodinium 240 t DHA oil USA DHA oil
Schizohytrium 10 t DHA oil USA DHA oil

9.3.2  Future Development of Microalgal Applications

Market potential: Microalgae have been exploited by man for millennia and
the BEAM network, supported by Murdoch University, Australia, believes
that microalgae biotechnology has grown and diversified significantly over
the past 30 years. Although, some progress has been made and there are
commercial microalgae applications, including pigments, fatty acids and
health foods, only a few hundred of the thousands of species of microalgae
have been studied and, just a handful are cultivated on an industrial scale.
It would appear that the potential of commercial applications of microalgae
are enormous, but, despite development over last 50 years, the number of
commercially available products are still fairly limited and, although there
are a number of types of closed bioreactor, being investigated and available,
the majority of microalgal production is in open ponds. The main commercial
product, despite the enormous range of biochemicals potentially available
from microalgae, appears to be ‘‘health food’’ that may produce health
benefits, but may be subject to fashion and fad. The second current key area
of commercial application of microalgae appears to be food additives in form
of carotenes, pigments and fatty acids. These can have functional advantages
over synthetic products or products from other natural sources, but can be
at a cost disadvantage. The challenge in the application of microalgae for
commercial ends is to focus only on those products with a large market and/
or profit potential where the use of microalgae leads to clear competitive
advantages. Fuel and food would appear to offer the largest markets. The
growth of microalgae to solve the world food shortage has, in the past, been
considered, but the wide-scale commercial growth of algae for human food is
restricted mainly to health foods, food supplements and food additives. The
health food market is the branch of algae production with the highest sales,
but the market is dependent on a number of claims of health benefits without
the necessary scientific proof of efficacy. There has also been some market
sentiment that Spirulina was being overproduced.
Food additives, such as b-carotene, pigments and fatty acids, from
microalgae, can be superior to synthetic products and products from other
natural sources, such as fish oil, but unfortunately, they are often also
considerably more expensive. There appears to be very considerable potential
Algae-Biotechnology 9.27

for the discovery of new therapeutic biochemicals from microalgae, but

an immediate breakthrough for algae-based products does not appear
imminent.
Current commercially viable exploitation of microalgae products is
limited to products other than fuel and, the immediate future for the
commercialization of microalgae may be with non-fuel products, but the
‘‘lessons’’ learned from microalgal non-fuel products, together with their
potential coproduction with fuel, may lead to the more rapid commercial
realization of microalgal biofuel.
Growth systems: The commercial growth of algae in open ponds, despite
over 30 years of research, is still only currently viable for three taxi, Spirulina,
Dunaliella and Chlorella, mainly due to the suppression of the growth of
competitive species by use of highly selective environments. Currently, the
majority of microalgal production occurs in outdoor ponds. Concerns have
also been expressed about the possible contamination of food from microalgae
grown in open ponds. Bioreactors are considerably more expensive than open
ponds and their use will probably be restricted to very high value products.
It has been argued that lower extraction costs, due to the higher algae
content, can make bioreactors more competitive. A study on the production of
the valuable fatty acid, EPA, found that 60% of the costs arise from the recovery
process and the cost of algae biomass production in bioreactors is high and
needs to be reduced. It has also been concluded that, although some products
are now being produced in bioreactors commercially, the development of
microalgae biotechnology has been slowed by the limited performance of
bioreactors. Open ponds are likely to remain the major means of production,
but efficient closed bioreactors may be viable in the production for high value
products where purity is essential or a ‘sensitive’ algae species is required.
Species selection: Many consider that the genetic modification of
microalgae is the best way to improve the yield of valuable products at
reduced cost. The absence of cell differentiation in microalgae can make
genetic manipulation simpler than in higher plants, but progress on the genetic
engineering of microalgae was relatively slow, until recently. The NREL in the
USA spent considerable time, effort and resources on the genetic modification
of microalgae. The NREL work on genetic modification may have diverted
their resources from algae selection and process optimization that may have
yielded more commercial benefit. Genetic modification has a considerable
image problem, particularly in Europe and, genetically modified algae may
also be seen as a potential environmental threat, particularly, if open pond
systems are to be used. Part of the appeal of microalgae substances for food
and therapeutic use is their ‘‘natural image” and genetic modification may
have a negative effect on this. Algae strain selection and process optimization,
rather than genetic engineering, may be the key areas for future development.
9.28 Environmental Biotechnology

9.4  MASS CULTIVATION OF MICROALGAE AS A SOURCE OF

PROTEIN AND FEED
Microalgae are microscopic photosynthetic organisms that are found in both
marine and freshwater environments. Their photosynthetic mechanism is
similar to landbased plants, but being due to a simple cellular structure, and
submerged in an aqueous environment where they have efficient access to
water, CO2 and other nutrients, they are generally more efficient in converting
solar energy into biomass.
In terms of biomass, microalgae form the world’s largest group of primary
producers and they occur in benthic, epithelic, symbiotic and pelagic forms.
The microalgal biomass contains all the essential amino acids, unsaturated
fatty acids, carbohydrates, dietary fiber, and a whole range of vitamins and
other bioactive compounds, so that, it can be a highly suitable alternative in
livestock feeding, human nutrition and perhaps, also in biofuel industry.
Photo bioreactors: Photo bioreactors are different types of tanks or closed
systems in which microalgae are cultivated. Microalgal cultures consist of a
single or several specific strains optimized for producing the desired product.
Water, necessary nutrients and CO2 are provided in a controlled way, while
oxygen has to be removed. Microalgae receive sunlight either directly through
the transparent container walls or, via light fibers or tubes, that channel it
from sunlight collectors. A great amount of developmental work to optimize
different photobioreactor systems for algae cultivation has been carried out
by many researchers. It has also been suggested to grow heterotrophic algae
in conventional fermenters instead of photobioreactors for production of
high-value products. Instead of light and photosynthesis, heterotrophic algae
are relying on utilizable carbon sources in the medium for their carbon and
energy generation.
Open pond systems: Open pond systems are shallow ponds in which
algae are cultivated. Nutrients can be provided through runoff water from
nearby land areas or by channeling the water from sewage/water treatment
plants. The water is typically kept in motion by paddle wheels or rotating
structures, and some mixing can be accomplished by appropriately designed
guides. Algal cultures can be defined (one or more selected strains), or are
made up of an undefined mixture of strains.
• The high capital cost associated with producing microalgae in closed
culture systems is the main challenge for commercialization of such
systems. Open systems do not require expenses associated with
sterilization of axenic algal cultures. However, this leads to high
risk of contamination of the culture by bacteria or other unwanted
microorganisms. A common strategy, therefore, to achieve monocultures
in an open pond system is to keep them at extreme culture conditions
such as high salinity, nutrition or alkalinity. Consequently, this strictly
limits the species of algae that can be grown in such systems. From
the public literature, currently only Dunaliella (high salinity), Spirulina
Algae-Biotechnology 9.29

(high alkalinity) and Chlorella (high nutrition) have been successfully

grown in commercial open pond systems.
• The necessity for a large cultivation area has been pointed out as a
limitation in using open ponds to grow microalgae for mitigating the
CO2 released from power generating plants. It has been estimated that
a raceway pond requires 1.5 km2 to fix the CO2 emitted from a 150 MW
thermal power plant. The large area requirements are partly due to the
comparable lower productivity of open pond systems. It was pointed
out that improving the control of limiting parameters in open ponds
such as culture medium temperature and contamination, and thereby,
increasing productivity, could be accomplished by using a transparent
cover over the ponds, such as a greenhouse.
• Selection of a suitable production system clearly depends on the
purpose of the production facility. For example, closed bioreactors will
not be suitable for wastewater treatment, because the costs for treating
wastewater in this system will be too high in relation to the low value
added during the production process. On the other hand, high quality/
value products that are produced only in small amounts might require
production in bioreactors.
Harvesting of microalgae: Conventional processes used to harvest
microalgae include concentration through centrifugation, foam fractionation,
flocculation, membrane filtration and ultrasonic separation. Harvesting costs
may contribute 20 – 30% to the total cost of algal biomass. The micro-algae
are typically small with a diameter of 3–30 mm, and the culture broths may be
quite dilute at less than 0.5g L–I. Thus, large volumes must be handled. The
harvesting method depends on the species, on the cell density, and often also
on the culture conditions.
Applications of microalgae: Microalgae find uses as food and as live
feed in aquaculture for production of bivalve molluscs, for juvenile stages
of abalone, crustaceans and some fish species and, for zooplankton used in
aquaculture food chains. Therapeutic supplements from microalgae comprise
an important market in which compounds such as b-carotene, astaxanthin,
polyunsaturated fatty acid (PUFA) such as DHA and EPA and polysaccharides
such as b-glucan, dominate. Exploitation of microalgae for bioenergy
generation (biodiesel, biomethane, biohydrogen), or combined applications
for biofuels production and CO2-mitigation, by which CO2 is captured and
sequestered, are under research. The dominating species of micro-algae in
commercial production includes Isochrysis, Chaetoceros, Chlorella, Arthrospira
(Spirulina) and Dunaliella.

9.5  MICROALGAE AS A SOURCE OF FEED

Microalgae aquaculture feeds: The largest current application of microalgae
feeds is in aquaculture. Microalgae are used fresh (e.g. live, or at least not
9.30 Environmental Biotechnology

dried) in bivalve, shrimp and fish fry and fingerling production (in the latter
case, via an intermediate food source, such as zooplankton or brineshrimp).
Several companies produce aquaculture feeds using Chlorella and Spirulina, or
a mixture thereof. Some examples of the use of microalgae for aquaculture:
• Microalgae species Hypneacervicornis and Cryptonemia crenulata
particularly, rich in protein, were tested in shrimp diets. Algae were
collected, rinsed, dried and ground up for the feed formulations.
Larvae shrimps were fed daily with one of four diets prepared with
different percentages of seaweed powder: 39%, 26%, 13%, 0%. The
results suggest that there is an increase in feed conversion when the
levels of algae are increased. Amount of algae in fish meal resulted in
significant increase in shrimp growth rates.
• A large number of marine nitrogen-fixing cyanobacteria have been
tested for their nutritional value with the hybrid Tilapia fish fry; a
majority were acceptable as single ingredient feeds. Very high growth
rates of Tilapia fish using marine cyanobacteria, with indoor and
outdoor cultures, have been reported. The marine cyanobacterium
Phormidium valderianum was shown to serve as a complete aquaculture
feed source, based on the nutritional qualities and non-toxic nature
with animal model experiments.
• More than 40 species of microalgae are used in aquaculture worldwide,
depending on the special requirements of local seafood production.
• Apart from feeding larvae and zooplankton, often with special
microalgal species, the addition of Spirulina and Chlorella to common
fish feed compositions seems to be a promising market. Initially, the
colour-enhancing effects of phycocyanin-containing Spirulina biomass
or carotenoides from Dunaliella were exploited in ornamental fish.
• In recent years, questions of feed utilization and health status in the
dense aquacultural fish populations became more important. Here,
the addition of microalgae can, depending on concentration, directly
enhance the immune system of fish, as investigations on carp have
shown.
The addition of microalga-derived astaxanthin to feed formulations
enhances the colour of the muscles of salmonids. This has a high
biotechnological potential and culture techniques for Haematococcus pluvialis
are well developed for this purpose.
Microalgae as poultry feeds: In poultry rations, algae up to a level of 5-10%
can be used safely as partial replacement for conventional proteins. The yellow
colour of broiler skin and shanks, as well as of egg yolk, is the most important
characteristics that can be influenced by feeding algae. Moreover, the Institut
für Getreideverarbeitung (Bergholz-Rehbrücke, Germany) produces a natural
feed with the algae Chlorella and Arthrospira, called Algrow.
Algae-Biotechnology 9.31

• Ginzberg and his group in the year 2000 studied role of algae,
Porphyridium sp. as feed supplement on metabolism of chicken. Earlier
results in the same laboratory showed a reduction in serum cholesterol
and triglyceride-levels in rodents fed with red algal biomass. In this
study, lyophilized algae biomass was fed to chickens at a proportion
of 5% or 10% of the standard chicken diet. Chickens fed with algae
biomass consumed 10% less food and their serum cholesterol levels
were significantly lower (by 11% and 28%, for the groups fed with 5%
and 10% supplement, respectively) as compared with the respective
values of the control group (with unsupplemented diet).
• Egg yolk of chickens fed with algae tended to have reduced cholesterol
levels (by 10%) and increased linoleic acid and arachidonic acid
levels (by 29% and 24%, respectively). In addition, the colour of egg
yolk became darker, indicating that higher carotenoid was produced
(2.4 fold higher).
• Other poultry feeding studies with Spirulina (up to 30%) showed that,
both protein and energy efficiency of this alga were similar to other
conventional protein carriers up to a level of 10%. Significantly higher
growth rates and lower non-specific mortality rate were observed in
turkey poults fed with Spirulina at the level of 1-10 g.kg -1 diet.
Microalgae as swine feeds: Abril and his group studied the potential
toxicity of DHA-rich microalgae (DRM) from Schizochytrium sp., administered
in the diet of growing swine. The only DHA-rich microalgae treatment-
related changes were higher weight gain and feed conversion efficiency. The
administration of DRM (at up to five times the anticipated commercial dose)
did not produce any treatment-related adverse effects in commercial strains
of swine.
Microalgae trials in pet feeds: Another very promising application
for microalgal biomass is the pet food market, where not only the health-
promoting effects but also, effects on the external appearance of the pet (shiny
hair, beautiful feathers) are of consumer importance. Studies on minks and
rabbits provide evidence of such effects for pets.
Other microalgae trials: Belay and his group assessed potential of
Arthrospira (Spirulina) in animal feed. About 30% of the current world
production of 2000 tõn  Spirulina is sold for animal feed applications. Some
of these positive effects of Spirulina like, increased growth rate, colour
enhancement and general tissue quality may be nutritional effects. However,
the fact that growth rates are improved even at 0.1% Spirulina supplementation
may suggest the presence of substances that may mimic the effects of or
stimulate production of growth hormones. The most promising application
may be its immune enhancement effects and through this, its anti-viral
and anti-bacterial properties, since these effects are exhibited at very low
supplemental concentrations in the feeds.
9.32 Environmental Biotechnology

• Spirulina or its extracts may accelerate development of the immune

system of many animals, especially, during the early stages of their
lives. Presently, Arthrospira is widely used as food additive and can
replace 50% of protein diets in existing feeds.
• Astaxanthin, from H. pluvialis, is therefore, of interest comparing with
other sources. More than 80% of astaxanthin from this microalgae, is
found in an esterified form, whereas in synthetic astaxanthin, it is in the
free form. In birds, astaxanthin in an esterified form has been shown to
be more efficiently absorbed than free astaxanthin.
• In mice infected with Helicobacter pylori, treatment with H. pluvialis
algal meal, significantly reduced the bacterial load of H. pylori in the
stomach. This was explained by the effects of astaxanthin on cytokines
produced by H. pylori-specific T- cells.
 CHAPTER

10 Concepts and Scope of

Plant Biotechnology

Plant biotechnology may be defined as the application of knowledge obtained

from study of the life sciences to create technological improvements in plant
species. By this very broad definition, plant biotechnology has been conducted
for more than ten thousand years.
• The roots of  plant biotechnology  can be traced back to the time
when humans started collecting seeds from their favorite wild plants
and began cultivating them in tended fields. It appears that when the
plants were harvested, the seeds of the most desirable plants were
retained and replanted the next growing season.
• While these primitive agriculturists did not have extensive knowledge
of the life sciences, they evidently did understand the basic principles of
collecting and replanting the seeds of any naturally-occurring variant
plants with improved qualities, such as those with the largest fruits
or the highest yield, in a process that we call artificial selection. This
domestication and controlled improvement of plant species was the
beginning of plant biotechnology. 
• This very simple process of selectively breeding naturally-occurring
variants with observably improved qualities served as the basis
of agriculture for thousands of years and resulted in thousands of
domesticated plant cultivars that no longer resembled the wild plants
from which they descended. The second era of plant biotechnology began
in the late 1800s as the base of knowledge derived from the study of the
life sciences increased dramatically. 
• In the 1860s Johannes Gregor Mendel, using data obtained from
controlled pea breeding experiments, deduced some basic principles
of genetics and presented these in a short monograph modestly
titled “Versuche über Pflanzenhybriden” (in Verhandlungen des
naturforschenden Vereins, 1866; Experiments with Plant-Hybridisation,
1910). 
10.2 Environmental Biotechnology

• In this publication, Mendel proposed that heritable genetic factors

segregate during sexual reproduction of plants and that factors for
different traits assort independently of each other. 
• Mendel’s work suggested a mechanism of heritable factors that could
be manipulated by controlled breeding of plants through selective
fertilization and, also suggested that, the pattern of inheritance for
these factors could be analyzed or, in some cases, predicted by the use
of mathematical statistics.
• These findings complemented the work of Charles Darwin, who
expounded the principles of descent with modification and selection as
the chief factor of evolutionary change in his 1859 book on the Origin
of Species by Means of Natural Selection.
The application of these principles to agriculture resulted in deliberately-
produced hybrid varieties for a large number of cultivated plants via
selective fertilization. These artificially selected hybrids soon began to benefit
humankind with tremendous increases in both the productivity and the
quality of food crops.

10.1 APPLICATIONS OF GENETIC ENGINEERING TECHNOLOGY

FOR CROP IMPROVEMENT
The third era of plant biotechnology involves a drastic change in the way
crop improvement may be accomplished, by direct manipulation of genetic
elements (genes). This process is known as genetic engineering and results in
plants that are called genetically modified organisms (GMOs), to distinguish
them from plants that are produced by conventional plant-breeding methods.
Genetically modified plants can contribute desirable genes from outside
traditional breeding boundaries. Even genes from outside the plant kingdom
can now be brought into plants. For example, animal genes, including human
genes, have been transferred into plants, a feat not replicated in nature.
• Our ancestors have been improving crops and livestock for thousands
of years through selective breeding or crossbreeding to produce
desired traits. Biotechnology is just an extension of this process. Genes
are added, deleted or temporarily silenced to produce desired results.
• Genetic engineering involves cutting and moving snippets of DNA
from one plant to another. Permanently integrating new DNA into
a plant’s original DNA forms what’s known as a transgenic plant or
genetically modified organism (GMO).
Steps in Genetic Engineering: The first genetically engineered plants,
tobacco plants, were reported in the scientific literature in 1984. Since 1984,
there have been thousands of genetically engineered plants produced in
laboratories worldwide. The process of genetically engineering a plant
involves several key steps:
• Isolating the genetic sequence (gene) to be placed from its biological
source.
Concepts and Scope of Plant Biotechnology 10.3

• Placing the gene in an appropriate vehicle to facilitate insertion into

plant cells.
• Inserting the gene into the plant by a process known as plant
transformation.
• Selecting the few plant cells that contain the new gene (transformed
cells) out of all the plant cells in the explant.
• Multiplying the transformed cells in sterile tissue culture.
• Regenerating the transformed cells into a whole plant that can grow
outside the tissue culture vessel.
The gene or genes to be placed in the plant may be obtained from virtually
any biological source: animals, bacteria, fungi, viruses, or other plants.
Placing genes into an appropriate vehicle for transfer into a plant involves
using various molecular biology techniques, such as restriction enzymes
and ligation, to essentially “cut and paste” the gene or genes of interest into
another DNA molecule, which serves as the transfer vehicle (vector).
Major goals of genetic engineering of plants:
• Produce crops with less impact on environment.
• Reduce expense of food production.
• Produce crops less vulnerable to insects, diseases, weeds and harsh
environments.
• Develop crops with more nutrients.
• Develop crops for production of medicines and vaccines.
Major genetically engineered traits in plants:
• Insect resistance,
• Herbicide resistance,
• Virus resistance,
• Delayed fruit ripening,
• Altered oil content,
• Pollen control.
Public Concern: It is perhaps this lack of natural boundaries for genetic
exchange that seems so foreign to conventional scientific thought and that
makes plant genetic engineering controversial.
• The thought of taking genes from animals, bacteria, viruses, or any other
organism and putting them into plants, especially plants consumed for
food, has raised a host of questions among concerned scientists and
public alike. 
• Negative public perception of genetically modified crops has
affected the development and commercialization of many  plant
biotechnology products, especially food plants. While there are dozens
10.4 Environmental Biotechnology

of genetically engineered plants ready for field production, public

pressure has delayed the release of some of these plants and has caused
the withdrawal of others from the marketplace.
• This public concern also appears to be driving increased government
review of products and decreased government funding for  plant
biotechnology  projects in Europe. Negative public perceptions do
not seem to be as strong in Asia, since the pressures of feeding large
populations tend to outweigh the perceived risks. 
• The social climate of the United States toward biotechnology, although
guarded, appears to be less apprehensive than that of most European
countries. Therefore, many agricultural biotechnology projects have
moved from European countries to U.S. laboratories.
Economic Goals: To what end are humans genetically engineering plants?
This is an  essential question for researchers, executives of biotechnology
companies, and consumers, at large. 
Before addressing technical questions about how to apply biotechnology,
the desired goals must be clearly defined. The general goals of  plant
biotechnology appear to be:
1. economic improvement of existing products, 
2. improvement of human nutrition, and 
3. development of novel products from plants.
Economic improvements include increases in yield, quality, pest resistance,
nutritional value, harvest ability, or any other change that adds value to an
established agricultural product. 
Examples of this category include insect-protected tomatoes, potatoes,
cotton, and corn; herbicide-resistant canola, corn, cotton, flax, and soybeans;
canola and soybeans with geneticallyaltered oil compositions; virus-resistant
squash and papayas; and improved ripening tomatoes. All these examples
were introduced to agriculture in the later half of the 1990s.
Nutritional Goals: Additionally, some products appearing in the scientific
literature but awaiting commercialization have the potential to dramatically
improve human nutritional deficiencies, which are especially prevalent in
developing countries. 
• These products include “golden rice,” genetically modified rice that
produces carotenoids, a dietary source of vitamin A. Golden rice has
the potential to prevent vitamin A deficiency in developing countries,
where this vitamin deficiency is a leading cause of blindness.
• Researchers are also using genetic engineering to increase the amount
of the iron-storing protein ferritin in seed crops such as legumes. Iron
deficiency, which affects 30 per cent of the human population, can
impair cognitive development and cause other health problems. This
Concepts and Scope of Plant Biotechnology 10.5

proposed enhancement of iron content in consumable plant products

could help more than a billion people who suffer from chronic iron
deficiency.

10.2 PRODUCTION OF TRANSGENIC PLANTS WITH IMPROVED

YIELDS AND NUTRITIONAL QUALITY
During the last decades, a tremendous progress has been made in the
development of transgenic plants using various techniques of genetic
engineering. The plants, in which a functional foreign gene has been
incorporated by any biotechnological methods, that generally are not present
in the plant, are called transgenic plants. As per estimates recorded in 2002,
transgenic crops are cultivated world-wide on about 148 million acres (587
million hectares) land by about 5.5 million farmers. Transgenic plants have
many beneficial traits like insect resistance, herbicide tolerance, delayed fruit
ripening, improved oil quality, weed control, etc.
Some of the commercially grown transgenic plants in developed countries
are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High-lauric’ rapeseed
(canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full
registration was granted to genetically engineered Bt gene containing insect-
resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA.
Some of the traits introduced in these transgenic plants are as follows:
Stress tolerance: Biotechnology strategies are being developed to
overcome problems caused due to biotic stresses (viral, bacterial infections,
pests and weeds) and abiotic stresses (physical actors such as temperature,
humidity, salinity, etc).
Abiotic stress tolerance: The plants show their abiotic stress response
reactions by the production of stress related osmolytes like sugars (e.g.
trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline,
glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic
plants have been produced which, overexpress the genes for one or more of
the above mentioned compounds. Such plants show increased tolerance to
environmental stresses. Resistance to abiotic stresses includes stress induced
by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone
and intense light. These environmental stresses result in the destruction and
deterioration of crop plants which leads to low crop productivity. Several
strategies have been used and developed to build resitance in the plants
against these stresses.
Herbicide tolerance: Weeds are unwanted plants which decrease the
crop yields and by competing with crop plants for light, water and nutrients.
Several biotechnological strategies for weed control are being used e.g., the
overproduction of herbicide-target enzyme (usually in the chloroplast) in the
plant which makes the plant insensitive to the herbicide. This is done by the
introduction of a modified gene that encodes for a resistant form of the enzyme
10.6 Environmental Biotechnology

targeted by the herbicide in weeds and crop plants. Roundup Ready crop
plants, tolerant to herbicide—Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop
herbicide resistant plants are:
(a) Overexpression of the target protein by integrating multiple copies of
the gene or by using a strong promoter.
(b) Enhancing the plant detoxification system which helps in reducing the
effect of herbicide.
(c) Detoxifying the herbicide by using a foreign gene, and
(d) Modification of the target-protein by mutation.
Some of the examples are:
Glyphosate resistance—Glyphosate is a glycine derivative and is a
herbicide which is found to be effective against the 76 of the world’s worst
78 weeds. It kills the plant by being the competitive inhibitor of the enzyme
5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic
acid pathway. Due to it’s structural similarity with the substrate phosphoenol
pyruvate, glyphosate binds more tightly with EPSPS and thus, blocks the
shikimic acid pathway.
Certain strategies were used to provide glyphosate resistance to plants.
• It was found that EPSPS gene was overexpressed in Petunia due to gene
amplification. EPSPS gene was isolated from Petunia and introduced
into the other plants. These plants could tolerate glyphosate at a dose
of 2–4 times higher than that required to kill wildtype plants.
• By using mutant EPSPS genes—A single base substitution from C to
T resulted in the change of an amino acid from proline to serine in
EPSPS. The modified enzyme cannot bind to glyphosate and thus,
provides resistance.
• The detoxification of glyphosate by introducing the gene (isolated from
soil organism—Ochrobactrum anthropi) encoding for glyphosate oxidase
into crop plants. The enzyme glyphosate oxidase converts glyphosate
to glyoxylate and amino methylphosponic acid. The transgenic plants
exhibited very good glyphosate resitance in the field.

Another example is of Phosphinothricin resistance

Phosphinothricin is a broad spectrum herbicide and is effective against
broad-leafed weeds. It acts as a competitive inhibitor of the enzyme
glutaminesynthase which results in the inhibition of the enzyme glutamine
synthase and accumulation of ammonia and finally the death of the plant.
The disturbance in the glutamine synthesis also inhibits the photosynthetic
activity.
The enzyme phosphinothricin acetyl transferase (which was first observed
in Streptomyces sp. in natural detoxifying mechanism against phosphinothricin)
Concepts and Scope of Plant Biotechnology 10.7

acetylates phosphinothricin, and thus inactivates the herbicide. The gene

encoding for phosphinothricin acetyl transferase (bar gene) was introduced
in transgenic maize and oil seed rape to provide resistance against
phosphinothricin.
Other abiotic stresses: The abiotic stresses due to temperature, drought,
and salinity are collectively also known as water-deficit stresses. The plants
produce osmolytes or osmo protectants to overcome the osmotic stress. The
attempts are on to use genetic engineering strategies to increase the production
of osmoprotectants in the plants. The biosynthetic pathways for the production
of many osmoprotectants have been established and genes coding the key
enzymes have been isolated. E.g., Glycine betaine is a cellular osmolyte which
is produced by the participation of a number of key enzymes like choline
dehydrogenase, choline monooxygenase, etc. The choline oxidase gene from
Arthrobacter sp. was used to produce transgenic rice with high levels of glycine
betaine giving tolerance against water-deficit stress.
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis,
when this plant was gradually exposed to slowly declining temperature.
By introducing the coordinating gene (it encodes a protein which acts as
transcription factor for regulating the expression of cold-tolerant genes),
expression of cold-tolerant genes was triggered, giving protection to the plants
against the cold temperatures.
Insect resistance: A variety of insects, mites and nematodes significantly
reduce the yield and quality of the crop plants. The conventional method is to
use synthetic pesticides, which also have severe effects on human health and
environment. The transgenic technology uses an innovative and eco-friendly
method to improve pest control management. About 40 genes obtained from
microorganisms of higher plants and animals have been used to provide insect
resistance in crop plants.
The first genes available for genetic engineering of crop plants for pest
resistance were Cry genes (popularly known as Bt genes) from a bacterium
Bacillus thuringiensis. These are specific to particular group of insect pests,
and are not harmful to other useful insects like butterflies and silk worms.
Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal,
cauliflower, cabbage, etc.) have been developed. This has proved to be an
effective way of controlling the insect pests and has reduced the pesticide use.
The most notable example is Bt cotton (which contains CrylAc gene) that is
resistant to a notorious insect pest Bollworm (Helicoperpa armigera). There
are certain other insectresistant genes from other microorganisms which have
been used for this purpose. Isopentenyl transferase gene, from Agrobacterium
tumefaciens, has been introduced into tobacco and tomato. The transgenic
plants with this transgene were found to reduce the leaf consumption by
tobacco hornworm and decrease the survival of peach potato aphid.
Certain genes from higher plants were also found to result in the synthesis
of products possessing insecticidal activity. One of the examples is the Cowpea
10.8 Environmental Biotechnology

trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and
oilseed rape for developing transgenic plants. Earlier it was observed that the
wild species of cowpea plants growing in Africa were resistant to attack by
a wide range of insects. It was observed that the insecticidal protein was a
trypsin inhibitor that was capable of destroying insects belonging to the orders
Lepidoptera, Orthaptera, etc. Cowpea trypsin inhibitor (CpTi) has no effect on
mammalian trypsin, hence, it is non-toxic to mammals.
Virus resistance: There are several strategies for engineering plants for
viral resistance, and these utilize the genes from virus itself (e.g. the viral coat
protein gene). The virus-derived resistance has given promising results in a
number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya.
The induction of virus resistance is done by employing virus-encoded gene,
virus coat proteins, movement proteins, transmission proteins, satellite RNA,
antisense RNAs, and ribozymes. The virus-coat protein—mediated approach
is the most successful one to provide virus resistance to plants. It was in 1986,
when the transgenic tobacco plants expressing tobacco mosaic virus (TMV)
coat protein gene were first developed. These plants exhibited high levels of
resistance to TMV.
The transgenic plant providing coat protein-mediated resistance to virus
are rice, potato, peanut, sugar beet, alfalfa, etc. The viruses that have been
used include Alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), Potato
virus X (PVX), potato virus Y (PVY) etc.
Resistance against Fungal and Bacterial infections: As a defense strategy
against the invading pathogens (fungi and bacteria), the plants accumulate
low molecular weight proteins which are collectively known as pathogenesis-
related (PR) proteins.
Several transgenic crop plants with increased resistance to fungal
pathogens are being raised with genes coding for different compounds.
One of the examples is the Glucanase enzyme that degrades the cell wall of
many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene
encoding for beta-1,4 glucanase has been isolated from barley, introduced, and
expressed in transgenic tobacco plants. This gene provided good protection
against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way
fungal infection can be reduced. Transgenic potato plants with lysozyme gene
providing resistance to Eswinia carotovora have been developed.
Delayed fruit ripening: The gas hormone, ethylene, regulates the ripening
of fruits therefore, ripening can be slowed down by blocking or reducing
ethylene production. This can be achieved by introducing ethylene, forming
gene(s) in a way that will suppress its own expression in the crop plant. Such
fruits ripen very slowly (however, they can be ripened by ethylene application)
and this helps in exporting the fruits to longer distances without spoilage due
to longer-shelf life.
Concepts and Scope of Plant Biotechnology 10.9

The most common example is the ‘Flavr Savr’ transgenic tomatoes,

which were commercialized in U.S.A in 1994. The main strategy used was the
antisense RNA approach. In the normal tomato plant, the PG gene (for the
enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme
polygalacturonase which is involved in the fruit ripening. The complimentary
DNA of PG encodes for antisense mRNA, which is complimentary to normal
(sense) mRNA. The hybridization between the sense and antisense mRNAs
renders the sense mRNA ineffective. Consequently, polygalacturonase is
not produced causing delay in the fruit ripening. Similarly, strategies have
been developed to block the ethylene biosynthesis, thereby, reducing the
fruit ripening. E.g., transgenic plants with antisense gene of ACC oxidase
(an enzyme involved in the biosynthetic process of ethylene) have been
developed. In these plants, production of ethylene was reduced by about 97%
with a significant delay in the fruit ripening.
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC
and removes amino group) has been transferred and expressed in tomato
plants which showed 90% inhibition in the ethylene biosynthesis.
Male Sterility: The plants may inherit male sterility either from the
nucleus or cytoplasm. It is possible to introduce male sterility through genetic
manipulations while the female plants maintain fertility. In tobacco plants,
these are created by introducing a gene coding for an enzyme (barnase, which
is a RNA–hydrolyzing enzyme, that inhibits pollen formation. This gene is
expressed specifically in the tapetal cells of anther using tapetal specific
promoter TA29 to restrict its activity only to the cells involved in pollen
production. The restoration of male fertility is done by introducing another
gene barstar that suppresses the activity of barnase at the onset of the breeding
season. By using this approach, transgenic plants of tobacco, cauliflower,
cotton, tomato, corn, lettuce, etc. with male sterility have been developed.
Nutritional quality: Transgenic crops with improved nutritional quality
have already been produced by introducing genes involved in the metabolism
of vitamins, minerals and amino acids.
• A transgenic Arabidopsis thaliana that can produce ten-fold higher
vitamin E (alpha-tocopherol) than the native plant has been developed.
The biochemical machinery to produce a compound close in structure
to alpha-tocopherol is present in A. thaliana. A gene that can finally
produce alpha-tocopherol is also present, but is not expressed. This
dormant gene was activated by inserting a regulatory gene from a
bacterium which resulted in an efficient production of vitamin E.
• Glycinin is a lysine-rich protein of soybean and the gene encoding
glycinin has been introduced into rice and successfully expressed. The
transgenic rice plants produced glycinin with high contents of lysine.
• Using genetic engineering, Prof. Potrykus and Dr. Peter Beyer have
developed rice, which is enriched in pro-vitamin A, by introducing
10.10 Environmental Biotechnology

three genes involved in the biosynthetic pathway for carotenoid, the

precursor for vitamin A. The aim was to help millions of people who
suffer from night blindness due to Vitamin A deficiency, especially,
whose staple diet is rice. The presence of beta-carotene in the rice
gives a characteristic yellow/orange colour, hence this pro-vitamin A
enriched rice is named as Golden Rice.
• The genetic engineering is also being used to improve the taste of food
e.g., a protein ‘monellin’ isolated from an African plant (Dioscorephyllum
cumminsii) is about 100,000 sweeter than sucrose on molar basis.
Monellin gene has been introduced into tomato and lettuce plants to
improve their taste.

10.3  TRANSGENIC PLANTS FOR THE PRODUCTION OF VIRAL

ANTIGENS
A viral Antigen is an antigen with multiple antigenicities that is protein in
nature, strain-specific, and closely associated with the virus particle. A viral
antigen is a protein, encoded by the viral genome. A viral protein is an antigen
specified by the viral genome that can be detected by a specific immunological
response.
Viruses are infectious pathogens that cause serious diseases & major
threats for global public health, such as influenza, hepatitis, & AIDS. Virus is a
sub-micrometer particle that has DNA or RNA packed in a shell called capsid.
Viral antigens protrude from the capsid and often fulfill important function in
docking to the host cell, fusion, and injection of viral DNA/RNA. Antibody-
based immune responses form a first layer of protection of the host from
viral infection; however, in many cases a vigorous cellular immune response
mediated by T-cells and NK-cells is required for effective viral clearance.
When cellular immunity is unable to clear the virus, the infection can become
chronic, and serum antibodies to the viral pathogen are used as first indicator
for the diagnosis of the disease.
ELISAs provide a valuable tool in the detection and diagnosis of virus
infection. The ability to produce recombinant viral proteins will ensure that
future ELISAs are safe, specific and rapid. Even when a virus cannot be
cultured, provided gene sequence is available, it is possible to rapidly respond
to emerging viruses and new viral strains of existing pathogens.
Recombinant viral antigens contain part of viral sequence, meaning
that, the recombinant antigen contains a region which can be recognized by
different antibodies produced by different individuals. This reduces the risk
of false negatives which can occur with synthetic peptides, which contain
only a small portion of the entire protein. If an individual infected with a viral
antigen makes antibodies to a part of the protein not included in the synthetic
peptides, a false negative results.
Recombinant viral protein usually contains a fusion protein/partner
which produces superior attachment to assay surfaces such as wells. For this
Concepts and Scope of Plant Biotechnology 10.11

reason, smaller amounts of recombinant protein will produce the same results
as larger amounts of infused protein. The choice of fusion partner prevents
false positives, allowing superior adhesion without incorrect results.
Recombinant Viral proteins are expressed in bacteria, yeast, mammalian
cells, and viruses. E. coli cells were first to be used for this purpose but the
expressed proteins were not glycosylated, which was a major drawback
since many of the immunogenic proteins of viruses such as the envelope
glycoproteins, were glycosylated. Nevertheless, in many instances, it was
demonstrated that the non-glycosylated protein backbone was just as
immunogenic. The obvious advantage of recombinant viral antigens is that
they are available in unlimited quantities and the production and quality
control processes is simple.
Advantages of using recombinant viral antigens:
• Production and quality control is simple.
• No nucleic acids or other viral or external proteins, therefore less toxic.
• Safer in cases where viruses are oncogenic or establish a persistent
infection.
• Feasible even if virus cannot be cultivated
Disadvantages:
• May be less immunogenic than conventional inactivated whole-virus
vaccines.
• Requires adjuvant.
• Fails to elicit CMI.
Facts about Viral Antigens:
• A Viral Protein Mimics its Way into cells.
• Viral Protein Helps Infected T Cells Stick To Uninfected Cells.
• The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through
a Nuclear Factor of Activated T Cell-dependent Trans-activation
Pathway.
• Viral Protein is an effective preventative against ear infection.
• HIV-1 Viral Protein R Induces Apoptosis via a Direct Effect on the
Mitochondrial Permeability Transition Pore.
• The Level of Viral Antigen, presented by Hepatocytes, Influences CD8
T-Cell Function.
• Antigen-presenting cells from calves persistently infected with bovine
viral diarrhea virus, a member of the Flaviviridae, are not compromised
in their ability to present viral antigen.
• There is a difference in the distribution and spread of a viral antigen,
development of lesions and correlation between presence of viral
antigen and lesions.
10.12 Environmental Biotechnology

• The absence of viral antigens on the surface of equine herpesvirus-

1-infected peripheral blood mononuclear cells is a strategy to avoid
complement-mediated lysis.
• Viral Protein Influences Key Cell-signaling Pathway.
• A viral protein produced by cancer-causing virus influences a key
signaling pathway in the immune cells that the virus infects. This
stimulates the cells to divide, helping the virus spread through the
body.
• Protection by recombinant viral proteins against a respiratory virulent
avian metapneumovirus has been achieved.
• Viral O-acetylesterases are found in Influenza C viruses and Corona-
viruses. Viral O-acetylesterases remove cellular receptors from the
surface of target cells which destroys the receptor. Recombinant viral
O-acetylesterases, derived from Sf9 insect cells as chimeric proteins
fused to eGFP, specifically hydrolyze 9-O-acetylated sialic acids
while that of Sialodacryoadenitis virus, a rat coronavirus related to
mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The
recombinant esterases were shown to specifically de-O-acetylate sialic
acids on glycoconjugates. The recombinant viral proteins can be used
to unambiguously identify O-acetylated acids.
To date, many plant species have been used for vaccine production. Early
studies used tobacco and potato but now tomato, banana, corn, lupine, lettuce
and others are being used for this purpose. The choice of the plant species
(and tissue, in which the protein accumulates) is important and is usually
determined through, how the vaccine is to be applied in the future. For
example, an edible, palatable plant is necessary if the vaccine is planned for
raw consumption. This limitation is overcome in non-edible plants by vaccine
antigen extraction and purification. Antigen extraction is often performed
when using tobacco, a plant that offers considerable experimental advantages
such as ease of transformation and extensive genomic sequence knowledge.
Heat treatment is feasible only if there is no deleterious effect on antigen
stability. Recently, a “cooked” GM corn snack that accumulates the E. coli heat-
labile enterotoxin has been proposed. In the case of vaccines for animal use,
the plant should preferentially be selected among those consumed as normal
component of the animals’ diet.
The production of a vaccine in plants depends upon the availability of
a DNA sequence coding for a protective antigen and on the construction
of an expression “cassette” suitable for plant transformation. Stable plant
transformation currently offers two options: insertion of the foreign gene
into the nuclear genome or into the chloroplast genome. Transient plant
transformation has also been used for plant expression of vaccine antigens
through integration of the gene of interest into a plant virus and subsequent
Concepts and Scope of Plant Biotechnology 10.13

infection of susceptible plants. Plants producing two or more antigens may also
be obtained through transformation with multiple gene constructs or through
sexual crossing. The strategies for plant expression cassette construction and
plant transformation depend on the desired goal.

10.3.1  Edible Vaccines

Crop plants offer cost-effective bioreactors to express antigens which
can be used as edible vaccines. The approach is to isolate genes encoding
antigenic proteins from the pathogens and then expressing them in plants.
Such transgenic plants or their tissues producing antigens can be eaten for
vaccination/immunization (edible vaccines). The expression of such antigenic
proteins in crops like banana and tomato are useful for immunization of
humans, since banana and tomato fruits can be eaten raw.
Transgenic plants (tomato, potato) have been developed for expressing
antigens derived from animal viruses, e.g. rabies virus, herpes virus. In 1990,
the first report of the production of edible vaccine (a surface protein from
Streptococcus) in tobacco at 0.02% of total leaf protein level was published in
the form of a patent application under the International Patent Cooperation
Treaty (Mason and Arntzen,1995). The first clinical trials in humans, using
a plant derived vaccine were conducted in 1997 and were met with limited
success. This involved the ingestion of transgenic potatoes with a toxin of
E.coli causing diarrhea.
The process of making of edible vaccines involves the incorporation of a
plasmid carrying the antigen gene and an antibiotic-resistance gene, into the
bacterial cells, e.g. Agrobacterium tumefaciens. The small pieces of potato leaves
are exposed to an antibiotic which can kill the cells that lack the new genes.
The surviving cells with altered genes multiply and form a callus. This callus
is allowed to grow and subsequently transferred to soil to form a complete
plant. In about a few weeks, the plants bear potatoes with antigen vaccines.
The bacteria E.coli, V. cholerae cause acute watery diarrhea by colonizing
the small intestine and by producing toxins. Chloera toxin (CT) is very similar
to E.coli toxin. The CT has two subunits, A and B. Attempt was made to
produce edible vaccine by expressing heatlabile enterotoxin (CT-B) in tobacco
and potato.
Another strategy adopted to produce a plant-based vaccine, is to infect
the plants with recombinant virus carrying the desired antigen that is fused
to viral coat protein. The infected plants are reported to produce the desired
fusion protein in large amounts in a short duration. The technique involves,
either placing the gene downstream a subgenomic promoter, or fusing the
gene with capsid protein, that coats the virus.
10.14 Environmental Biotechnology

Technical and social benefits envisaged in plant-derived edible vaccines

No. Benefit Characteristics
1. Oral delivery The plant cell wall, consisting essentially of
cellulose and sugars, provides protection in the
stomach and gradual release of the antigen in
the gut.
2. Use as raw food or The vaccinogenic plant tissue may be used as
dry powder raw food, dried or, alternatively, proteins may
be partially or fully purified and administered
in capsules as dry powder.
3. No need for “cold The vaccinogenic plant parts or plant extracts
chain” can be stored and shipped at room temperature.
4. Mucosal and serum Plant-derived vaccines are primarily designed
immune response to trigger the mucosal immune system (IgA),
thus preventing pathogen entry at mucosal
surfaces; they also elicit serum and, possibly,
cytotoxic responses.
5. Cost efficiency Production cost will be reduced 100–1000 times
as compared with that of traditional vaccines.
6. Optimised Plants may be engineered to accumulate
expression system the antigenin – convenient intracellular
compartments (endoplasmic reticulum,
chloroplast)
7. Ease of genetic Procedures essentially rely on established
manipulation molecular and genetic manipulation protocols;
these are already available in developing
countries.
8. Ease of production GM-plants can be stored as seeds. Unlimited
and scale-up vaccine quantity can be produced from these
in limited time; production and management is
suitable for developing countries.
9. Safer than Lack of contamination with mammalian
conventional pathogens.
vaccines
10. Ideal for ace bio-A. Safety and cost-efficiency propose, plants
weapons plant-derived vaccines, as an ideal tool to face
bio-terrorism.
11. Ideal for veterinary Cost-affordable, Ready for use as food additive.
use
Concepts and Scope of Plant Biotechnology 10.15

Advantages of edible vaccines: The edible vaccines produced in

transgenic plants will solve the storage problems, will ensure easy delivery
system by feeding and will have low cost as compared to the recombinant
vaccines produced by bacterial fermentation. Vaccinating people against
dreadful diseases like cholera and hepatitis B, by feeding them banana, tomato,
and vaccinating animals against important diseases, will be an interesting
development.
Safety and public acceptance: Plant-derived vaccines are certified
free from animal pathogen contaminants. Furthermore, plant DNA is not
known to interact with the animal DNA and plant viral recombinants do not
invade mammalian cells. Further safety of plant-derived vaccines is obtained
through following the same regulations established for traditional vaccines.
Nevertheless, the present concern over the use of GM-plants is now affecting
research in this important field, especially in Europe. One of the fears is that
GM-pollen may outcross with sexually compatible plants (related crops
or weeds) and affect biodiversity. In order to address this alarm, several
pollen-containment approaches have been developed. These are essentially
based on the exploitation of different forms of male sterility (suicide genes,
infertility barriers, apomixis). An alternative way of solving the problem is
engineering vaccines into the cpDNA, which is not transmitted to the sexual
progeny through the pollen grains. An additional safety feature would be
the recognition of GM-plants that produce vaccines by the addition of genes
encoding coloured plant pigments. It is important to recognize that plants that
produce vaccines are medicinal plants and should be grown, processed and
regulated as pharmaceutical products. It is thought that pharmaceutical crops
will be able to be grown on relatively small extensions of land, preferably
contained within greenhouses, using controlled environmental conditions. In
the majority of earlier papers, level of antigen accumulation in the plant organ
was in the order of 0.1–0.4% of total soluble protein, while the more recent
developments on cpDNA integration promises to increase this value to 30%
or more. At the latter value, land requirements for industrial plant-derived
vaccine-production will be in the order of a few thousand square meters. This
will definitely enable vaccine-producing plants to be set apart from field-
grown crop plants and offer added safety when engineered plant viruses are
used for transient antigen expression. A further point of public concern in GM-
plants is the presence of antibiotic-resistance genes (used as selective marker
in most transgenic plants). Approaches have now been developed to generate
GM-plants (with both nuclear or cpDNA integration) that do not carry these
genes.

10.4 BIOTECHNOLOGY IN AGRICULTURE—MERITS AND

DEMERITS
Biotechnology is the application of scientific techniques to modify and improve
plants, animals, and microorganisms to enhance their value. Agricultural
biotechnology is the area of biotechnology involving applications to agriculture.
10.16 Environmental Biotechnology

Agricultural biotechnology has been practiced for a long time, as people

have sought to improve agriculturally important organisms by selection
and breeding. An example of traditional agricultural biotechnology is the
development of disease-resistant wheat varieties by cross-breeding different
wheat types, until the desired disease resistance is present in a resulting new
variety.
In the 1970s, advances in the field of molecular biology provided scientists
with the ability to manipulate DNA—the chemical building blocks that
specify the characteristics of living organisms—at the molecular level. This
technology is called Genetic engineering. It also allows transfer of DNA between
more distantly related organisms than was possible with traditional breeding
techniques. Today, this technology has reached a stage where scientists can
take one or more specific genes from nearly any organism, including plants,
animals, bacteria, or viruses, and introduce those genes into another organism.
An organism that has been transformed using genetic engineering techniques
is referred to as a transgenic organism, or a genetically engineered organism.
Many other terms are in popular use to describe these aspects of today’s
biotechnology. The term “genetically modified organism” or “GMO” is
widely used, although genetic modification has been around for hundreds,
if not thousands–of years, since deliberate crosses of one variety or breed
with another results in offspring that is genetically modified compared to
the parents. Similarly, foods derived from transgenic plants have been called
“GMO foods,” “GMPs” (genetically modified products), and “biotech foods.”
While some refer to foods developed from genetic engineering technology as
“biotechnology-enhanced foods,” others call them “frankenfoods.” For the
reasons discussed later in this publication, controversy affects various issues
related to the growing of genetically engineered organisms and their use as
foods and feeds.

Genetic engineering Vs traditional biotechnology

In traditional breeding, crosses are made in a relatively uncontrolled manner.
The breeder chooses the parents to cross; but at the genetic level, the results
are unpredictable. DNA from the parents recombines randomly, and desirable
traits such as pest resistance are bundled with undesirable traits, such as lower
yield or poor quality.
Traditional breeding programs are time-consuming and labor-intensive. A
great deal of effort is required to separate undesirable from desirable traits, and
this is not always economically practical. For example, plants must be back-
crossed again and again over many growing seasons to breed out undesirable
characteristics produced by random mixing of genomes.
Current genetic engineering techniques allow segments of DNA, that code
genes for a specific characteristic, to be selected and individually recombined
in the new organism. Once the code of the gene that determines the desirable
Concepts and Scope of Plant Biotechnology 10.17

trait is identified, it can be selected and transferred. Similarly, genes that code
for unwanted traits can be removed. Through this technology, changes in a
desirable variety may be achieved more rapidly than with traditional breeding
techniques. The presence of the desired gene controlling the trait can be tested
for at any stage of growth, such as in small seedlings in a greenhouse tray.
The precision and versatility of today’s biotechnology enable improvements
in food quality and production to take place more rapidly than when using
traditional breeding.

10.4.1 Transgenic Crops in the U.S. Market

Although genetically engineered organisms in agriculture have been available
for only 10 years, their commercial use has expanded rapidly. Recent estimates
are that, more than 60–70 percent of food products on store shelves may
contain at least a small quantity of crops produced with these new techniques.
Major crop plants produced by genetic engineering techniques have been
so welcomed by farmers, that, currently a third of the corn and about three-
quarters of the soybean and cotton grown in the USA are varieties developed
through genetic engineering (see: http://usda.mannlib.cornell.edu/reports/
nassr/field/pcp-bbp/pspl0302.pdf). Twelve transgenic crops (corn, tomato,
soybean, cotton, potato, rapeseed [canola], squash, beets, papaya, rice, flax, and
chicory) have been approved for commercial production in the USA. The most
widely grown are “Bt” corn and cotton and glyphosate-resistant soybeans.
Bt corn and cotton have had DNA from a naturally-occurring insecticidal
organism, Bacillus thuringiensis, incorporated into their genome; it kills some of
the most serious insect pests of these crops (European and southwestern corn
borers, and cotton budworms and bollworms) after they feed on the plant,
while beneficial insects are left unaffected. Glyphosate-resistant soybeans are
unharmed by the broad-spectrum herbicide glyphosate, a characteristic that
allows farmers to kill yield-reducing weeds in soybean fields without harming
the crop.

Benefits of genetic engineering in agriculture

Everything in life has its benefits and risks, and genetic engineering is no
exception. Much has been said about potential risks of genetic engineering
technology, but so far there is little evidence from scientific studies that these
risks are real. Transgenic organisms can offer a range of benefits above and
beyond those that emerged from innovations in traditional agricultural
biotechnology. Following are a few examples of benefits resulting from
applying currently available genetic engineering techniques to agricultural
biotechnology.
Increased crop productivity: Biotechnology has helped to increase crop
productivity by introducing such qualities as, disease resistance and increased
drought tolerance to the crops. Now, researchers can select genes for disease
resistance from other species and transfer them to important crops. For
10.18 Environmental Biotechnology

example, researchers from the University of Hawaii and Cornell University

developed two varieties of papaya, resistant to papaya ring spot virus,
by transferring one of the virus’ genes to papaya to create resistance in the
plants. Seeds of the two varieties, named ‘SunUp’ and ‘Rainbow’, have been
distributed under licensing agreements to papaya growers since 1998.
Further examples come from dry climates, where crops must use water as
efficiently as possible. Genes from naturally drought-resistant plants can be
used to increase drought tolerance in many crop varieties.
Enhanced crop protection: Farmers use crop-protection technologies
because they provide cost-effective solutions to pest problems which, if left
uncontrolled, would severely lower yields. As mentioned above, crops such as
corn, cotton, and potato have been successfully transformed through genetic
engineering to make a protein that kills certain insects when they feed on the
plants. The protein is from the soil bacterium Bacillus thuringiensis, which has
been used for decades as the active ingredient of some “natural” insecticides.
In some cases, an effective transgenic crop-protection technology can
control pests better and more cheaply than existing technologies. For example,
with Bt engineered into a corn crop, the entire crop is resistant to certain pests,
not just the part of the plant to which Bt insecticide has been applied. In these
cases, yields increase as the new technology provides more effective control.
In other cases, a new technology is adopted because it is less expensive than a
current technology with equivalent control.
There are cases in which new technology is not adopted because, for
one reason or another, it is not competitive with the existing technology. For
example, organic farmers apply Bt as an insecticide to control insect pests in
their crops, yet, they may consider transgenic Bt crops to be unacceptable.
Improvements in food processing: The first food product resulting from
genetic engineering technology to receive regulatory approval, in 1990, was
chymosin, an enzyme produced by genetically engineered bacteria. It replaces
calf rennet in cheese-making and is now used in 60 per cent of all cheese
manufactured. Its benefits include increased purity, a reliable supply, a 50
percent cost reduction, and high cheese yield efficiency.
Improved nutritional value: Genetic engineering has allowed new options
for improving the nutritional value, flavor, and texture of foods. Transgenic
crops in development include soybeans with higher protein content, potatoes
with more nutritionally available starch and an improved amino acid content,
beans with more essential amino acids, and rice with the ability to produce
beta-carotene, a precursor of vitamin A, to help prevent blindness in people
who have nutritionally inadequate diets.
Better flavor: Flavor can be altered by enhancing the activity of plant
enzymes that transform aroma precursors into flavoring compounds.
Transgenic peppers and melons with improved flavor are currently in field
trials.
Concepts and Scope of Plant Biotechnology 10.19

Fresher produce: Genetic engineering can result in improved keeping-

properties to make transport of fresh produce easier, giving consumers access
to nutritionally-valuable whole foods and preventing decay, damage, and loss
of nutrients. Transgenic tomatoes with delayed softening can be vine-ripened
and still be shipped without bruising. Research is underway to make similar
modifications to broccoli, celery, carrots, melons, and raspberry. The shelflife
of some processed foods such as peanuts has also been improved by using
ingredients that have had their fatty acid profile modified.
Environmental benefits: When genetic engineering results in reduced
pesticide dependence, we have less pesticide residues on foods, we reduce
pesticide leaching into groundwater, and we minimize farm worker exposure
to hazardous products. With Bt cotton’s resistance to three major pests, the
transgenic variety now represents half of the cotton crop and has thereby
reduced total world insecticide use by 15 per cent! Also, according to the U.S.
Food and Drug Administration (FDA), “increases in adoption of herbicide-
tolerant soybeans were associated with small increases in yields and variable
profits but significant decreases in herbicide use”.
Benefits for developing countries: Genetic engineering technologies can
help to improve health conditions in less developed countries. Researchers
from the Swiss Federal Institute of Technology’s Institute for Plant Sciences
inserted genes from a daffodil and a bacterium into rice plants to produce
“golden rice,” which has sufficient beta-carotene to meet total vitamin A
requirements in developing countries with rice-based diets. This crop has
potential to significantly improve vitamin uptake in poverty-stricken areas
where vitamin supplements are costly and difficult to distribute and vitamin
A deficiency leads to blindness in children.

10.4.2 Possible Risks Associated with using Transgenic Crops in

Agriculture
Some consumers and environmentalists feel that inadequate effort has been
made to understand the dangers in the use of transgenic crops, including their
potential long-term impacts. Some consumer-advocate and environmental
groups have demanded the abandonment of genetic engineering research
and development. Many individuals, when confronted with conflicting
and confusing statements about the effect of genetic engineering on our
environment and food supply, experience a “dread fear” that inspires great
anxiety. This fear can be aroused by only a minimal amount of information or,
in some cases, misinformation. With people thus concerned for their health
and the well-being of our planetary ecology, the issues related to their concerns
need to be addressed. These issues and fears can be divided into three groups:
health, environmental, and social.

10.4.3 Health-related Issues

Allergens and toxins: People with food allergies have an unusual immune
reaction when they are exposed to specific proteins, called allergens, in food.
10.20 Environmental Biotechnology

About 2 per cent of people across all age groups have a food allergy of some
sort. The majority of foods do not cause any allergy in the majority of people.
Food-allergic people usually react only to one or a few allergens in one or
two specific foods. A major safety concern raised with regard to genetic
engineering technology is the risk of introducing allergens and toxins into,
otherwise safe, foods. The Food and Drug Administration (FDA) checks to
ensure that the levels of naturally-occurring allergens in foods made from
transgenic organisms have not significantly increased above the natural range
found in conventional foods. Transgenic technology is also being used to
remove the allergens from peanuts, one of most serious causes of food allergy.
Antibiotic resistance: Antibiotic resistance genes are used to identify and
trace a trait of interest that has been introduced into plant cells. This technique
ensures that a gene transfer during the course of genetic modification was
successful. Use of these markers has raised concerns that new antibiotic-
resistant strains of bacteria will emerge. The rise of diseases that are resistant
to treatment with common antibiotics is a serious medical concern of some
opponents of genetic engineering technology.
The potential risk of transfer from plants to bacteria is substantially less
than the risk of normal transfer between bacteria, or between us and the
bacteria that naturally occur within our alimentary tracts. Nevertheless, to
be on the safe side, FDA has advised food developers to avoid using marker
genes that encode resistance to clinically important antibiotics.

10.4.4 Environmental and Ecological Issues

Potential gene escape and super weeds: There is a belief among some
opponents of genetic engineering technology that transgenic crops might
cross-pollinate with related weeds, possibly resulting in “super weeds”
that become more difficult to control. One concern is that pollen transfer
from glyphosate-resistant crops to related weeds can confer resistance to
glyphosate. While the chance of this happening, although extremely small,
is not inconceivable; resistance to a specific herbicide does not mean that the
plant is resistant to other herbicides, so, affected weeds could still be controlled
with other products.
Some people are worried that genetic engineering could conceivably
improve a plant’s ability to “escape” into the wild and produce ecological
imbalances or disasters. Most crop plants have significant limitations in their
growth and seed dispersal habits that prevent them from surviving long
without constant nurture by humans, and they are thus, unlikely to thrive in
the wild as weeds.
Impacts on “non-target” species: Some environmentalists maintain that
once transgenic crops have been released into the environment, they could have
unforeseen and undesirable effects. Although transgenic crops are rigorously
tested before being made commercially available, not every potential impact
Concepts and Scope of Plant Biotechnology 10.21

can be foreseen. Bt corn, for instance, produces a very specific pesticide

intended to kill only pests that feed on the corn. In 1999, however, researchers
at Cornell University found that pollen from Bt corn could kill caterpillars
of the harmless Monarch butterfly. When they fed Monarch caterpillars
milkweed, dusted with Bt corn pollen in the laboratory, half of the larvae died.
But follow-up field studies showed that, under real-life conditions, Monarch
butterfly caterpillars are highly unlikely to come into contact with pollen from
Bt corn that has drifted onto milkweed leaves—or, to eat enough of it to harm
them.
Insecticide resistance: Another concern related to the potential impact
of agricultural biotechnology on the environment involves the question of
whether insect pests could develop resistance to crop-protection features of
transgenic crops.
There is fear that large-scale adoption of Bt crops will result in rapid build-
up of resistance in pest populations. Insects possess a remarkable capacity to
adapt to selective pressures, but to date, despite widespread planting of Bt
crops, no Bt tolerance in targeted insect pests has been detected.
Loss of biodiversity: Many environmentalists, including farmers, are
very concerned about the loss of biodiversity in our natural environment.
Increased adoption of conventionallybred crops raised similar concerns in the
past century, which led to extensive efforts to collect and store seeds of as
many varieties as possible of all major crops. These “heritage” collections in
the USA, and elsewhere, are maintained and used by plant breeders. Modern
biotechnology has dramatically increased our knowledge of how genes
express themselves and highlighted the importance of preserving genetic
material, and agricultural biotechnologists also want to make sure that we
maintain the pool of genetic diversity of crop plants needed for the future.
While transgenic crops help ensure a reliable supply of basic foodstuffs, U.S.
markets for specialty-crop varieties and locally-grown produce, appear to
be expanding rather than diminishing. Thus, the use of genetically modified
crops is unlikely to negatively impact biodiversity.

10.4.5 Social Issues

Labeling: Some consumer groups argue that foods derived from genetically
engineered crops should carry a special label. In the USA, these foods currently
must be labeled only if they are nutritionally different from a conventional
food.
“Terminator” technology: Most farmers in the USA and elsewhere buy
fresh seeds each season, particularly of such crops as corn, green peppers, and
tomatoes. Anyone growing hybrid varieties must buy new seeds annually,
because seeds from last year’s hybrids grown on the farm will not produce
plants identical to the parent. For the same reason—to avoid random genetic
diversity due to open pollination—farmers do not plant mango, avocado, or
10.22 Environmental Biotechnology

macadamia from seed; instead, they clone individual plants of known quality
through techniques such as grafting.
In developing countries, many farmers who are not growing hybrids save
harvested seeds for replanting the next year’s crop. A technology has been
developed that might be used to prevent purchasers of transgenic crop seeds
from saving and replanting them. Such “terminator” seeds are genetically
engineered, along with other improvements more acceptable to farmers, to
produce plants with seeds that have poor germination. This forces farmers,
who otherwise save seed, to purchase it, if they wish to use these improved
commercial varieties. And, in the USA, the crops engineered with various
characters are sold alongside non-transgenic alternatives for which, growers
also, typically, purchase seeds annually.
Despite these mitigating circumstances, this is a serious issue among
organic-growers and in developing countries, where the practice of saving
seeds is the norm for farmers who are not growing hybrid crops. Inclusion
of “terminator” genes means that these farmers cannot take advantage of
improvements brought about by genetic engineering without being brought
into the economic cycle that profits the seed companies. Without profit
incentive, however, these companies are unlikely to invest in improving crops.
This issue is analogous to that faced by pharmaceutical companies developing
new medications against human diseases. Clearly, it is a difficult and divisive
social issue.

Safety regulations in India

A major contribution of Department of Biotechnology, Government of India
has been to set up, in collaboration with other government departments, a
framework for the evaluation and eventual clearance of transgenic crops for
field cultivation. The release of transgenic crops is governed by the Indian
Environment Protection Act (EPA) – 1986 which came into force from May,
1986. The Act provides a framework for the protection and improvement
of environment. Later, the rules and regulations for the manufacture, use,
import, export and storage of hazardous microorganisms, genetically
engineered organisms or cells were notified under EPA, 1986 on 5th December,
1989. A mechanism based on interaction between committees and different
departments of Government of India has been set up. Such materials will have
to meet with the approval of the following Committees: Institutional Biosafety
Committee (IBSC), Review Committee on Genetic Manipulation (RCGM)
and Genetic Engineering Approval Committee (GEAC). Under the EPA 1986,
the GEAC examines from the viewpoint of environmental safety and issues
clearance or the release of genetically engineered organisms/transgenic crops
and products into the environment. The GEAC also grants permits to conduct
experimental and large-scale field trials which are beyond the limit of 20 acres.
In case of transgenic crops, applicants are also required to seek clearance from
the Ministry of Agriculture.
Concepts and Scope of Plant Biotechnology 10.23

Responsible scientists, farmers, food manufacturers, and policy makers

recognize that the use of transgenic organisms should be considered very
carefully to ensure that they pose no environmental and health risks or at least
no more than the use of current crops and practices. Modern biotechnology
represents unique applications of science that can be used for the betterment
of society through development of crops with improved nutritional quality,
resistance to pests and diseases, and reduced cost of production. Biotechnology,
in the form of genetic engineering, is a facet of science that has the potential
to provide important benefits if used carefully and ethically. Society should
be provided with a balanced view of the fundamentals of biotechnology and
genetic engineering, the processes used in developing transgenic organisms,
the types of genetic material used, and the benefits and risks of the new
technology.
 CHAPTER

11 Animal Biotechnology

An important aspect of any biotechnological processes is the culture of animal

cells in artificial media. These animal cells in culture are used in recombinant
DNA technology, genetic manipulations and in a variety of industrial
processes. Now-a-days it has become possible to use the cell and tissue
culture in the areas of research which have a potential for economic value
and commercialization. The animal cell cultures are being extensively used in
production of vaccines, monoclonal antibodies, pharmaceutical drugs, cancer
research, genetic manipulations etc.
Animal cells, e.g. egg cells, are used for multiplication of superior
livestock using a variety of techniques like cloning of superior embryonic
cells, transformation of cultured cells, leading to the production of transgenic
animals. The animal cells are also used, in vitro fertilization and transfer of
embryos to surrogate mothers. Hence, the establishment and maintenance
of a proper animal culture is the first step towards using them as tools for
biotechnology.

11.1  HISTORY OF ANIMAL CELL CULTURE

• It was Jolly, who (1903) showed for the first time that the cells can
survive and divide in vitro. Ross Harrison (1907) was able to show the
development of nerve fibers from frog embryo tissue, cultured in a
blood clot. Later, Alexis Carriel (1912) used tissue and embryo extracts
as cultural media to keep the fragments of chick embryo heart alive.
• In the late 1940s, Enders, Weller and Robbins grew Poliomyelitis virus
in culture which paved way for testing many chemicals and antibiotics
that affect multiplication of virus in living host cells. The significance of
animal cell culture was increased when viruses were used to produce
vaccines on animal cell cultures in late 1940s.
• For about 50 years, mainly tissue explants rather than cells, were used
for culture techniques; although, later, after 1950s, mainly dispersed
11.2 Environmental Biotechnology

cells in culture were utilized. In 1966, Alec Issacs discovered Interferon

by infecting cells in tissue culture with viruses. He took filtrates from
virus-infected cells and grew fresh cells in the filtered medium. When
the virus was reintroduced in the medium, the cells did not get infected.
He proposed that cells infected with the virus secreted a molecule
which coated onto uninfected cells and interfered with the viral entry.
This molecule was called “Interferon”.
• Chinese Hamster Ovary (CHO) cell lines were developed during
1980s. Recombinant erythropoietin was produced on CHO cell lines
by AMGEN (U.S.A.). It is used to prevent anaemia in patients with
kidney failure who require dialysis. After this discovery, the Food and
Drug Administration (U.S.A) granted the approval for manufacturing
erythropoietin on CHO cell lines. In 1982, Thilly and co-workers used
the conventional conditions of medium, serum, and O2 with suitable
beads as carriers and grew certain mammalian cell lines to densities as
high as 5x106 cells/ml.
• A lot of progress has been also made in the area of stem cell technology
which will have their use in the possible replacement of damaged and
dead cells. In 1996, Wilmut and co-workers successfully produced a
transgenic sheep named Dolly through nuclear transfer technique.
Thereafter, many such animals (like sheep, goat, pigs, fishes, birds, etc.)
were produced.
• For animals, if the explant maintains its structure and function in
culture, it is called as an ‘organotypic culture’. If the cells in culture
re-associate to create a three dimensional structure irrespective of the
tissue from which it was derived, it is described as a ‘histotypic culture’.

11.2  ANIMAL CELL CULTURE

Salient Features of Animal cell culture
• Animal cells can grow in simple glass or plastic containers in nutritive
media but they grow only to limited generations.
• Animal cells exhibit contact inhibition. In culture the cancer cells
apparently differ from the normal cells. Due to uncontrolled growth
and more rounded shape, they loose contact inhibition and pile over
each other.
• There is a difference in the in vitro and in vivo growth pattern of cells.
For example,
— there is an absence of cell-cell interaction and cell-matrix interaction,
— there is a lack of three-dimensional architectural appearance, and
— there is changed hormonal and nutritional environment. The way
of adherence to glass or plastic container in which they grow, cell
proliferation and shape of cell, results in alterations.
Animal Biotechnology 11.3

— The maintenance of growth of cells under laboratory conditions in

suitable culture medium is known as primary cell culture.
— Cells are dissociated from tissues by mechanical means and by
enzymatic digestion using proteolytic enzymes.
— Cells can grow as adherent cells (anchorage-dependent) or as
suspension cultures (anchorage-independent).
— The primary culture is subcultured in fresh media to establish
secondary cultures.
— The various types of cell lines are categorized into two types as
Finite cell line and Continuous cell line. Finite cell lines are those
cell lines which have a limited life span and grow through a limited
number of cell generations. The cells normally divide 20 to 100
times (i.e., is 20–100 population doublings) before extinction. Cell
lines transformed under in vitro conditions, give rise to continuous
cell lines. The continuous cell lines are transformed, immortal and
tumorigenic.
— The physical environment includes the optimum pH, temperature,
osmolality and gaseous environment, supporting surface and
protects the cells from chemical, physical, and mechanical stresses.
— Nutrient media is the mixture of inorganic salts and other nutrients
capable of sustaining cell survival in vitro.
— Serum is essential for animal cell culture and contains growth factors
which promote cell proliferation. It is obtained as exuded liquid
from blood undergoing coagulation and filtered using Millipore
filters.
— Cryopreservation is storing of cells at very low temperature
(–180° C to –196° C), using liquid nitrogen. DMSO is a cryopreser-
vative molecule which prevents damage to cells.
— In order to maintain the aseptic conditions in a cell culture, a LAF
hood is used. Based on the nature of cells and organism, the tissue
culture hoods are grouped into three types: Class I, Class II, and
Class III.
— CO2 incubators are used and designed to mimic the environmental
conditions of the living cells.
— An inverted microscope is used for visualizing cell cultures in situ.
— For most animal cell cultures, low-speed centrifuges are needed.
— Neuronal cells constitute the nervous system. In culture, the
neuronal cells cannot divide and grow.
— The cells that form connective tissue (skin) is called fibroblast. The
fibroblast can divide and grow in culture upto some generations
after which they die. All normal animal cells are mortal.
11.4 Environmental Biotechnology

— Organ culture: The culture of native tissue that retains most of the in
vivo histological features is regarded as organ culture.
— Histotypic culture: The culturing of the cells for their re-aggregation
to form a tissue-like structure represents histotypic culture.
— Organotypic culture: This culture technique involves the
recombination of different cell types to form a more-defined tissue
or an organ.
There are certain terms that are associated with the cell lines.
These are as follows:
(i) Split ratio: The divisor of the dilution ratio of a cell culture at
subculture.
(ii) Passage number: It is the number of times that the culture has been
cultured.
(iii) Generation number: It refers to the number of doublings that a cell
population has undergone.
In fact, these parameters help us to distinguish the cancer cells in culture
from the normal cells because the cancer cells in culture, change shape (more
rounded), loose contact inhibition, pile on each other due to overgrowth and
have uncontrolled growth.

11.3  REQUIREMENTS FOR ANIMAL CELL CULTURE

Among the essential requirements for animal cell culture are, special incubators
to maintain the levels of oxygen, carbon dioxide, temperature, humidity as
present in the animal’s body, the synthetic media with vitamins, amino acids,
and fetal calf serum. Following parameters are essential for successful animal
cell culture:
(a) Temperature: In most of the mammalian cell cultures, the temperature
is maintained at 37°C in the incubators, as the body temperature of
Homo sapiens is 37°C.
(b) Culture media: The culture media is prepared in such a way that it
provides—
(1) The optimum conditions of factors like pH, osmotic pressure, etc.
(2) It should contain chemical constituents which the cells or tissues
are incapable of synthesizing. Generally, the media is the mixture
of inorganic salts and other nutrients capable of sustaining cells in
culture such as amino acids, fatty acids, sugars, ions, trace elements,
vitamins, cofactors, and ions. Glucose is added as energy source—
it’s concentration varying, depending on the requirement. Phenol
Red is added as a pH indicator of the medium. There are two types
of media used for culture of animal cells and tissues—the natural
media and the synthesized media.
(3) Natural Media: The natural media are the natural sources of
nutrient sufficient for growth and proliferation of animal cells and
Animal Biotechnology 11.5

tissues. The Natural Media used to promote cell growth fall in three
categories.
I. Coagulant, such as plasma clots. It is now commercially available in the
form of liquid plasma kept in silicon ampoules or lyophilized plasma.
Plasma can also be prepared in the laboratory by taking out blood from
male fowl and adding heparin to prevent blood coagulation.
II. Biological fluids such as serum. Serum is one of the very important
components of animal cell culture which is the source of various amino
acids, hormones, lipids, vitamins, polyamines, and salts containing
ions such as calcium, ferrous, ferric, potassium, etc. It also contains
the growth factors which promotes cell proliferation, cell attachment
and adhesion factors. Serum is obtained from human adult blood,
placental-cord blood, horse blood, calf blood. The other forms of
biological fluids used are coconut water, amniotic fluid, pleural fluid,
insect haemolymph serum, culture filtrate, aqueous humour, from
eyes, etc.
III. Tissue extracts, for example, Embryo extracts: Extracts from tissues
such as embryo, liver, spleen, leukocytes, tumour, bone marrow, etc.,
are also used for culture of animal cells.

11.3.1  Synthetic Media

Syntheic media are prepared artificially by adding several organic and
inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors,
etc. Different types of synthetic media can be prepared for a variety of cells and
tissues to be cultured. Synthetic media are of two types—Serum containing
media (media containing serum) and serum-free media (media with out
serum). Examples of some media are: minimal essential medium (MEM),
RPMI 1640 medium, CMRL 1066, F12, etc.
Advantages of serum in culture medium are:
(i) serum binds and neutralizes toxins,
(ii) serum contains a complete set of essential growth factors, hormones,
attachment and spreading factors, binding and transport proteins,
(iii) it contains the protease inhibitors,
(iv) it increases the buffering capacity,
(v) it provides trace elements.
Disadvantages of serum in culture medium are:
(i) it is not chemically defined and therefore it’s composition varies a lot,
(ii) it is sometimes source of contamination by viruses, mycoplasma,
prions, etc.
(iii) it increases the difficulties and cost of downstream processing,
(iv) it is the most expensive component of the culture medium.
11.6 Environmental Biotechnology

(4) pH—Most media maintain the pH between 7 and 7.4. A pH below 6.8
inhibits cell growth. The optimum pH is essential to maintain the proper ion
balance, optimal functioning of cellular enzymes and binding of hormones and
growth factors to cell surface receptors in the cell cultures. The regulation of
pH is done using a variety of buffering systems. Most media use a bicarbonate-
CO2 system as its major component.
(5) Osmolality: A change in osmolality can affect cell growth and function.
Salt, glucose and amino acids in the growth media determine the osmolality
of the medium. All commercial media are formulated in such a way that their
final osmolality is around 300 mOsm.

11.4  CELL-BASED THERAPY

The animal cell culture techniques are used in replacing the damaged and
dead cells with normal and healthy cells using the stem cell technology.
This therapy is called Cell-Based therapy which involves the use of stem
cell technology involving the replacement of damaged and dead cells with
normal and healthy cells. This is used to treat blood cancer, and other neuro-
degenerative diseases, etc.

11.5  APPLICATIONS OF ANIMAL CELL CULTURE

The animal cell cultures are used for a diverse range of research and
development. These areas are:
(a) Production of antiviral vaccines, which requires the standardization of
cell lines for the multiplication and assay of viruses.
(b) Cancer research, which requires the study of uncontrolled cell division
in cultures.
(c) Cell fusion techniques.
(d) Genetic manipulation, which is easy to carry out in cells or organ
cultures.
(e) Production of monoclonal antibodies requires cell lines in culture.
(f) Production of pharmaceutical drugs, using cell lines.
(g) Chromosome analysis of cells derived from womb.
(h) Study of the effects of toxins and pollutants using cell lines.
(i) Use of artificial skin.
(ii) Study the functions of the nerve cells.

11.5.1  Somatic Cell Fusion

One of the applications of animal cell culture is the production of hybrid
cells by the fusion of different cell types. These hybrid cells are used for the
following purposes:
(i) study of the control of gene expression and differentiation,
Animal Biotechnology 11.7

(ii) study of the problem of ‘malignancy’,

(iii) viral application,
(iv) gene-mapping,
(v) production of hybridomas for antibody production.
In 1960s, in France, for the first time, hybrid cells were successfully
produced from mixed cultures of two different cell lines of mouse. Cells
growing in culture are induced by some of the viruses such as ‘Sendai virus’
to fuse and form hybrids. This virus induces two different cells first, to form
heterokaryons. During mitosis, chromosomes of heterokaryon move towards
the two poles, and later on, fuse to form hybrids. It is important to remove the
surface carbohydrates to bring about cell fusion. Some other chemicals like
polyethylene glycol also induce somatic cell fusion.
Many commercial proteins have been produced by animal cell culture and
their medical application is being evaluated.
Tissue Plasminogen activator (t-PA) was the first drug that was produced
by the mammalian cell culture by using rDNA technology. The recombinant
t-PA is safe and effective for dissolving blood clots in patients with heart
diseases and thrombotic disorders.

Production of T-PA

11.5.2  Blood Factor VIII

Haemophilia A is a blood disorder which is a sex-linked genetic disease in
humans. The patients suffering from Haemophilia A lack factor VIII, which
plays an important role in the clotting of blood. This factor VIII is secreted by
a gene present on X-chromosome but this gene undergoes mutations in people
suffering from Haemophilia. Current therapy for this disease is the transfusion
of blood factor VIII into patients. Using rDNA technology, factor VIII has been
produced from mammalian cell culture, e.g., Hamster kidney cell.
11.8 Environmental Biotechnology

11.5.3  Erythropoietin (EPO)

The EPO is a glycoprotein consisting of 165 amino acids and is formed in the
foetal liver and kidneys of the adults. It causes proliferation and differentiation
of progenitor cells into erythrocytes (erythroblasts) in the bone marrow.
Erythropoietin is hormone-like in nature and is released by the kidney under,
hypoxic or anoxic conditions caused by anaemia.
Amgen Inc. holds US patent for preparation of, eErythropoietin, by
recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin
(EPO) is a hormone-like substance released by the kidney under hypoxic or
anoxic conditions caused by anaemia. r-HUEPO- recombinant human erythro-
poitein has been effectively used to treat anemia associated with AIDS, renal
failure etc.

11.5.4 The production of Monoclonal Antibodies using Hybridoma

Technology
Antibodies are proteins synthesized in blood against antigens and are collected
from the blood serum. The antibodies, which are heterogenous and non-
specific in action are called polyclonal antibodies. If a specific lymphocyte,
after isolation and culture in vitro becomes capable of producing a single
type of antibody bearing specificity against specific antigen, it is known as
monoclonal antibody. The monoclonal antibodies are used in the diagnosis
of diseases because of the presence of desired immunity. However, these
antibody-secreting cells cannot be maintained in culture. It was observed that
the myeloma cells (bone marrow tumour cells, due to cancer) grow indefinitely
and also produce immunoglobulins which are, in fact, monoclonal antibodies.
In 1974, George Kohler and Milstein isolated clones of cells from the fusion
of two parental cell lines – lymphocytes from spleen of mice immunized with
red blood cells from sheep and myeloma cells. These cells were maintained
in vitro and produced antibodies. The hybrid cells maintained the character
of lymphocytes to secrete the antibodies, and of myeloma cells to multiply
in culture. These hybrid cell lines are called “Hybridoma” and are capable
of producing unlimited supply of antibodies. Hybridoma are obtained by
using an antibody, producing lymphocytes cell and a single myeloma cell.
Monoclonal antibodies bind very specifically to an epitope (specific domains)
on an antigen and, by using them, it is possible to detect the presence of
specific antigens.
The monoclonal antibodies are used for the treatment of patients with
malignant leukaemia cells, B cell lymphomas and allograft rejection after
transplantation. CD3 is an antigen present on the surface of mature T-cells
lymphocytes. If T-cell population is depleted or controlled, the transplanted
organ will not be rejected. An antibody that acts against CD3 surface antigen
of T-cells is called OKT3, i.e., anti-CD3 Moab. OKT3 is a monoclonal antibody
which has been licensed for clinical use for the treatment of acute renal allograft
rejection. OKT3 removes antigen-bearing cells from circulation, thereby, helps
in accepting the graft.
Animal Biotechnology 11.9

Steps Involved in the production of monoclonal antibodies

When monoclonal antibodies are used as enzymes using the technique of
enzyme engineering, then they are called abzymes.
Using animal cell cultures, it is also possible to produce Polyclonal
Antibodies. Polyclonal antisera are derived from many cells; therefore,
contains heterogeneous antibodies that are specific for several epitopes or an
antigen.

11.6 SCALE-UP OF ANIMAL CELL CULTURE

Modifying a laboratory procedure, so that it can be used on an industrial
scale is called scaling up. Laboratory procedures are normally scaled up via
intermediate models of increasing size. The larger the plant, the greater the
running costs, as skilled people are required to monitor and maintain the
machinery. The first pre-requisite for any large scale cell culture system and
its scaling up is the establishment of a cell bank. Master cell banks (MCB)
are first established and they are used to develop Master Working Cell Banks
(MWCB). The MWCB should be sufficient to feed the production system at
a particular scale for the predicted life of the product. The cell stability is an
important criteria, so MWCB needs to be repeatedly sub-cultured and each
generation should be checked for changes. A close attention should be paid to
the volume of cultured cells as the volume should be large enough to produce
a product in amounts which is economically viable. The volume is maintained
by:
• increasing the culture volume,
11.10 Environmental Biotechnology

• by increasing the concentration of cells in a reactor by continuous

perfusion of fresh medium, so that the cells keep on increasing in
number without the dilution of the medium.
A fully automated bioreactor maintains the physicochemical and biological
factors to optimum level and maintains the cells in suspension medium. The
most suitable bioreactor used is a compact-loop bioreactor consisting of
marine impellers. The animal cells unlike bacterial cells, grow very slowly.
The main carbon and energy sources are glucose and glutamine. Lactate and
ammonia are their metabolic products that affect growth and productivity of
cells. So, the on-line monitoring of glucose, glutamate, and ammonia is carried
out by on line flow injection analysis (FIA) using gas chromatography (GC),
high performance liquid chromatography (HPLC), etc.
In batch cultures, mainly Roller Bottles with Micro Carrier Beads (for
adherent cells) and spinner flasks (for suspension cultures) are used in Scale-
up of animal cell culture process.

11.6.1  Roller Bottles

The Roller bottles provide total curved surface area of the micro carrier beads
for growth. The continuous rotation of the bottles in the CO2 incubators helps
to provide medium to the entire cell monolayer in culture. The roller bottles
are well attached inside a specialized CO2 incubators. The attachments rotate
the bottles along the long axis which helps to expose the entire cell monolayer
to the medium during the one full rotation. This system has the following
advantages over the static monolayer culture:
(a) it provides increase in the surface area,
(b) provides constant gentle agitation of the medium,
(c) provides increased ratio of surface area of medium to its volume, which
allows gas exchange at an increased rate through the thin film of the
medium over the cells. Typically, a surface area of 750-1500 cm2 with
200-500 ml medium will yield 1–2 × 108 cells.

The roller bottle cell culture

Animal Biotechnology 11.11

11.6.2  Micro Carrier Beads

Micro carrier beads are small spherical particles with diameter 90-300
micrometers, made up of dextran or glass. Micro Carrier beads, increase the
number of adherent cells per flask. These dextran or glass-based beads come
in a range of densities and sizes. The cells grow at a very high density which
rapidly exhausts the medium, and therefore, the medium has to be replaced
for the optimum cell growth. At the recommended concentration when the
microcarriers are suspended, they provide 0.24 m2 area for every 100 ml of
culture flask.

11.6.3  Spinner cultures

The spinner flask, was originally developed to provide the gentle stirring of
micro-carriers, but are now used for scaling up the production of suspension
cells. The flat surface glass flask is fitted with a Teflon paddle that continuously
turns and agitates the medium. This stirring of the medium improves gas
exchange in the cells in culture. The spinner flask used at commercial scale
consists of one or more side arms for taking out samples and decantation, as
well.

11.7  TYPES OF CELL CULTURES

Primary cell culture: The maintenance of growth of cells dissociated from
the parental tissue (such as kidney, liver) using the mechanical or enzymatic
methods, in culture medium using suitable glass or plastic containers, is called
Primary Cell Culture.
The primary cell culture could be of two types depending upon the kind
of cells in culture.
(a) Anchorage Dependent/Adherent cells: Cells shown to require
attachment for growth are set to be Anchorage Dependent cells. The
Adherent cells are usually derived from tissues of organs such as
kidney where they are immobile and embedded in connective tissue.
They grow adhering to the cell culture.
(b) Suspension Culture/Anchorage Independent cells: Cells which do
not require attachment for growth or, do not attach to the surface of the
culture vessels, are anchorage independent cells/suspension cells. All
suspension cultures are derived from cells of the blood system because
these cells are also suspended in plasma in vitro e.g. lymphocytes.
Secondary cell cultures :When a primary culture is sub-cultured, it
becomes known as secondary culture or cell line. Subculture (or passage)
refers to the transfer of cells from one culture vessel to another culture vessel.
Subculturing—Subculturing or splitting cells is required to periodically
provide fresh nutrients and growing space for continuously growing cell
lines. The process involves removing the growth media, washing the plate,
disassociating the adhered cells, usually enzymatically. Such cultures may be
called secondary cultures.
11.12 Environmental Biotechnology

Cell Line: A Cell Line or Cell Strain may be finite or continuous depending
upon whether it has limited culture life span or it is immortal in culture. On
the basis of the life span of culture, the cell lines are categorized into two types:
(a) Finite Cell Lines: The cell lines which have a limited life span and go
through a limited number of cell generations (usually, 20–80 population
doublings) are known as finite cell lines. These cell lines exhibit the
property of contact inhibition, density limitation and anchorage
dependence. The growth rate is slow and doubling time is around
24–96 hours.
(b) Continuous Cell Lines: Cell lines transformed under laboratory
conditions or, in vitro culture conditions, give rise to continuous
cell lines. The cell lines show the property of ploidy (aneuplacdy or
heteroploidy), absence of contact inhibition and anchorage dependence.
They grow in monolayer or suspension form. The growth rate is rapid
and doubling time is 12–24 hours.
(c) Monolayer cultures: When the bottom of the culture vessel is covered
with a continuous layer of cells, usually one cell in thickness, they are
referred to as monolayer cultures.
(d) Suspension cultures: Majority of continuous cell lines grow as
monolayers. Some of the cells which are non-adhesive, e.g. cells of
leukemia or, certain cells which can be mechanically kept in suspension,
can be propagated in suspension. There are certain advantages in
propagation of cells by suspension culture method.
These advantages are:
(a) The process of propagation is much faster,
(b) The frequent replacement of the medium is not required,
(c) Suspension cultures have a short-lag period,
(d) treatment with trypsin is not required,
(e) a homogenous suspension of cells is obtained,
(f) the maintenance of suspension cultures is easy and bulk production of
the cells is easily achieved,
(g) scale-up is also very convenient.
The cell lines are known by:
(a) A code, e.g., NHB for Normal Human Brain.
(b) A cell line number—This is applicable when several cell lines are
derived from the same cell culture source, e.g. NHB1, NHB2.
(c) Number of population doublings the cell line has already undergone
e.g., NHB2/2 means two doublings.
Animal Biotechnology 11.13

Salient features of cell culture with volution of a cell line

11.8  CHARACTERIZATION OF CELL LINES

The cell lines are characterized by their—(a) growth rate and (b) karyotyping.
(a) Growth Rate: A growth curve of a particular cell line is established
taking into consideration the population doubling time, a lag time, and a
saturation density of a particular cell line. A growth curve consist of:
(1) Lag Phase: The time the cell population takes to recover from such sub
culture, attach to the culture vessel and spread.
(2) Log Phase: In this phase, the cell number begins to increase exponentially.
(3) Plateau Phase: During this phase, the growth rate slows or stops due to
exhaustion of growth medium or confluency.
(b) Karyotyping: Karyotyping is important as it determines the species of
origin and determine the extent of gross chromosomal changes in the line. The
cell lines with abnormal karyotype are also used if they continue to perform
normal function. Karyotype is affected by the growth conditions used, the
way in which the cells are subcultured, and whether or not the cells are frozen.
(c) There are certain terms that are associated with the cell lines.
11.14 Environmental Biotechnology

These are as follows:

(i) Split ratio: The divisor of the dilution ratio of a cell culture, at subculture.
(ii) Passage number: It is the number of times that the culture has been
cultured.
(iii) Generation number: It refers to the number of doublings that a cell
population has undergone.
Some Animal Cell Lines and the Products obtained from them
Cell line Product
Human tumor Angiogenic factor
Human leucocytes Interferon
Mouse fibroblasts Interferon
Human Kidney Urokinase
Transformed human kidney Single chain urokinase-type plasminogen
cell line, TCL-598 activator (scu-PA)
Human kidney cell (293) Human protein (HPC)
Dog kidney Canine distemper vaccine
Cow kidney Foot and Mouth Disease (FMD) vaccine
Chick embryo fluid Vaccines for influenza, measles and pumps
Duck embryo fluid Vaccines for rabies and rubella
Chinese Hamster Ovary 1. Tissue-type plasminogen activator (t-PA)
(CHO) cells 2. b-and gamma interferons
3. Factor VIII

11.9  STEM CELL TECHNOLOGY

Stem cells retain the capacity to self-renew as well as to produce progeny
with a restricted mitotic potential and restricted range of distinct types of
differentiated cells, they give rise to. The formation of blood cells, also called
haematopoiesis, is the classical example of concept of stem cells. Indirect
assay methods were developed to identify the haematopoietic stem cells. The
process of haematopoeis occurs in the spleen and bone marrow in mouse. In
human beings, about 100,000 haematopoietic stem cells produce one billion
RBCs, one billion platelets, one million T-cells, one million B-cells per kg body
weight per day.
Several methods have been developed to study haematopoiesis and stem
cells:
(a) Repopulation assay: Edmens Snell’s group created mice which were
genetically identical by mating of sibling mice after 21 generations.
Two groups of mice were lethally X- irradiated to destroy their blood
cell-forming capacity. One of this group was injected with marrow
Animal Biotechnology 11.15

cells from the femur bone of a normal and healthy albino mice. It was
observed that this group survived, whereas the mice in the other group
died. The spleen of mice which survived had the colonies of the bone
marrow cells just like bacterial colonies on a petri plate. This came to be
known as colony-forming units of spleen (CFU-S) and the technique is
known as repopulation assay.
(b) The in vitro clonal assay: In this assay, the stem cells proliferate to form
colonies of differentiated cells on semi-solid media. This assay helps in
identifying growth factors required for the formation of blood cells from
the primitive stem cells. One of the first commercialized biotechnology
product – erythropoietin, was assayed by this procedure.
(c) Long-term marrow culture: In this method, the marrow cells from
femur bone were grown under in vitro conditions on plastic surfaces.
These techniques were helpful in bone marrow transplantation and
treatment of blood cancer by releasing immature blood cells into the
blood stream.
(d) Embryonic stem cell culture: Embryonic stem cells are cell lines derived
from the inner cell mass of fertilized mouse embryo without the use
of immortalizing or transforming agents. The Inner Cell Mass (ICM)
are the cells that are maintained in tissue culture in the presence of
irradiated fibroblast cells. These cells are often used in creating chimeric
mice. In 1998, J.A. Thomson developed the method to multiply the
human embryonic stem cells. Human ICM can also be now derived
either by IVF or from germ cell precursors and cultured on a petri plate.
The differentiation of these cells into lineage restricted (neuronal and
glial) cells can be accomplished by altering the media in which the cells
grow.

Scheme of obtaining chimeras

11.16 Environmental Biotechnology

(e) The ICM cells could be used to create chimeric mice. In chimeric mice,
it was possible to take ES cells from a black mouse and implant it into
the embryo of an albino mouse (white). The progeny, so developed,
had skin colour of black and white (a chimera).

11.9.1  Genetic Engineering of Animal Cells and their Applications

The mammalian cells are genetically modified by introducing the genes needed
for specific purposes such as production of specific proteins or to improve the
characteristics of a cell line. The methods used to introduce the foreign genes/
DNA into mammalian cells are: Electroporation, Lipofection, Microinjection
and/or fusion of mammalian cells with bacteria or viruses.
After the integration of the foreign DNA into the mammalian cells, the
transfected/transformed cells are selected by using suitable markers. Some
of such markers in use are: Viral thymidine kinase, Bacterial dihydrofolate
reductase, Bacterial neomycin phosphotransferase. It has been possible
to overproduce several proteins in mammalian cells through genetic
manipulations, e.g. tissue plasminogen activator, erythropoietin, interleukin-2,
interferon-beta, clotting factors VIII and IX, tumor necrosis factors. The
recombinant mammalian cells are also conveniently used for the production
of monoclonal antibodies.

11.9.2  Manipulation of Gene Expression in Eukaryotes

The eukaryotic organisms have the capability to bring about the post-
translational modifications such as glycosylation, phosphorylation, proteolytic
cleavage, etc., which ultimately help in the production of stable and biologically-
active proteins. Due to these reasons, the use of eukaryotic expression system
is preferred, however, it is difficult to conduct experiments with eukaryotic
cells. The introduction of a foreign DNA into animal cells is called transfection.
The insert, DNA in the eukaryotic cells may be associated with vector or
integrated into the host chromosomal DNA. Among the various hosts used
for the expression of cloned genes, the common yeast Saccharomyces cerevisiae
is the most extensively used. Besides this, the cultured insect cells are in use
for expressing cloned DNAs. Baculoviruses exclusively infect insect cells. The
DNA of these viruses encode for several products and their productivity in cells
is very high to the extent of more than 10,000 times compared to mammalian
cells. The Baculoviruses not only carry a large number of foreign genes but
can also express and process the products formed. By using baculovirus as an
expression vector system, a good number of mammalian and viral proteins
have been synthesized. The most commonly used baculovirus is Autographa
californica multiple nuclear polyhedrosis virus (AcMNPV). It grows on the insect
cell lines and produces high levels of polyhedrin or a recombinant protein.
The mammalian cell expression vectors are used for the production of
specific recombinant proteins and to study the function and regulation of
Animal Biotechnology 11.17

mammalian genes. However, large-scale production of recombinant proteins

with engineered mammalian cells is costly. The mammalian vector contains a
eukaryotic origin of replication from an animal virus such as Simian virus 40 (SV
40) and a prokaryotic origin of replication. It has a multiple cloning site and a
selectable marker gene, both of which remain under the control of eukaryotic
promoter and polyadenylation sequences. These sequences are obtained
from either animal viruses (SV40, herpes simplex virus) or mammalian genes
(growth hormone, metallothionein). The promoter sequences facilitate the
transcription of cloned genes (at the multiple cloning site) and the selectable
marker genes. On the other hand, the polyadenylation sequences terminate
the transcription.

11.9.3  Collection and purification process of Recombinant proteins

As the recombinant proteins start accumulating in the host cells, it becomes
important to collect and purify them. This is a tricky process since and many
times, the recombinant protein is a foreign body for the host cells the enzyme
machinery of the host cell becomes activated to degrade the outside protein.
One of the strategies adopted is the use of bacterial strains, deficient in
proteases or, alternatively, the recombinant proteins are fused with the native-
host proteins. The fusion proteins are resistant to protease activity. Sometimes,
the foreign proteins accumulate as aggregates in the host organism which
minimizes the protease degradation. The best way out is to quickly export
and secrete out the recombinant proteins into the surrounding medium. The
recovery and the purification of foreign proteins is easier from the exported
proteins. The efforts have been made to develop methods to increase the
export of recombinant proteins.
Some of the species of the bacterium, Bacillus subtilis, normally secrete
large quantities of extracellular proteins. A short DNA sequence, called signal
sequence from such species, is introduced into other B. subtilis. These bacteria
produce recombinant DNA tagged with signal peptide, which promotes export
and secretion. This signal peptide is removed after the purification of foreign
protein. The techniques used for the purification of recombinant proteins
from the mixture of secreted proteins are affinity-tagging, immunoaffinity
purification, etc.

11.9.4  Organ culture and Histotypic cultures

The cell-cell interaction leads to a multistep events in in vivo situations. For
example, hormone stimulation of fibroblasts is responsible for the release
of surfactant by the lung alveolar cells. Androgen-binding to stomal cells
stimulates the prostrate epithelium. In other words, hormones, nutritional
factors and xenobiotics exert stimulating effects on the cells to function in a
coordinated manner. Xenobiotics broadly refers to the unnatural, foreign, and
synthetic chemicals such as pesticides, herbicides, refrigents, solvents and
11.18 Environmental Biotechnology

other organic compounds. It is impossible to study these cellular interactions

that occur in the in vivo system with isolated cells or, cells in culture. This has
lead to the attempts to develop organ and histotypic culture with the aim of
creating in vitro models comparable to the in vivo system. The three types of
such cultures are:
(a) Organ culture: In this type of culture, the whole organs or small
fragments of the organs, with their special and intrinsic properties
intact, are used in culture.
(b) Histotypic culture: The cell lines grown in three dimensional matrix to
high density represent histotypic cultures.
(c) Organotypic cultures: A component of an organ is created by using
cells from different lineages in proper ratio and spatial relationship
under laboratory conditions.

11.9.5  Organ culture

In the organ culture, the cells are integrated as a single unit which helps to
retain the cell to cell interactions found in the native tissues or organs. Due
to the preservation of structural integrity of the original tissue, the associated
cells continue to exchange signals through cell adhesion or communications.
Due to the lack of a vascular system in the organ culture, the nutrient supply
and gas exchange of the cells become limited. In order to overcome this
problem, the organ cultures are placed at the interface between the liquid and
gaseous phases. Sometimes, the cells are exposed to high O2 concentration
which may also lead to oxygen-induced toxicity. Due to the inadequate supply
of the nutrients and oxygen, some degree of necrosis at the central part of
the organ may occur. In general, the organ cultures do not grow except some
amount of proliferation that may occur on the outer cell layers.

11.9.6  Techniques and Procedure for organ culture

In order to optimize the nutrient and gas exchanges, the tissues are kept at
gas-limited interface using the support material which ranges from semi-solid
gel of agar, clotted plasma, micropore filter, lens paper, or strips of Perspex
or plexiglass. The organ cultures can also be grown on top of a stainless steel
grid. Another popular choice for growing organ cultures is the filter-well
inserts. Filter-well inserts with different materials like ceramic, collagen,
nitrocellulose, are now commercially available. Filter well inserts have been
successfully used to develop functionally-integrated thyroid epithelium,
stratified epidermis, intestinal epithelium, and renal epithelium.
The procedure for organ cultures has the following steps:
(a) The organ tissue is collected after the dissection.
(b) The size of the tissue is reduced to less than 1mm in thickness.
(c) The tissue is placed on a gas medium interface support.
Animal Biotechnology 11.19

(d) Incubation in a CO2 incubator.

(e) M199 or CMRL 1066 medium is used and changed frequently.
(f) The techniques of histology, autoradiography, and immunochemistry
are used to study the organ cultures.

11.9.7  The advantages of organ culture

The organ cultures can be used to study the behavior of an integrated tissue in
the laboratory. It provides an opportunity to understand the biochemical and
molecular functions of an organ/tissue.

11.9.8  Limitations of organ culture

It is a difficult and expensive technique. The variations are high with low
reproducibility. For each experiment, a new or fresh organ is needed, as organ
cultures are not propagated.

11.9.9  Histotypic cultures

Using histotypic culture, it is possible to use dispersed monolayers to
regenerate tissuelike structures. It is the growth and propagation of cell lines
in three-dimensional matrix to high cell density that contributes to this. The
techniques used in histotypic cultures are:
(a) Gel and sponge technique: In this method, the gel (collagen) or sponges
(gelatin) are used which provides the matrix for the morphogenesis
and cell growth. The cells penetrate these gels and sponges, while
growing.
(b) Hollow fibers technique: In this method, hollow fibers are used
which helps in more efficient nutrient and gas exchange. In recent
years, perfusion chambers with a bed of plastic capillary fibers have
been developed to be used for histotypic type of cultures. The cells get
attached to capillary fibers and increase in cell density to form tissue
like structures.
(c) Spheroids: The re-association of dissociated cultured cells leads to
the formation of cluster of cells called spheroids. It is similar to the
reassembling of embryonic cells into specialized structures. The
principle followed in spheroid cultures is that the cells in heterotypic
or homotypic aggregates have the ability to sort themselves out and
form groups which form tissue-like architecture. However, there is a
limitation of diffusion of nutrients and gases in these cultures.
(d) Multicellular tumour spheroids: These are used as an in vitro
proliferating models for studies on tumor cells. The multicellular
tumor spheroids have a three-dimensional structure which helps in
performing experimental studies related to drug therapy, penetration
of drugs besides using them for studying regulation of cell proliferation,
11.20 Environmental Biotechnology

immune response, cell death, and invasion and gene therapy. A size
bigger than 500 mm leads to the development of necrosis at the centre
of the MCTS. The monolayer of cells or aggregated tumour is treated
with trypsin to obtain a single cell suspension. The cell suspension is
inoculated into the medium in magnetic stirrer flasks or roller tubes.
After 3–5 days, aggregates of cells representing spheroids are formed.
Spheroid growth is quantified by measuring their diameters regularly.
The spheroids are used for many purposes. They are used as models
for a vascular tumour growth. They are used to study gene expression
in a three-dimensional configuration of cells. They are also used to
study the effect of cytotoxic drugs, antibodies, radionucleotides, and
the spread of certain diseases like, rheumatoid arthritis.

11.9.10  Organotypic cultures

These cultures are used to develop certain tissues or tissue models, for
example, skin equivalents have been created by culturing dermis, epidermis
and intervening layer of collagen, simultaneously. Similarly, models have
been developed for prostrate, breast, etc. Organotypic culture involves the
combination of cells in a specific ratio to create a component of an organ.

11.10  CELL AND TISSUE ENGINEERING

Tissue engineering refers to the application of the principles of engineering
to cell culture for the construction of functional anatomical units—tissues/
organs. The aim of tissue engineering is nothing but to supply the various
body parts for the repair or replacement of damaged tissues or organs. It is
now possible to grow skin cells, blood cells, cardiac cells, etc. by using the
ability of stem cells to proliferate and differentiate.
During the last decade, the tissue culture work in animals demonstrated
that virtually any human tissue or organ can be grown in culture. This became
possible only after it became known that the ability of cultured cells to undergo
differentiation can be restored. ‘Skin’ was the first organ to be cultured in
artificial media and could be successfully used for transplantation following
serious skin burns. For past few years, some of the biotech companies like, ATS
(Advanced Tissue Science, USA), Biosurface Technology (BTI, Cambridge)
and Organogenesis, are developing artificial skins to the stage of clinical trials.
In the field of tissue replacement, focus of attention is the Artificial cartilage.
As it is not vascularized, it is not rejected due to immunogenic response. This
will have lots of implications in the treatment of sports related injuries and
diseases like, arthritis.

11.10.1  Design and Engineering of Tissues

The design and tissue engineering should essentially cause minimal discomfort
to the patient. The damaged tissues should be easily fixed with the desired
Animal Biotechnology 11.21

functions, quickly restored. Another important factor controlling the designing

of tissue culture is the source of donor cells. The cells from the patient himself, is
always preferred as it considerably reduces the immunological complications.
However, under certain situations, allogeneic cells (cells taken from a person
other than the patient), are also used. The other important factors are – the
support material, it’s degradation products, cell adhesion characteristics, etc.
It was demonstrated in 1975 that human keratinocytes could be grown in the
laboratory in a form suitable for grafting. A continuous sheet of epithelial cells
can be grown now; however, there is still difficult to grow TE skin with the
dermal layer with all the blood capillaries, nerves, sweat glands, and other
accessory organs.
Some of the implantable skin substitutes which are tissue engineering
skin constructs with a limited shelf life of about 5 days are:
(a) Integra TM—A bioartificial material composed of collagen-
glycosaminoglycan and is mainly used to carry the seeded cells.
(b) Dermagraft TM—This is composed of polyglycolic acid polymer-mesh
seeded with human dermal fibroblasts from neonatal foreskins.
(c) Apligraf TM—It is constructed by seeding human dermal fibroblasts
into collagen gel with the placement of a layer of human keratinocytes
on the upper surface.
These tissue-constructs integrate into the surrounding normal tissue and
form a good skin cover with minimum immunological complications.
The urothelial cells and smooth muscle cells from bladder are now being
cultured and attempts are on to construct TE urothelium. Some progress
has also been made in the repair of injured peripheral nerves using tissue-
engineered peripheral nerve implants. The regeneration of the injured nerve
occurs from the proximal stump to rejoin at distal stump.
The regeneration process requires substances like-
(a) Conduct material: The conduct material is composed of collagen-
glycosaminoglycans, PLGA (poly lactic-co-glycolicacid), hyaluronan
and fibronectin and forms the outer layer.
(b) Filling material: The filling material contains collagen, fibrin,
fibronectin and agarose. This supports the neural cells for regeneration.
And,
(c) Additives: A large number of other factors are also added, e.g. growth
factors, neurotrophic factors such as fibroblast growth factor (FGF),
nerve growth factor (NGF).
  The other important applications of tissue engineering are in gene therapy,
pseudo-organs and as model cell systems for developing new therapeutic
approaches to human diseases. The attempts are on to create tissue models
in the form of artificial organs using tissue engineering. The artificial liver is
being created using hepatocytes cultured as spheroids and held suspended in
11.22 Environmental Biotechnology

artificial support system such as porous gelatin sponges, agarose or collagen.

Some progress has been made in the area of creating the artificial pancreas
using spheroids of insulin-secreting cells which have been developed from
mouse insulinoma beta cells. Three-dimensional brain cell cultures have
been used for the study of neural myelination, neuronal regeneration, and
neurotoxicity of lead. The aggregated brain cells are also being used to study
Alzheimer’s disease and Parkinson’s disease. Thyroid cell spheroids are being
used to study cell adhesion, motility, and thyroid follicle biogenesis.
Table depicting the technological goals and areas of
research in tissue engineering
Growth of cells in three-dimensional systems
Delivery systems for protein therapeutics
Cell cultivation methods for culturing ‘recalcitrant cells’
Expression of transgenic proteins in transplantable cells
To develop vehicles for delivering transplantable cells
Development of markers for tracking transplanted cells
Avoiding immunogenicity in transplantable cells
Development of in vivo and ex vivo biosensors for monitoring cell behaviour
during tissue production

Downstream Processing: Downstream processing or down-streaming is

the extraction and purification of the desired end products of fermentation
processes. Such products might include cells, solvents or solutes. Various
processes are available for the separation of cells from the fermentation broth
in which they are grown, including flocculation, filtration, centrifugation,
sedimentation or flotation. The procedure adopted depends on whether it
is the cells, or the solution surrounding them, that contains the desired end-
products.
Bioethics in Animal Genetic Engineering: There are some serious issues
related to genetic modification of animals using animal genetic engineering
techniques. One is not sure of the consequences of these genetic modifications
and the further interaction with the environment. Proper clinical trials are also
necessary before one can use it for commercial purposes. In the recent past,
people have raised objections on some of the methods used, e.g., the transfer
of a human genes into food animals, use of organisms containing human genes
as animal feed. Some religious groups have expressed their concern about the
transfer of genes from animals whose flesh is forbidden for use as food into
the animals that they normally eat. Transfer of animal genes into food plants
may be objectionable to the vegetarians.
Besides this, there are several other aspects of this issue that have to be
sorted out.
Animal Biotechnology 11.23

(a) What will be the consequences, if a modified animal will breed with
other domestic or wild animals, thereby, transferring the introduced
genes to these populations?
(b) What are the health risks to human on consumption of genetically
modified animals and their products?
(c) With the production of disease-resistant animals, what will be the effect
on ecology?
(d) There is also widespread concern about the risks of human recipients
getting infected with animal viral diseases after a xenotransplantation.,
which might infect the population at large.
(e) There are also concerns about the risk that drug resistance gene
markers used in genetic engineering procedures might inadvertently
be transferred and expressed.
The need of the hour is to formulate clear guidelines which should be
followed while using genetic engineering techniques in bio-medical research
e.g., products from transgenic organisms should be clearly marked to give
choice to people who follow dietary restrictions due to religious beliefs. In
fact, all the ethical and moral issues raised by some aspects of biotechnology
should be addressed by open discussion and dialogue.
 CHAPTER

12 Biotechnology of Aquaculture

Aquaculture, also known as aquafarming, is the farming of aquatic organisms

such as fish, crustaceans, molluscs and aquatic plants. Aquaculture involves
cultivating freshwater and saltwater populations under controlled conditions,
and can be contrasted with commercial fishing, which is the harvesting of wild
fish. Mariculture refers to aquaculture practiced in marine environments.
• According to the Food and Agriculture Organization (FAO), aquaculture
“is understood to mean the farming of aquatic organisms including
fish, molluscs, crustaceans and aquatic plants. Farming implies some
form of intervention in the rearing process to enhance production, such
as regular stocking, feeding, protection from predators, etc. Farming
also implies individual or corporate ownership of the stock being
cultivated.
Biotechnology provides powerful tools for the sustainable development of
aquaculture, fisheries, as well as the food industry. Increased public demand
for seafood and decreasing natural marine habitats have encouraged scientists
to study ways that biotechnology can increase the production of marine food
products, and making aquaculture as a growing field of animal research.
• Biotechnology allows scientists to identify and combine traits in fish
and shellfish to increase productivity and improve quality.
• Scientists are investigating genes that will increase production of
natural fish growth factors as well as the natural defense compounds,
marine organisms use, to fight microbial infections.
• Modern biotechnology is already making important contributions and
poses significant challenges to aquaculture and fisheries development.
• It perceives that modern biotechnologies should be used as adjuncts
to, and not as substitutes for, conventional technologies in solving
problems, and that their application should be need-driven rather than
technology-driven.
12.2 Environmental Biotechnology

• The use of modern biotechnology to enhance production of aquatic

species holds great potential not only to meet demand but also to
improve aquaculture.
• Genetic modification and biotechnology also holds tremendous
potential to improve the quality and quantity of fish reared in
aquaculture.
• There is a growing demand for aquaculture; biotechnology can help
to meet this demand. As with all biotech-enhanced foods, aquaculture
will be strictly regulated before approved for market.
• Biotech aquaculture also offers environmental benefits. When
appropriately integrated with other technologies for the production
of food, agricultural products and services, biotechnology can be
of significant assistance in meeting the needs of an expanding and
increasingly urbanized population.
• Successful development and application of biotechnology are possible
only when a broad research and knowledge base in the biology,
variation, breeding, agronomy, physiology, pathology, biochemistry
and genetics of the manipulated organism exists.
• Benefits offered by the new technologies cannot be fulfilled without a
continued commitment to basic research.
• Biotechnological programmes must be fully integrated into a research
background and cannot be taken out of context if they are to succeed.
Biotechnology in fish breeding: Gonadotropin releasing hormone (GnRH)
is now the best available biotechnological tool for the induced breeding of
fish. GnRH is the key regulator and central initiator of reproductive cascade
in all vertebrates. It is a decapeptide  and was first isolated from pig and ship
hypothalami with the ability to induce pituitary release of luteinising  hormone
(LH) and follicle stimulating hormone (FSH). Since then, only one form of
GnRH has been identified in most placental mammals including, human
beings, as the sole neuropeptide causing the release of LH and FSH. However,
in non-mammalian species (except guinea pig) twelve GnRH variants have now
been structurally elucidated, among them seven or eight different forms have
been isolated from fish species. Depending on the structural variant and their
biological activities, number of chemical analogues have seen prepared and,
one of them is salmon GnRH analogue, profusely used now in fish breeding
and marked commercially throughout the world. The induced breeding of
fish is now successfully achieved by development of GnRH technology.
  Transgenesis: Transgenesis or transgenics may be defined as the
introduction of exogenous gene/DNA into host genome resulting in its stable
maintenance, transmission and expression. The technology offers an excellent
opportunity for modifying or improving the genetic traits of commercially
important fishers, molluscs and crustaceans for aquaculture. The idea of
Biotechnology of Aquaculture 12.3

producing transgenic animals became popular when Palmitter and his group
in 1982, first produced transgenic mouse by introducing metallothionein
human growth hormone fusion gene (mT-hGH) into mouse egg, resulting
in dramatic increase in growth. This triggered a series of attempts on gene
transfer in economically important animals, including fish.
The first transgenic fish was produced by Zhu and his group in 1985 in
China, who claimed the transient expression n putative transgenics, although
they gave no molecular evidence for the integration of the transgene. The
technique has now seen successfully applied to a number of fish species.
Dramatic growth enhancement has been shown using this technique,
especially in salmonids. Some studies have revealed enhancement of growth
in adult salmon to an average of 3 Ð, 5 times the size of non-Ð transgenic
controls, with some individuals, especially during the first few months
of growth, reaching as much as 10 Ð, 30 times the size of the controls. The
introduction of transgenic technique has simultaneously put more emphasis
on the need for production of sterile progeny in order to minimize the risk of
transgenic stocks mixing in the wild populations. The technical development
has expanded the possibilities for producing either sterile fish or those whose
reproductive activity can be specifically turned on or off using inducible
promoters. This would clearly be of considerable value allowing both optimal
growth and controlled reproduction of the transgenic stocks while ensuring
that any escaped fish would be unable to breed. An increased resistance of fish
to cold temperatures has been another subject of research in fish transgenics
for the past several. Cold water temperatures pose a considerable stressor to
many fish and few are able to survive water temperatures much below 0–1°C.
This is often a major problem in aquaculture in cold climates. Interestingly,
some marine teleosts have high levels (10 Ð 25 mg/ml) of serum antifreeze
proteins (AFP) or glycoproteins (AFGP) which effectively reduce the freezing
temperature by preventing ice-crystal growth. The isolation, characterization
and regulation of these antifreeze proteins, particularly, of the inter-flounder
Pleuronectas americanus, has been the subject of research for a considerable
period in Canada. Consequently, the gene encoding the liver AFP from winter
flounder was successfully introduced into the genome of Atlantic salmon
where it became integrated into the germ line and then passed onto the off
Ð spring F3, where it was expressed specifically in the liver. The introduction
of AFPs to gold fish also increased their cold tolerance, to temperatures at
which all the control fish died. Similarly, injection or oral administration of
AFP to juvenile milkfish or tilapia led to an increase in resistance to a 26 to
13°C. drop in temperature. The development of stocks harboring this gene
would be a major benefit in commercial aquaculture in countries where winter
temperatures often border the physiological limits of these species.
The most promising tool for the future of transgenic fish production
is  undoubtedly in the development of the embryonic stem cell (ESC)
technology. There cells are undifferentiated and remain totipotent, so, they
12.4 Environmental Biotechnology

can be manipulated in vitro, and subsequently reintroduced into early

embryos where they can contribute to the germ line of the host. This would
facilitate the genes to be stably introduced or deleted. Although significant
progress has been made in several laboratories around the world, there are
numerous problems to be resolved before the successful commercialization of
the transgenic brood stock for aquaculture. To realize the full potential of the
transgenic fish technology in aquaculture, several important scientific break
through are required. These include:
(i) more efficient technologies for mass gene transfer,
(ii) targeted gene transfer technologies such as embryonic stem cell gene
transfer,
(iii) suitable promoters to direct the expression of transgenes at optimal
levels during the desired developmental stages,
(iv) identified genes of desirable traits for aquaculture and other
applications,
(v) information on the physiological, nutritional, immunological and
environmental factors that maximize the performance of the transgenics,
(vi) safety and environmental impacts on transgenic fish.
Chromosome Engineering: Chromosome sex manipulation techniques to
induce polyploidy (triploidy and tetraploidy) and uniparental chromosome
inheritance (gynogenesis and androgenesis) have been applied extensively
in cultured fish species. These techniques are important in the improvement
of fish breeding as they provide a rapid approach for gonadal sterilization,
sex control improvement of hybrid viability and clonation. Most vertebrates
are diploid, meaning that, they possess two complete chromosome sets in
their somatic cells. Polyploidy individuals possess one or more additional
chromosome sets, bringing the total to three in triploids, four in tetraploids and
so on. Induced triploidy is widely accepted as the most effective method for
producing sterile fish for aquaculture and fisheries management. The methods
used to induce triploids and other types of chromosome set manipulations in
fishes and the applications of these biotechnologies to aquaculture and fisheries
management are well described by many scientists. Tetraploid breeding
lines are of potential benefit to aquaculture, by providing a convenient way
to produce large numbers of sterile triploid fish through simple interploidy
crosses between tetraploids   and diploids. Although tetraploidy has been
induced in many finfish species, the viability of tetraploids was low in most
instances.
In teleosts, technique for inducing sterility, include exogenous hormone
treatment and triploidy induction. The use of hormone treatments, however,
could be limited by governmental regulation and a lack of consumer acceptance
of hormone-treated fish products. Triploidy can be induced by exposing eggs
Biotechnology of Aquaculture 12.5

to physical or chemical treatment shortly after fertilization to inhibit extrusion

of the second polar body triploid; fish are expected to be sterile because of
the failure of homologous chromosomes to synapse correctly during the first
meiotic division. Methods of triploidy induction included exposing fertilized
eggs to temperature shock (hot or cold), hydrostatic pressure shock or
chemicals such an colchicines, cytochalasin-B or nitrous oxide. Triploid can
also be produced by crossing teraploids and diploids. Tetraploid induction
involves fertilizing eggs with normal sperm and exposing the diploid zygote
for physical or chemical treatment to suppress the first mitotic division.
Gynogenesis is the process of animal development with exclusive maternal
inheritance. The production of gynogenetic individuals is of particular interest
to fish breeders because a high level of inbreeding can be induced in single
generation. Gynogenesis may also be used to produce all Ð female populations
in species with female homogamety and to reveal the sex determination
mechanisms in fish. It is convenient to use all female gynogenetic progenies
(instead of normal bisexual progenies) for sex inversion experiments.
Methodologies combining use of induced gynogenesis with hormonal sex
inversion have been developed for several aquaculture species. Androgenesis
is the process which have commercial application in aquaculture. It can also
be used in generating homozygous lines of fish and in the recovery of lost
genotypes from the crypreserved sperms. Androgenetic individuals have
been produced in a few species of cyprinids, cichlids and salmonids.
Biotechnology and fish health management:  Disease problem area is a
major constraint for development of aquaculture. Biotechnological tools such
as molecular diagnostic methods, use of vaccines and immunostimulants are
gaining popularity for improving the disease resistance in fish and shelfish
species world over for viral diseases, avoidance of the pathogen is very
important. In this context, there is a need to rapid method for detection of the
pathogen. Biotechnological tools such as gene probes and polymerase chain
reaction (PCR) are showing great potential in this area. Gene probes and PCR-
based diagnostic methods have been developed for a number of pathogens
affecting fish and shrimp. In case of finfish aquaculture, number of vaccine
against bacteria and viruses have been developed. Some of these have been
conventional vaccines consisting of killed microorganism but new generation
of vaccine consisting of protein subunit vaccine, genetically engineered
organism, and DNA vaccine, are currently under development.
In the vertebrate system, immunization against disease is a common
strategy. However, the immune system of shrimp is rather poorly developed;
biotechnological tools are helpful for development of molecule, which can
stimulate this immune system of shrimp. Recent studies have shown that the
non-specific defense system can be stimulated using microbial product such as
lipopolysacharides, peptidoglycans or glucans. Among the immunostimulants
known to be effective in fish, glucan and levamisole enhance phagocytic
activities and specific antibody responses.
12.6 Environmental Biotechnology

Cryopreservation of gametes or gene banking:   Cryopreservation is a

technique, which involve long-term preservation and storage of biological
material at a very low temperature, usually at -196°C, the temperature of liquid
nitrogen. It is based on the principle that very low temperature tranquilize
or immobilize the physiological and biochemical activities of cell, thereby,
making it possible to keep them viable for very long period.
The technology of cryopreservation of fish spermatozoa (milt)  has been
adopted for animal husbandary . The first success in preserving fish sperm
at low temperature was reported by Blaxter (1953) who fertilized Herring
(Clupea herengus) eggs with frozen, thawed semen. The spermatozoa of almost
all cultivable fish species has now been cryopreserved. Cryopreservation
overcomes problems of male maturing before female, allows selective breeding
and stock improvement and enables conservation. One of the emerging
requirements for that can be used by breeders for evolving new strains. Most
of the plant varieties that have been produced are based on the gene bank
collections. Aquatic gene bank, however, suffers from the fact that, at present,
it is possible to cryopreserve only the male gametes of finfishes and there in no
viable technique for finfish eggs and embryos..

Conclusion
Biotechnological research and development are growing at a very fast
rate. Biotechnology has assumed greatest importance in recent years in
the development of fisheries, agriculture and human health. The science of
biotechnology has endowed us with new tools and tremendous power to
create novel genes and genotypes of plants, animals and fish. The application of
biotechnology in the fisheries sector is a relatively recent practice. Nevertheless,
it is a promising area to enhance fish production. The increased application of
biotechnological tools can certainly revolutionize our fish farming besides its
role in biodiversity conservation.  

12.1  PRODUCTION OF TRANSGENIC FISH

By using different transgenic techniques, researchers are seeking to improve
the genetic traits of the fish used in aquaculture. Researchers are trying to
develop fish which are: larger and grow faster, more efficient in converting
their feed into muscle, resistant to diseases, tolerant of low-oxygen levels in
the water, and tolerant to freezing temperatures.
• For example, some species of fish make a protein which allows them to
survive in the Arctic. This “antifreeze” gene has been transplanted into
other species of fish so, they also can survive in very cold waters.
Growing fish, that are longer and heavier, is the goal of researchers, who
are experimenting with applying various types of growth hormone to fish.
One method of doing this is to dip the fish in a solution which contains the
hormone. However, there are some problems with this technique. First, it
Biotechnology of Aquaculture 12.7

may be difficult to produce large quantities of purified growth hormone, the

method is labor-intensive, and it’s difficult to determine whether the fish are
getting the right amount of growth hormone. Therefore, researchers want to
develop new strains of transgenic fish which naturally produce just the right
amount of growth hormone to speed their growth. Such fish would be more
cost-effective since, they would produce higher levels of growth hormone on
their own, and they would pass this trait to their offspring.
There has been some success in this area. For example, a researcher at
the University of Connecticut has developed a tilapia fish that grows twice as
fast and up to five times as large as wild strains. The scientist introduced an
extra copy of the growth hormone gene into fish embryos at a very early stage,
resulting in the unique growth characteristics.
There are two main techniques which researchers use to transfer genetic
material in fish.
• One is called microinjection, in which the genetic material is injected into
newly-fertilized fish eggs. However, this method is time-consuming, so
researchers may prefer to use electroporation.
• Electroporation involves transferring the genetic material, or DNA,
into fish embryos through the use of an electrical current.
• Besides these two techniques, Retroviral vectors, containing the
envelope protein of vesicular stomatitis virus, have been developed,
and used to produce transgenic fish.
Microinjection: Gene transfer research with fish began in the mid 1980s
utilizing microinjection. Zhu and his group, in 1980, published the first report
of transgenes microinjected into the fertilized eggs of goldfish. In almost
all fish gene transfer research, the foreign gene was microinjected into the
cytoplasm of one-to-four cell embryos, as pronuclei are extremely difficult to
visualize in live one-cell fish embryos. Microinjection is a tedious and slow
procedure and can result in high egg mortality. After the initial development of
microinjection, new techniques such as electroporation, retroviral integration,
liposomal-reverse-phase-evaporation, sperm-mediated transfer and high
velocity micro-projectile bombardment were developed that sometimes can
more efficiently produce large quantities of transgenic individuals in a shorter
time period.
Electroporation: It involves placing the eggs in a buffer solution containing
DNA and applying short electrical pulses to theoretically create a transient
openings of the cell membrane, allowing the transfer of genetic material
from solution into the cell. The efficiency of the electroporation is affected
by a variety of factors including voltage, number of pulses and frequency of
pulses. The first successful gene transfer utilizing electroporation produced
integration rates and survival similar to that for microinjection. It is proved
experimentally that electroporation can be more efficient than microinjection
with integration rates sometimes as high as 30–100%. Hatching rates are higher
12.8 Environmental Biotechnology

for electroporated embryos than for microinjected channel catfish embryos,

and post-fertilization electroporation treatments had higher hatching rates
than electroporation of sperm and then eggs, prior to fertilization.
Efficiency of gene transfer is determined by several factors including:
hatching percentage, gene integration frequency, the number of eggs which
can be manipulated in a given amount of time and the quantity of effort
required to manipulate the embryos. In this regard, electroporation is a
powerful technique for mass production of transgenic fish.
Retroviral vectors: Retroviral vectors containing the envelope protein
of vesicular stomatitis virus have been developed, and used to produce
transgenic fish. Integration rates may be increased because of active infection.
Unfortunately, these vectors are prone to unstable expression or even complete
silencing of transgene expression. Sarmasik and his group (2001) successfully
utilized retroviral constructs to produce transgenic crayfish and topminnows,
Poeciliposis lucida. The pantropic retroviral vectors were derived from the
Hepatitis B virus and the Vesicular stomatitis virus, a pathogen similar to hoof
and mouth disease which infects mammals, insects and possibly, plants. The
vector sticks to most cell membranes of any species. Transgenic crayfish and
topminnows were produced by injecting immature gonads with a solution
of the vector, about one month before the normal age of first reproduction.
Matured injected individuals were mated with normal individuals, and
produced 50% transgenic offspring. Integration, expression and transmission
of the pantropic retroviral-reporter transgene were observed, for at least three
generations. This is a very good gene transfer technique for live-bearers and
fish, in general, but, introduction of viral sequences into food fish may not be
accepted by the public. Use of transposases to enhance integration rates may be
a more viable option than retroviral vectors for oviparous aquatic organisms,
but does not solve the problem of live-bearers. Theoretically, inactivation of a
gene can be accomplished by knockout of the gene by replacing the original
gene with a mutated copy of the gene, or by disruption of gene expression
using the antisense approach or the ribozyme technology. Although, the later
two approaches are currently feasible, the knockout approach is the ultimate
method for gene inactivation because it will eliminate the gene products
completely. Another technique for post-transcriptional gene silencing is
utilization of RNA antisense constructs. Both double-stranded RNA and
antisense RNA were effective in disrupting the expression of GFP in transgenic
zebra fish.
Various antisense technologies appear feasible. Regardless of the method
of transfer, the foreign DNA introduced to the developing embryo, it appears
to initially replicate and amplify rapidly in the cytoplasm of the developing
embryo, and then disappears, as development proceeds. Integration at the
one-cell stage has never been observed, thus the delayed integration causes
mosaicism, and, not all tissues contain the transgene and, not all cells within
Biotechnology of Aquaculture 12.9

the transgenic tissues, harbor the transgene. Copy numbers can range from
one to several thousand at a single locus, and, in contrast to the head-to-tail
organization observed in the mouse system, in some but not all cases, the DNA
can also be found organized in all possible concatemeric forms, suggesting
random end-to-end ligation of the injected DNA prior to integration.
Transgenes can integrate at single or multiple chromosomal locations
for individual transgenic fish. For salmonids, the frequency of transgene
transmission from founder animals averages about 15%, suggesting that
integration of the foreign DNA occurs on average at the two-to-four cell stage
of development. Transmission of transgenes to F2, or later progeny, occurs at
Mendelian frequencies, indicating that the DNA is stably integrated into the
host genome and passes normally through the germ line.

12.2  PEARL OYSTER CULTURE

The pearl oyster industry, traditionally, relied upon spat collection in the field
to supply its needs for farm requirements. As the industry has expanded,
the need for a more predictable source of the spat has emerged. Likewise,
the thought of controlling some desirable characteristics of the pearl oysters
has resulted in the setup of hatchery and nursery facilities. The hatchery and
nursery will give the farmer a predictable supply of spat and will allow greater
manipulation of genetic traits. This is the road to proper animal husbandry.
Every hatchery and nursery has site-specific conditions which require
fine-tuning of the initiating protocol used at the facility. This development of
the specific protocol for a hatchery will take place as the start-up begins and,
as spat are produced. In the end, the protocol for the given hatchery will be the
most efficient possible for that hatchery. Good observation and thoroughness
are necessary to fine-tune the initiating protocol. The hatchery/nursery staff
must exhibit these qualities in their work. It is the intent of the consultant
that the start-up and training stage of this new hatchery and nursery will be
greatly facilitated by the information contained in a manual which is prepared
for each hatchery.
Broodstock: Central to larval culture is, the availability of good quality
broodstock. Broodstock Pinctada margaritifera may be obtained from shell
that have settled on collectors set in the lagoons of atolls or, in some cases,
from wildstock shell. Freshly-collected shell, which are potential broodstock,
should be assessed for DVM (Dorso-Ventral Measurement) size and shape of
the shell. If the shell looks suitable for broodstock-use, the shell should be
drilled in the appropriate place and hung on chaplets from a long line or they
may be placed into 8–pocket panel nets and suspended from a long line. They
should remain on the long line at least 4–6 months before they may be used
during a spawning for the hatchery. This allows time for the shell to acclimate
to the lagoon conditions, and for gametogenesis to proceed toward ripe
gametes. Another very suitable source of broodstock are seeded shell which
12.10 Environmental Biotechnology

are already hanging on long lines. Although the farmer must be careful not
to have excessive handling of these seeded shell, it is normal in most places
that cleaning of these shell is carried out on a regular basis to reduce fouling
organisms growing on the outside of the shell. Cleaning of these shells can be
combined with their use as broodstock for a planned spawning in the hatchery.
Large quantities of good quality eggs can be obtained by this method because
shells which have been hanging on chaplets and long lines generally are in
good condition and release gametes readily upon handling. The numbers of
shell being cleaned are generally large, so there is no problem with obtaining
sufficient eggs for stocking into the hatching tanks.
Spawning: Other than the thermal method of spawning stimulation,
chemical methods can be used to induce spawning. These include different
concentrations [1.532, 3.064, or 6.128 millimolars] of hydrogen peroxide,
either in normal seawater or alkaline seawater (pH 9.1). The pH media can
be prepared using Tris buffer or Sodium hydroxide pellets. The Pearl Oyster
Farming and Pearl Culture Manual in India [Central Marine Fisheries Research
Institute at Tuticorin, India, published, February 1981] stated that, when
inducing spawning chemically, “A pH value of 9.0 in the case of Tris buffer
and 9.5 in NaOH, gives 78.6% and 68.4% of spawning, respectively.” Further,
they say, “Injection of 0.2 ml of N/10 ammonium hydroxide solution into the
adductor muscle of the pearl oyster results in 48% spawning.” It should be
noted here that serotonin-induced spawning such as used with giant clams
(1–4 ml of 2 millimolar serotonin solution) may also be a possibility with pearl
oysters. However, information from the James Cook University Blacklip Pearl
Oyster Project indicates that serotonin is not so effective with pearl oysters as
it is with giant clams or other bivalves.
Although, initial spawnings may utilize relatively large numbers of brood
stock that will not be identified as the parents, eventually, the hatchery will be
used for crosses between broodstock shell with desirable traits. In this case, the
broodstock should be kept on tagged chaplets or 8–pocket panel nets so that
the parents can be identified. DVM measurements would also be recorded at
each spawning date that the shells are used as broodstock.
Do pearl oysters which produce good quality, round pearls, at the first
harvest, possess a genetic trait for round pearls? This question could be tested
by using these shell for a special spawning after they have been re-seeded a
second time and had 4–6 months rest in the lagoon. The growth, survival, and
general development of their offspring would be carefully recorded up, until
the time, they were old enough for their first seeding. The result will then
come out in the first harvest. There are other characteristics of the shell shape,
the nacre, etc., which are likewise important for genetic manipulation.
Larval Rearing in the Hatchery: A most important factor in larval rearing
success is cleanliness. It is essential that egg and sperm collection materials
have been chlorine-cleaned and are stored dry for use during a spawning and
during the larval cycle.
Biotechnology of Aquaculture 12.11

The following details on the development of embryos and larvae are taken
from the Pearl Oyster Farming and Pearl Oyster Culture Manual:

12.2.1  Early Development and Larval Rearing

Cleavage: The first cell division is seen 45 minutes after fertilization resulting
in the formation of a micromere and a macromere. The polar body is placed at
the cleavage furrow. During the second cleavage the micromere divides into
two and the macromere divides unequally into a micromere and macromere.
The stage with three micromeres and a macromere is called Trefoil stage.
The macromere does not take part in further divisions. Micromeres divide
repeatedly, thus becoming smaller and smaller and passing through 8-cell,
and so on, until the morula stage. Each micromere develops a small cilium
which helps in the movement of the embryo.
Blastula: The embryo is ball-like with transparent cells and a blastocoel.
The embryos lift themselves in the water column and congregate at the
surface. The floating embryos are siphoned out to clean containers and the
residues at the bottom, containing broken tissues, undeveloped embryos,
unfertilized eggs, sperm, etc., are discarded. Reorientation of cells starts and
the blastocoel and blastopore are formed. The blastula stage is reached 5 hours
after fertilization.
Gastrula: Gastrulation takes place by epiboly. The cells convolute and
differentiate into different dermal layers. The archenteron is formed. The
embryo is bean-shaped as there is convolution of cells. The gastrula exhibits
negative phototropism. The stage is reached in 7 hours.
Trochophore larva: The minute cilia present in the gastrula stage
disappear and the pre-oral and post-oral tufts of cilia develop, thus marking
antero-posterior differentiation of the embryo. A single apical flagellum is
developed at the anterior side. The anterior portion of the larva is broader
while the posterior end is tapering like an inverted triangle. The movement of
the larva is affected by the propulsive movement of the flagellum. The dorsal
ectodermal cells secrete the embryonic shell, known as the prodissoconch I.
Veliger: A definite ‘D’ shape is obtained by the secretion of the
prodissoconch I having a hinge line, mantle and rearrangement of the pre-oral
tuft of cilia into a velum. The single flagellum, pre-oral and post-oral tufts of
cilia disappear. The veliger larva [of Pinctada fucata] measures 67.5 um along
the antero-posterior axis and 52.5 um along the dorso-ventral axis. This stage
is reached in 20 hours.
A Quick Reference to Feeding Schedules for Larvae and Spat are prepared
in the manual. This useful reference should be placed on the Algal Lab wall and
used daily during the larval feeding. Another part of the protocol shown in the
manual are, days of larval life shown against the columns of Stocking Density
(of larvae), Algal Feed, Flushing requirements, Draindown requirements,
12.12 Environmental Biotechnology

Microscope checks on size of larvae, % feeding, and Spat collectors. This table
will allow a quick visual check over the larval cycle by technicians to see what
is required.
Technician Duty Schedule: Along with the above tables which help the
technicians to keep up with the protocol, there need to be forms for Weekly
Duty Schedules for, 1.) the Hatchery Phase, 2.) the Nursery Stage, and 3.) the
Algal Production. These schedules list the most important duties that the
technicians need to do over a weekly period. Some duties are required daily,
whilst others are required less regularly.
Land Nursery Culture of Spat: The manual discusses settling materials
to use for late stage larvae and the potential positive effects of conditioning
the collector materials. Spat can be left on their collectors until they are large
enough to be safely removed and placed into trays with the appropriate size
mesh to retain the spat. The minimum protocol required in the raceways is
discussed in the manual.
Algal Culture: The usual food of bivalve larvae such as blacklip pearl
oysters is unicellular algae ranging from 2–10 microns (um). Generally, it is
wise to be careful in feeding new veliger larvae with too much unicellular
algae as the gut may only just be in the process of completion and the
possibility exists of the gut becoming plugged up with algal cells that cannot
be completely digested. The protocol on the feeding density over days of the
larval cycle are shown in the manual.
Monospecific cultures: Whether a hatchery is located in a temperate or
tropical area, the monospecific unicellular algal cultures are required for larval
rearing needs. A considerable amount of time is needed to set up and maintain
these cultures. The trained technicians handling the algae cultures must keep
careful attention to detail and hygiene. The manual shows the steps involved
in starting with stock cultures of unicellular algae (= microalgae) to large
mass cultures of 60–250-L. The f/2 medium is one of the standard microalgal
culture mediums in use around the world. This will be the medium to be used
at hatchery for cultures from 50 ml flasks to the 250-L cylinder cultures, but
where the budget is restricted, Aquasearch has it’s own cheap medium which
works nearly as well, as the f/2 medium. The stocks must be cared for because
all the cultures come from the stocks. It should be noted here that sodium
metasilicate is only added to the media which will be used to grow diatoms in.
Diatoms have an outside shell (like a jewelry box that fits neatly into top and
bottom) made of silicate, so, this material becomes limiting in dense cultures.
Mass microalgal cultures that will be grown in the outdoor algal culture
area will grow well with other culture media that are tested for mass culture.
These media are cheaper than f/2 and quite suitable for the large volumes of
algae being grown to feed spat in the land nursery raceways.
Biotechnology of Aquaculture 12.13

12.3  SHRIMP FARMING

Shrimp are decapod crustaceans, that can be found in both fresh and salt
water. Shrimp are swimming crustaceans living close to the bottom of the
water bodies. With firm and translucent flesh and their delicious taste, shrimp
are one of the most popular seafood, just next to fish. Low in saturated fats and
calories, but high in essential fats and other nutrients, shrimp are a healthy
food, with innumerable health benefits. So let’s take a brief look at shrimp
nutritional value and benefits.
A shrimp farm is an aquaculture business for the cultivation of marine
shrimp or prawns for human consumption. Commercial shrimp farming
began in the 1970s, and production grew steeply, particularly to match the
market demands of the United States, Japan and Western Europe. The total
global production of farmed shrimp reached more than 1.6 million tonnes in
2003, representing a value of nearly 9 billion U.S. dollars. About 75% of farmed
shrimp is produced in Asia, in particular, in China and Thailand. The other
25% is produced mainly in Latin America, where Brazil, Ecuador, and Mexico
are the largest producers. The largest exporting nation is Thailand.
Shrimp farming has changed from traditional, small-scale businesses
in Southeast Asia into a global industry. Technological advances have led
to growing shrimp at ever higher densities, and broodstock is shipped
worldwide. Virtually, all farmed shrimp are of the family Penaeidae, and just
two species – Penaeus vannamei (Pacific white shrimp) and Penaeus monodon
(giant tiger prawn) – account for roughly 80% of all farmed shrimp. These
industrial monocultures are very susceptible to diseases, which have caused
several regional wipe-outs of farm shrimp populations. Increasing ecological
problems, repeated disease outbreaks, and pressure and criticism from both
NGOs and consumer countries, led to changes in the industry in the late 1990s
and, generally stronger regulation by governments. In 1999, a program aimed
at developing and promoting more sustainable farming practices was initiated,
including governmental bodies, industry representatives, and environmental
organizations.
Farming methods: When shrimp farming emerged to satisfy demand that
had surpassed the wild fisheries’ capacity, the subsistence farming methods of
old were rapidly replaced by the more productive practices required to serve a
global market. Industrial farming, at first, followed traditional methods, with
so-called “extensive” farms, compensating for low density with increased
pond sizes; instead of ponds of just a few hectares, ponds of sizes up to 100
hectares (1.0 km2) were used and huge areas of mangroves were cleared in
some areas. Technological advances made more intensive practices possible
that increased yield per area, helping reduce pressure to convert more land.
Semi-intensive and intensive farms appeared, where the shrimp were reared
on artificial feeds and ponds were actively managed. Although many extensive
farms remain, new farms typically are of the semi-intensive kind.
12.14 Environmental Biotechnology

Until the mid-1980s, most farms were stocked with young wild animals,
called ‘postlarvae’, typically, caught locally. Post larvae fishing became an
important economic sector in many countries. To counteract the depletion of
fishing grounds and to ensure a steady supply of young shrimp, the industry
started breeding shrimp in hatcheries.

Life cycle
• Shrimp mature and breed only in a marine habitat. The females lay
100,000 to 500,000 eggs, which hatch after some 24 hours into tiny
nauplii.
• These nauplii feed on yolk reserves within their bodies, and then
metamorphose into zoeae.
• Shrimp in this second larval stage, feed in the wild on algae, and after
a few days, morph again into myses.
• The myses look akin to tiny shrimp, and feed on algae and zooplankton.
• After another three to four days, they metamorphose a final time into
postlarvae—young shrimp, that have adult characteristics.
• The whole process takes about 12 days from hatching.
In the wild, post larvae then migrate into estuaries, which are rich in
nutrients and low in salinity. They migrate back into open waters when they
mature
Supply chain: In shrimp farming, this life cycle occurs under controlled
conditions. The reasons to do so include, more intensive farming, improved
size control resulting in more uniformly-sized shrimp, and better predator-
control, but also the ability to accelerate growth and maturation by controlling
the climate (especially, in farms in the temperate zones, using greenhouses).
There are three different stages:
• Hatcheries breed shrimp and produce nauplii or even post larvae, which
they sell to farms. Large shrimp farms maintain their own hatcheries
and sell nauplii or post larvae to smaller farms in the region.
• Nurseries grow post larvae and accustom them to the marine conditions
in the grow-out ponds.
• In the grow-out ponds the shrimp are grown from juveniles to marketable
size, which takes between three to six months.
Most farms produce one to two harvests a year; in tropical climates, even
three are possible. Because of the need for salt water, shrimp farms are located
on or near a coast. Inland shrimp farms have also been tried in some regions,
but, the need to ship salt water and competition for land with agricultural
users, led to problems.
Hatcheries: Small-scale hatcheries are very common throughout Southeast
Asia. Often run as family businesses and using a low-technology approach,
Biotechnology of Aquaculture 12.15

they use small tanks (less than ten tons) and often low animal densities. They
are susceptible to disease, but due to their small size, they can typically restart
production quickly after disinfection. The survival rate is anywhere between
zero and 90%, depending on a wide range of factors, including disease, the
weather, and the experience of the operator.
• Green water hatcheries are medium-sized hatcheries, using large tanks
with low animal densities. To feed the shrimp larvae, an algal bloom is
induced in the tanks. The survival rate is about 40%.
• Galveston hatcheries (named after Galveston, Texas, where they
were developed) are large-scale, industrial hatcheries using a closed
and tightly-controlled environment. They breed the shrimp at high
densities in large (15 – 30 t) tanks. Survival rates vary between 0% and
80%, but typically, achieve 50%.
• In hatcheries, the developing shrimp are fed on a diet of algae, and
later also, brine shrimp nauplii, sometimes (especially in industrial
hatcheries) augmented by artificial diets. The diet of later stages
also includes fresh or freeze-dried animal protein, for example, krill.
Nutrition and medication (such as antibiotics) fed to the brine shrimp
nauplii are passed on to the shrimp that eat them.
Nurseries: Farmers transferring post larvae from the tanks on the truck to
a grow-out pond Many farms have nurseries where the post-larval shrimp are
grown into juveniles for another three weeks in separate ponds, tanks, or so-
called raceways. A raceway is a rectangular, long, shallow tank through which
water flows continuously.
• In a typical nursery, there are 150 to 200 animals per square metre. They
are fed on a high-protein diet for at most three weeks before they are
moved to the grow-out ponds. At that time, they weigh between one
and two grams. The water salinity is adjusted gradually to that of the
grow-out ponds.
• Farmers refer to post larvae as “PLs”, with the number of days suffixed
(i.e., PL-1, PL-2, etc.). They are ready to be transferred to the grow-
out ponds after their gills have branched, which occurs around PL-
13 to PL-17 (about 25 days after hatching). Nursing is not absolutely
necessary, but is favored by many farms because it makes for better food
utilization, improves the size uniformity, helps use the infrastructure
better, and can be done in a controlled environment to increase the
harvest. The main disadvantage of nurseries is that some of the post-
larval shrimp die upon the transfer to the grow-out pond.
• Some farms do not use a nursery, but stock the post larvae directly in
the grow-out ponds after having acclimated them to the appropriate
temperature and salinity levels in an acclimation tank. Over the course
of a few days, the water in these tanks is changed gradually to match
12.16 Environmental Biotechnology

that of the grow-out ponds. The animal density should not exceed 500/
liter for young post larvae and 50/liter for larger ones, such as PL-15.
Feeding the shrimp: While extensive farms mainly rely on the natural
productivity of the ponds, more intensively managed farms rely on artificial
shrimp-feeds, either exclusively or as a supplement to the organisms that
naturally occur in a pond. A food chain is established in the ponds, based on
the growth of phytoplankton. Fertilizers and mineral conditioners are used to
boost the growth of the phytoplankton to accelerate the growth of the shrimp.
Waste from the artificial food pellets and shrimp excrement can lead to the
eutrophication of the ponds.
Artificial feeds come in the form of specially formulated, granulated
pellets that disintegrate quickly. Up to 70% of such pellets are wasted, as they
decay before the shrimp have eaten them. They are fed two to five times daily;
the feeding can be done manually either from ashore or from boats, or using
mechanized feeders distributed all over a pond. The feed conversion rate
(FCR), i.e., the amount of food needed to produce a unit (e.g., one kilogram)
of shrimp, is claimed by the industry to be around 1.2–2.0 in modern farms,
but this is an optimum value that is not always attained, in practice. For a farm
to be profitable, a feed conversion rate below 2.5 is necessary; in older farms
or under suboptimal pond conditions, the ratio may easily rise to 4:1. Lower
FCRs result in a higher profit for the farm.
Farmed species: Although there are many species of shrimp and prawn,
only a few of the larger ones are actually cultivated, all of which belong to the
family of penaeids (family Penaeidae), and within it, to the genus Penaeus.
Many species are unsuitable for farming: they are too small to be profitable,
or simply stop growing, when crowded together, or are too susceptible to
diseases. The two species dominating the market are:
• Pacific white shrimp (Litopenaeus vannamei, also called “whiteleg
shrimp”) is the main species cultivated in western countries. Native
to the Pacific coast from Mexico to Peru, it grows to a size of 23 cm. L.
vannamei accounts for 95% of the production in Latin America. It is easy
to breed in captivity.
• Giant tiger prawn (Penaeus monodon, also known as “black tiger shrimp”)
occurs in the wild in the Indian Ocean and in the Pacific Ocean from
Japan to Australia. The largest of all the cultivated shrimp, it can grow
to a length of 36 cm and is farmed in Asia. Because of its susceptibility
to white spot disease and the difficulty of breeding it in captivity, it is
gradually being replaced by L. vannamei since 2001.
Together, these two species account for about 80% of the whole farmed
shrimp production.
• Indian white shrimp (P. indicus) is a native of the coasts of the Indian
Ocean and is widely bred in India, Iran and the Middle East and along
the African shores.
Biotechnology of Aquaculture 12.17

Shrimp marketing: For commercialization, shrimp are graded and

marketed in different categories. From complete shrimp (known as “head-
on, shell-on” or HOSO) to peeled and deveined (P&D), any presentation is
available in stores. The animals are graded by their size uniformity and then
also, by their count per weight unit, with larger shrimp attaining higher prices.

12.3.1  Shrimp Nutritional Value Details

Shrimp Nutrition Facts: Shrimp are quite popular as a low calorie food source
of protein. About 85 g of shrimp provide just 84 calories. The same amount of
shrimp contain approximately—
• 0.9 g fats, with 0.2 g saturated fats,
• 0.2 g monounsaturated fats and
• 0.4 g poly unsaturated fats.
Apart from these, 85 g shrimp contain about 17.8 g proteins, 166 mg
cholesterol, 295 mg omega-3 fatty acids, 17.9 mg omega-6 fatty acids, 191 IU
of vitamin A, 1.9 mg vitamin C, 1.3 mcg vitamin B12, 2.2 mg niacin, 3.4 mcg
folate, 68.8 mg choline, 33.2 mg calcium, 2.6 mg iron, 28.9 mg magnesium, 155
mg potassium, 116 mg phosphorus, 1.3 mg zinc and 33.7 mg selenium.
Shrimp Nutritional Benefits: A mere look at the shrimp nutrition facts
can give you an idea about how nutritious this seafood is. The only thing that
can confuse people is that shrimp are quite high in cholesterol. Though shrimp
contains a high amount of cholesterol and increases the level of LDL cholesterol
in the body, it can also raise the level of HDL cholesterol, which is considered
as good for the health of the cardiovascular system. In fact, increase in HDL
cholesterol is more than the increase in LDL cholesterol, when a shrimp diet is
followed. Thus, eating shrimp can lower the ratio of LDL to HDL cholesterol,
which can prove beneficial for the heart health. In addition to increasing the
level of HDL cholesterol, shrimp can provide a number of health benefits,
which are explained below:
• Shrimp are rich in omega-3 and 6 fatty acids, the two types of essential
fatty acids that can boost heart and cardiovascular health. Eating
foods rich in these fatty acids can significantly lower the risk for
cardiovascular diseases, by reducing the level of cholesterol in the
body, and preventing blood clotting.
• Omega-3 essential fatty acids can also slow down or prevent the
development of diseases like rheumatoid arthritis, high blood pressure,
colorectal cancer and cancerous tumors.
• Being a very important dietary source of selenium, shrimp can provide
protection against degenerative diseases and cancer. Selenium is the
trace mineral, which can prevent the proliferation of cancerous cells
and neutralize the damaging effects of free radicals, which is associated
with a number of diseases including, cancer.
12.18 Environmental Biotechnology

• Vitamin B12, found in shrimp, ensures proper formation and maturation

of the blood cells. This vitamin is also essential for the functions of the
brain.
• Shrimp contain significant amount of vitamin D and the mineral
calcium. Vitamin D is required by the body for the proper absorption
of the mineral calcium, which is essential for the formation of strong
bones and teeth.
• Phosphorus, found in shrimp, can also contribute towards the
development of strong bones and teeth.
• Shrimp do not contain significant level of saturated fats, but contains
unsaturated fatty acids, which again can help to improve the health
of heart and the cardiovascular system. This seafood can also help to
lower the level of triglycerides, and thereby, reduce the risk of heart
disease and stroke.
To sum up, shrimp is one of the highly nutritious seafood around.
However, it is also common food allergens, and many individuals can
experience severe allergic reactions due to their intake. An allergic reaction to
shrimp can produce itching, hives, skin rash, breathing problems, wheezing
and unusual swelling—especially of the face, tongue, throat and the lips. To
avoid such severe reactions, it is generally advised not to take or consume
shrimp in its pure form. But those who are not allergic to shrimp, can realize
the shrimp nutritional value or health benefits, while enjoying the delectable
taste of this seafood. Shrimp can be prepared in a number of ways, but the
point to be kept in mind is that it should be cooked quickly, so as to preserve
its taste and flavor.

12.4  GROWTH AND REPRODUCTION OF EDIBLE CRUSTACEANS

Crustaceans (Crustacea) form a very large group of arthropods, usually treated
as a subphylum, which includes such familiar animals as crabs, lobsters,
crayfish, shrimp, krill and barnacles. The 67,000 described species range in
size from Stygotantulus stocki at 0.1 mm (0.004 in), to the Japanese spider crab
with a leg span of up to 12.5 ft (3.8 m) and a mass of 44 lb (20 kg). Like other
arthropods, crustaceans have an exoskeleton, which they moult to grow. They
are distinguished from other groups of arthropods, such as insects, myriapods
and chelicerates, by the possession of biramous (two-parted) limbs, and by the
nauplius form of the larvae.
Most crustaceans are free-living aquatic animals, but some are terrestrial
(e.g. woodlice), some are parasitic (e.g. Rhizocephala, fish lice, tongue worms)
and some are sessile (e.g. barnacles). The group has an extensive fossil record,
reaching back to the Cambrian, and includes living fossils such as Triops
cancriformis, which has existed apparently unchanged since the Triassic
period. More than 10 million tons of crustaceans are produced by fishery or
farming for human consumption, the majority of it being shrimps and prawns.
Biotechnology of Aquaculture 12.19

Krill and copepods are not as widely fished, but may be the animals with
the greatest biomass on the planet, and form a vital part of the food chain.
The scientific study of crustaceans is known as carcinology (alternatively,
malacostracology, crustaceology or crustalogy), and a scientist who works in
carcinology is a carcinologist.
Consumption by humans: Many crustaceans are consumed by humans,
and nearly 10,700,000 tons were produced in 2007; the vast majority of this
output is of DECAPOD CRUSTACEANS: crabs, lobsters, shrimp, and prawns.
Over 60% by weight of all crustaceans caught for consumption are shrimp and
prawns, and nearly 80% are produced in Asia, with China alone producing
nearly half the world’s total. Non-decapod crustaceans are not widely
consumed, with only 118,000 tons of krill being caught, despite krill having
one of the greatest biomasses on the planet
Decapod Crustaceans (edible crustaceans): The decapods or Decapoda
(literally “ten-footed”) are an order of crustaceans within the class Malacostraca,
including many familiar groups, such as crayfish, crabs, lobsters, prawns and
shrimp. Most decapods are scavengers. It is estimated that the order contains
nearly 15,000 species in around 2,700 genera, with approximately 3,300 fossil
species. Nearly half of these species are crabs, with the shrimp and Anomura
(including hermit crabs, porcelain crabs, squat lobsters), making up the bulk
of the remainder.
Classification within the order Decapoda depends on the structure of the
gills and legs, and the way in which the larvae develop, giving rise to two
suborders:
• Dendrobranchiata and
• Pleocyemata.
• Dendrobranchiata consists of prawns, including many species
colloquially referred to as “shrimp”, such as the “white shrimp”,
Litopenaeus setiferus.
• Pleocyemata includes the remaining groups, including true shrimp.
Those groups which usually walk rather than swim (Pleocyemata,
excluding Stenopodidea and Caridea) form a clade called Reptantia.
Decapods are primarily marine animals and are most abundant in warm,
shallow tropical waters, but they are exploited commercially throughout the
world. Some shrimp, for example, live in the open ocean and possess light
organs, or photophores, which are thought to aid in feeding, species recognition,
or camouflage (by counter-illumination). Approximately, 10 per cent of
known decapod species occur in freshwater or terrestrial habitats. Survival in
freshwater depends upon an organism’s ability to keep its blood concentration
at a level higher than the medium and to reduce the permeability of its body
surface. Those decapods that have colonized terrestrial environments, such
as some species of hermit and fiddler crab, have evolved mechanisms to
12.20 Environmental Biotechnology

protect against desiccation and overheating while regulating the internal

concentrations of their body fluids. Vascularization of the gill surfaces has
made respiration possible on land for some species of decapods. Terrestrial
decapods must usually return to the sea to spawn, while most freshwater
decapods spend their entire life cycle in fresh water, commonly hatching their
young as miniature adults.
Growth and reproduction: Decapods exist in a variety of relationships
with other organisms. Members of some hermit crab species, for example,
carry anemones or bryozoan colonies on the shell in a commensal relationship
(one, in which, the colonies do not feed on the host tissue). The pea crab
Pinnotheres ostreum, on the other hand, parasitically feeds on the American
oyster, causing gill damage. Some shrimp have symbiotic relationships with
fish; they remove parasites from the mouths and gills of the fish.
Decapods are behaviorally complex. Hermit crabs seek out empty shells to
use as a protective covering, selecting successively larger ones to accommodate
their growth. They discriminate between available shells based on each shell’s
size, species, weight, and degree of physical damage. The two basic types
of locomotion are swimming and crawling, though the macruran decapods
are able to move swiftly backward by flexing their abdomens. Burrowing is
accomplished by beating the leaf like swimmerets, or pleopods, or by digging
with the thoracic legs.
There is generally a separation between the sexes, although there are some
examples of simultaneous hermaphroditism (i.e., individuals with both male
and female reproductive organs). In most groups, fertilization is external,
although in some species it is internal. Variations in patterns of mating activity
are believed to be linked to the molting cycle. Male decapods can copulate
only when their exoskeleton is fully hardened, while some females are capable
of copulation only after a molt, when their shells are soft. In most decapods,
the fertilized eggs are carried cemented to the abdominal appendages until
they are hatched. After hatching, they can be classified as one of four basic
larval types, partly by their mode of locomotion: nauplius, protozoea, zoea,
and postlarva. Most decapod crustacean larvae hatch in the zoea stage.
Decapods have three distinct body regions, each made up of segments,
or somites: the head, thorax, and abdomen. The head and the thorax are
fused and are often referred to as the cephalothorax. A pair of appendages
is attached to each somite. The first two pairs, the first and second antennae,
consist of a segmented stalk and flagella, and serve such sensory functions
as olfaction, touch, and balance. The remaining three head appendages are
either the crushing and chewing mandibles or the flattened, multi-lobed
food manipulators. The anterior thoracic appendages serve as mouthparts,
while the posterior pairs are the walking legs, or pereiopods. The remaining
appendages may be modified for swimming, sperm transfer, pinching claws,
or even forming a tail fan with the telson.
Biotechnology of Aquaculture 12.21

A head shield, or carapace, covers the cephalothorax and extends over the
gills, which are attached to the body wall of the thorax. The heart is located
to the rear of the carapace above the gut, which is basically a straight tube
consisting of the stomodeum, or foregut, the mesenteron, or midgut, and
the proctodeum, or hindgut. The primary excretory organ is a gland (the
“green gland”) that opens at the base of the antennae. The central nervous
system consists of a supraesophageal ganglion with lateral connections to a
subesophageal ganglion. The eyes, which may be absent in some deep-sea
species, are usually well-developed with a pigmented, multifaceted cornea.
Reproduction: Crustaceans produce from eggs, which have been fertilized
by sperm in much the same manner as other animals. The eggs are produced
in the ovaries in the female and passed to the outside through oviducts. The
sperms are produced in tubular testes in the male. After the eggs have been
fertilized, they begin development and then hatch.
• When the eggs hatch, this can take several days to several weeks;
depending on the species, the young larvae are detached. From this
point on, they are on their own and must fee, grow, swim and survive.
After a series of transformations, the larvae becomes a miniature adult.
• Crustaceans cannot grow as many other animals do because of their
outer skeleton. Instead, they periodically shed the outer skeleton, grow
rapidly for a short time, and then, form another hard skeleton. While
this process is taking place, they hid in an isolated place.
• Another remarkable ability, the crustacean has is, to be able to break off
or to drop their appendages. This is called autotomy. They have special
breaking-off points near the body. If caught, they can quickly break-off
this appendages to get away. A new appendage is more easily grown.
Following table illustrates the nutritional value of edible crustaceans.
Nutritional value per 100 g
Energy 410 kJ (98 kcal)
Carbohydrates 0g
Sugars 0g
Dietary fiber 0g
Fat 0.59 g
saturated 0.107 g
monounsaturated 0.091 g
polyunsaturated 0.16 g
Protein 20.5 g
Thiamine (vit. B1) 0 mg (0%)
Riboflavin (vit. B2) 4 mg (333%)
12.22 Environmental Biotechnology

Niacin (vit. B3) 4 mg (27%)

Pantothenic acid (B5) 2 mg (40%)
Vitamin B6 4 mg (308%)
Folate (vit. B9) 2 mg (1%)
Vitamin C 0 mg (0%)
Calcium 6 mg (1%)
Iron 2 mg (15%)
Magnesium 8 mg (2%)
Phosphorus 15 mg (2%)
Potassium 0 mg (0%)
Zinc 15 mg (158%)

12.5  NEUROENDOCRINE PRINCIPLES INVOLVED IN THE REGULATION

OF GROWTH, REPRODUCTION AND METABOLISM OF PRAWNS
AND CRABS (EDIBLE CRUSTACEANS)
Programming of reproductive events of a species is being regulated by
interactions between various exogenous and endogenous factors. In the
tropical brachyurans, reproduction occurs round the year due to the relatively
stable environmental situations. Estuaries are characterized by fluctuating
conditions of salinity and temperature to the extent that both are considered
dominant ecological factors which may act either singly, or in concert, to
modify programming the reproduction and growth and distribution of
estuarine organisms. Significantly, many estuarine invertebrates require
specific temperature and salinity conditions at different developmental
stages. Many species use photoperiod as an external cue to initiate a series of
physiological events like molting, reproduction and hatching. Stephens (1955)
established photoperiod as a determinant factor in the induction of moulting
in the crayfish Procambarus sp. Photoperiod and temperature influence ovarian
development and spawning in the American lobster H.americanus.
The neurosecretory system of the eyestalk consists of a group of peptidergic
neurons clustered in the medulla terminalis of X-organ (XO) and their bulbous
axonic terminals that constitute the sinus gland (SG), which is a neurohaemal
organ that releases a number of peptide hormones. XO-SG complex is the
neurendocrine system located within the eyestalk of crustaceans that produces
hormonal factors that control physiological processes of gonads. Neurological
hormones controlling moult and reproduction come under two categories,
viz., inhibitory and stimulatory principles. Several neuropeptides, forming
the so-called crustacean hyperglycemic hormone family, have been isolated
from the Xorgan–SG complex: the moult-inhibiting hormone (MIH) involved
in moulting, the vitellogenesis (or gonad)-inhibiting hormone (VIH/GIH)
involved in reproduction, the mandibular organ-inhibiting hormone (MOIH)
involved in reproduction and development, and the crustacean hyperglycemic
Biotechnology of Aquaculture 12.23

hormone (CHH) involved in the regulation of hemolymph glucose level, are

examples.
Regulation of growth: The physiological process of growth is hormonally
co-ordinated and regulated. The rigid exoskeleton in crustaceans and insects
require periodic molts to accomplish growth and metamorphosis and this is
harmoniously regulated by a cascade of neuropeptides.
Stimulatory principles: In crustaceans, the ecdysial glands known as
the Y-organs are responsible for ecdysteroid synthesis. Y-organ is located in
the anterior branchial chamber in all crustaceans; it appears as a compact
mass in crabs and a less compact mass in crayfishes and lobsters. Moulting
is stimulated directly by the presence of moulting hormone, ecdysteroids
(a group of polyhydroxylated ketosteroids) circulating in the hemolymph.
The synthesized ecdysone is released into the hemolymph and converted
into active 20-hydroxyecdysone (20-HE) by peripheral tissues and promotes
the physiological changes associated with moulting. In Cancer antennarius,
additional ecdysteroids are released by the Y-organs, 3-dehydroxyecdysone
and 25-deoxyecdysone, a precursor for active ponasterone A. Ponasterone A
is the main serum ecdysone present during premoult in Carcinus maenas and
the land crab, Gecarcinus lateralis. In the fiddler crab, Uca pugilator and other
crustaceans, several ecdysteroids (e.g. 25-deoxyecdysone, ponasterone A, and
ecdysone, 20E) circulate in the hemolymph. Changes in these ecdysteroid
titers and ratios during the molt cycle are temporally correlated with major
physiological events involved in moulting and regeneration of lost limbs.
The haemolymph ecdysteroid titres are known to vary with stages in
the moulting cycle, with high levels during premoult and low levels during
postmoult and intermoult. However, no significant change in haemolymph
ecdysteroid titres was observed in relation to oocyte development in Carcinus
maenas. The Y-organ in the cephalothorax of crustaceans and the integument
of ticks has been reported to be the sources of secreted ecdysteroids in
adults, as in earlier stages, but the tissue source is not known for adults in
many arthropod groups. In arthropods, the ecdysteroid hormones regulate
growth, differentiation, and reproduction by influencing gene expression.
Ecdysteroids enhance the formation of ecdysteroid receptor–ultraspiracle
protein (EcR–USP) heterodimers which regulate gene transcription. The close
relationships between Y-organ activities and hemolymph ecdysteroid levels
suggest that the Y-organ activity directly affects the hemolymph ecdysteroid
levels in M. rosenbergii.

12.5.1  Inhibitory Principles

Moult-inhibiting hormone (MIH): The neurosecretory system of the eyestalk
consists of a group of peptidergic neurons clustered in the medulla terminalis
of X-organ and their bulbous axonic terminals that constitute the sinus gland,
which is a neurohaemal organ that releases a number of peptide hormones
including the complex of the eyestalks. Crustacean moult-inhibiting hormone
(MIH), a polypeptide secreted by the X-organ sinus gland complex of the
12.24 Environmental Biotechnology

eyestalks, regulates molting by inhibiting ecdysteroid synthesis of Y-organs.

The peptidic nature of MIH has been established for many brachyuran and
macruran species. Aguilar and his group (1996) isolated the MIH from the
SG of the Mexican crayfish, Procambarus bouvieri and compared its sequences
with other four known peptides from Homarus americanus, Carcinus maenas,
Callinectes sapidus and Penaeus vannamei and found that the length varied
between 72–78 residues and molecular mass between 8 and 9 KDa. All had six
cysteines that form three disulfide bonds.
Yang and his group (1996) isolated and sequenced a peptide with MIH
activity from the sinus gland of the kuruma prawn, P. japonicus. In the crabs
Carcinus maenas, Cancer pagurus, and Gecarcinus lateralis, the MIH gene
encodes a 113-amino acid prohormone (proMIH) composed of a 35-residue
signal peptide and a 78-amino acid mature peptide. The mature peptide had
the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine,
and one aspargine in identical positions in the highly conserved sequence,
characteristic of other crustacean MIHs. Scientists revealed that the MIH genes
of Cancer feriatus and C. pagurus consist of three exons and two introns, and
span approximately 4.3 kb. The hormone-binding domain of the MIH receptor
is likely to be highly conserved, and therefore, the MIH from one crab species
is capable of inhibiting ecdysteroid production by Y-organs in other. A cDNA
encoding the mature peptide was used to express recombinant MIH (rMIH)
using yeast, Pichia pastoris expression system.
Regulation of reproduction: Crustacean reproduction is under the
control of stimulatory and inhibitory principles. High titres of MIH and GSH
and low titres of GIH and MH always result in reproduction. While GSH acts
as a direct acceleratory principle, MIH acts indirectly as gonad acceleratory
principle by inhibiting the production of MH by Yorgan. Reproduction is
under endogenous regulation by nervous, endocrine and/or neurendocrine
systems. Regulation of crustacean reproduction is now thought to be based
on a multi-hormonal system in which factors of different chemical nature and
origin may play a direct role in gonad development, sexual differentiation and
mating behavior.
Maturation of the gonads in crustaceans is regulated by two antagonistically
acting neurological hormones; the gonad inhibiting hormone (GIH) from the
sinus gland and the gonad stimulating hormone (GSH) found in the brain and
thoracic ganglia. Decapod females depend mainly on neurological endocrine
centers outside the eyestalk for the gonad stimulatory principles and that
from the eyestalk for gonad inhibitory principles. Crustacean mandibular
organs are suggested to play a role in regulating reproduction in crustacean
females. Mandibular organ that control growth and reproduction is known to
be controlled by mandibular organ inhibiting hormone (MOIH), originating
from the eyestalk. Recent developments in crustacean endocrinology have
given an insight into the importance of ecdysteroids in female reproduction.
Biotechnology of Aquaculture 12.25

Eyestalk neuropeptides such as MIH is found to have an inhibitory control on

ecdysteroid synthesis by the Y-organ. Mandibular organ is also suggested to
have a stimulatory role for Y organ.

12.5.2  Stimulatory principles

Gonad stimulating hormone (GSH): The hormonal factors that have a
stimulatory effect on reproduction in crustaceans include, the vitellogenesis
stimulating ovarian hormone, methyl farnesoate, a JH analogue, and the
ecdysteroids. The existence of a female hormone of ovarian origin in the
Decapoda, as in the amphipods and isopods, has been reported to stimulate
development of secondary sexual characters. However, its role in stimulating
oogenesis has not been proved.
Vitellogenesis Stimulating Ovarian Hormone (VSOH): A possible
existence of an ovarian hormone or VSOH from the ovary and/or the follicle
cells surrounding the ovary was strongly considered by Charniaux-Cotton
(1960). However, the investigators who proposed the existence of VSOH were
not able to substantiate it. It has been suggested that estradiol-17b secreted
from ovarian follicle cells induces vitellogenin synthesis in the ovary as VSOH
in the Penaeid shrimp, P. vannamei.
Mandibular organ: In decapods, a glandular tissue is located at the base
of the tendon associated with the posterior abductor muscle of the mandibles,
the mandibular organ (MO). Mandibular organ produces methyl farnesoate
(MF), a juvenile hormone-related compound which is involved in crustacean
reproduction and development. Ultra-structural studies reveal the occurrence
of cells with extensive SER and abundant mitochondria. Initial studies by
several authors suggested that the mandibular organ might be involved in
regulating reproduction, and moulting.
Current evidence indicates that the MO is negatively regulated by
peptides present in the eyestalk, mandibular organ inhibiting factor
(MOIH). Methyl farnesoate was identified as a secretory product of MO.
The mandibular organ when implanted into immature females, stimulated
ovarian growth accompanied by morphological and ultra-structural signs of
active vitellogenesis.

12.5.3  Inhibitory principles

Gonad inhibiting hormone (GIH): GIH is secreted by the neurosecretory
cells of the XO in the eyestalks and is transported intra-axonally through the
XO-SG tract to the sinus gland (eyestalk). GIH is suggested to suppress the
ovarian development by blocking vitellogenin synthesis by hepatopancreas
and/or vitellogenin uptake by oocytes. GIH repress vitellogenin synthesis
in adipose tissue or vitellogenin uptake by oocytes. XO-SG extract when
injected to ablated animals suppressed ovarian development. It has also been
suggested that GIH inhibits only the secondary phase of the maturation of
12.26 Environmental Biotechnology

oocytes and probably, has no effect on the primary growth. The primary phase
has been suggested to be under the control of MH.
Mandibular organ inhibiting hormone (MOIH): The methyl farnesoate
(MF) secretion, by mandibular organ is suggested to be negatively controlled
by the mandibular organ inhibiting hormone produced by the eyestalk.
MOIH has been isolated and found to consist of 72–76 peptides and a MW of
8.0–8.5 Kd. MOIH peptides resemble that of the GIH. Removal of the eyestalk
removes MOIH inhibition and results in the hypertrophy of MO with increased
secretion of MF. Injection of sinus gland extracts decreases secretion of methyl
farnesoate by the mandibular organ. The amino acid sequences of these MOIH
peptides are similar to peptides in the crustacean hyperglycemic hormone
(CHH) family of neuropeptides.
In addition, there appears a compound in the eyestalk that lowers
hemolymph levels of methyl farnesoate in vivo, but does not directly affect
the mandibular organ in vitro. The inhibition of methyl farnesoate synthesis
by eyestalk peptides involves the inhibition of farnesoic acid O-methyl
transferase, the last enzyme in the methyl farnesoate biosynthetic pathway.
The activity of this enzyme is affected by cyclic nucleotides, suggesting
that these compounds may be involved in the signal transduction pathway
mediating the effects of MOIH. G-protein in the crustacean mandibular
organ participate in the signal transduction from the eyestalk neuropeptides
(MOIH) to the enzyme farnesoic acid O-methyl transferase responsible for
methyl farnesoate synthesis. Farnesoic acid O-methyl transferase (FAMeT),
directly or indirectly (through MF), modulates the reproduction and growth of
crustaceans by interacting with the eyestalk neuropeptides as a consequence
of its presence in the neurological secretory cells of the X-organ-sinus gland .
Interaction between growth and reproduction: Growth and reproduction
are two related processes among crustaceans. These processes are regulated
by two related endocrine axes that are governed by inhibitory neurological
hormones secreted from the X-organ-sinus gland complex (XO-SG) in the
eyestalk. Hormones involved in the two processes are related. MH is needed
for the prepubertal development. In adults, alternatively high levels of MIH
and GSH and low levels of MH and GIH bring about seasonal changes in the
moulting and reproduction. In brachyuran crabs and lobsters, these are two
antagonistc events wherein the animal enters into either moult or reproduction
at a particular period. Surgical extirpation of the eyestalks can induce both
moulting and vitellogenesis. The reproductive programming of a species or
population is the outcome of the interaction between various exogenous and
endogenous factors. In the tropical brachyurans, reproduction occurs round
the year due to the relatively stable environmental situations.
Much research has been conducted on the normal growth and reproductive
cycles of several crustaceans, simply because of their high reproductive potential
which enable successful culture for food purposes and the high nutritional
Biotechnology of Aquaculture 12.27

values. The general pattern of growth and reproduction of a population varies

even among the same species. Identification of gonad stimulating substances
is greatly used for domestication of commercially important species that do
not reproduce easily under captivity. Precocious development of gonad by
way of unilateral and bilateral eyestalk extirpation in the prawn and shrimp
species has been employed in aquaculture. The larvae produced by eyestalk
ablation have been found to be of low quality than those of the intact ones and
this warrants more research in this field. The methyl farnesoate produced by
mandibular organ stimulates and enhances reproduction. Detailed study of
mandibular organ and its role in stimulation of ovarian maturation in more
species may provide better understanding. Environmental variables play an
important role in growth and reproductive behavior. In estuarine species, the
behavior patterns are affected by hormones, temperature, tidal conditions and
photoperiod.
 CHAPTER

13 Nutrient Film Culture Techniques

Nutrient Film culture Technique or NFT is a hydroponic technique wherein a

very shallow stream of water containing all the dissolved nutrients required
for plant growth is recirculated past the bare roots of plants in a watertight
gully, also known as channels. In an ideal system, the depth of the recirculating
stream should be very shallow, little more than a film of water, hence the name
‘nutrient film’.
• This ensures that the thick root mat, which develops in the bottom of
the channel, has an upper surface, which, although moist, is in the air.
Subsequent to this, an abundant supply of oxygen is provided to the
roots of the plants. A properly designed NFT system is based on using
the right channel slope, the right flow rate, and the right channel length.
• The main advantage of the NFT system over other forms of hydroponics
is that the plant roots are exposed to adequate supplies of water,
oxygen and nutrients. In all other forms of production, there is a
conflict between the supply of these requirements, since excessive or
deficient amounts of one results in an imbalance of one or both of the
others. NFT, because of its design, provides a system wherein all three
requirements for healthy plant growth can be met at the same time,
provided that the simple concept of NFT is always remembered and
practiced.
• The result of these advantages is that higher yields of high-quality
produce are obtained over an extended period of cropping. A downside
of NFT is that it has very little buffering against interruptions in the
flow, e.g., power outages; but, overall, it is one of the most productive
techniques.
Though it is a simple hydroponic system, for NFT to function efficiently
and to get better results, one should need to carefully monitor the following:
13.2 Environmental Biotechnology

• Type of channels: There is a variety of channel available on the market,

and to select an appropriate one you need to consider factors such as
cost, availability on the market and requirements of your hydroponic
plants. They are available with pre-punched holes and removable lids
for placing plant seedlings and cleaning the system and the length
should be around 10–15 m. Ensure, that it should be flat and not curved
to maintain a considerable depth of liquid at the centre of a channel. It
is also important to have correct channel slope to get constant flow of
nutrients.
• Aeration: In NFT, since plant roots are submerged in water, it leads
to oxygen deficiency, at times. To eliminate this problem, you need to
install small compact air pumps or air stones to supply oxygen.
• Pathogens and Pest infestation: Moulds and pathogens are prone to
grow in moist environment, so a regular check on the condition of your
plants will help you to spot the early signs of pests and diseases. To
prevent your plants from Pythium infection, it is better to add H2O2 to
your solution.
• Nutrient check: One needs to make sure that one’s plants receive
balanced nutrients comprising all the essential micro- and
macronutrients that are available during the different growth phases
of one’s hydroponic plants. Inadequate nutrient supply can affect the
overall growth of plants. If your crops grow faster than normal, it can
be a sign of nitrogen depletion in the nutrient solution.
• Pump maintenance: One can come across pump failures at some point
of time as the submerged water pumps are continuously working.
However, these pumps are small and inexpensive and can be easily
replaced. You also need to clean your system on a regular basis to
ensure smooth flow of nutrients, without which, the crops might suffer.

13.1  PLANT DISEASES

Plant disease is the abnormal growth and development of a plant. Growth
and development of the plant does not live up to the normal expectations. A
diseased plant is incapable of carrying out its normal physiological functions
to the best of its genetic potential.
Many different living and non-living entities can have a negative effect on
plants.
A. Infectious Diseases (caused by biotic organisms):
(a) Fungi
(b) Prokaryotes:
( ) Bacteria
(ii) Mycoplasmas
Nutrient Film Culture Techniques 13.3

(c) Viruses and viroids

(d) Nematodes
(e) Parasitic Higher Plants
B. Non-Infectious (caused by abiotic factors)
(a) Temperature extremes
(b) Moisture extremes
(c) Light extremes
(d) Nutrient extremes
(e) Soil acidity or alkalinity (salt problems)
(f) Pesticide toxicity
(g) Air conditions: pollution, strong winds, etc.
(h) Improper cultural practices
2. A disease episode requires the interaction of three components, which
we call “The Disease Triangle”.
A. The host must be susceptible to the disease, at the proper age and
physiological state, for infection and development of disease to occur.
Healthy, strong-growing, non-­stressed plants are less susceptible to
disease than plants, under stress.
B. The pathogen must be virulent (able to cause disease), not in a state of
dormancy and must be present at a certain minimum population level.
C. The environment must be conducive (favorable) for the development
of disease: temperature, moisture, nutrients, wind, etc. must all favor
the pathogen.
D. The degree to which these three components interact, relates to the
severity of the disease episode (e.g., if the host is highly susceptible,
the pathogen, highly virulent and the environment, highly conducive,
then, the disease will be very severe).
E. Successful disease-control depends on the integrated use of available
control methods.

Symptoms and Signs

1. Symptoms are the response of the plant to attack by a disease causing
agent.
A. Examples: leaf spots, wilting, stunting, chlorosis, necrosis, etc.
2. Signs are the visual presence of some structure formed by the pathogen
on the host.
A. Examples: mycelium, spores, fruiting bodies, bacterial ooze, etc.

Organisms Associated with Diseased Tissue

1. Primary organism: The organism is directly responsible for the disease.
13.4 Environmental Biotechnology

2. Secondary organism: The organism(s) is(are) taking advantage of

weakened tissue.
3. Disease complexes and organism succession.
A. Disease complex refers to the situation where the disease is caused
by more than one organism. Disease complexes are especially
common in turf.
B. Organism succession refers to the fact that plants are colonized over
time by many different organisms. For example, when plants are
healthy, they are colonized by non­-pathogenic symbionts. When
the plants become diseased, they are first colonized by primary
pathogens, then by secondary organisms, and eventually, by other
saprophytes. Saprophytic organisms are in association with healthy
plants. The primary disease-causing agent is only operating by
itself for a short period of time. Secondary organisms may be weak-
pathogens or pathogens. Weak pathogens are organisms that are not
aggressive and (typically) do not cause disease by themselves.
C. Disease complexes add to difficulty in disease management, as
identification and control of one organism, may accelerate activity
of another organism in the complex.
D. Organism succession makes primary pathogen identification
difficult. The diseased specimen must be examined relatively quickly
after disease symptoms begin; otherwise, secondary pathogens or
saprophytes are all that can be found. Accurate diagnosis of the causal
agent is required for effective use of chemical control measures. Use
of an inappropriate chemical will not only be ineffective against the
disease agent, but can also lead to additional disease problems by
killing beneficial microorganisms in the environment.

13.2  PHYTODIAGNOSTICS BASED ON IMMUNOLOGICAL AND

MOLECULAR TECHNIQUES
Accurate detection and identification of plant pathogens are fundamental
to plant pathogen diagnostics, and thus, plant disease management. The
lack of rapid, accurate and reliable means by which plant pathogens can
be detected and identified has been one of the main limitations in plant
disease management and has prompted the search for alternative diagnostic
techniques. The advent of enzyme-linked Immunosorbent assay (ELISA)
and polymerase chain reaction (PCR) has caused a shift towards the use of
molecular approaches in modern plant pathogen diagnostics. Nowadays,
many techniques have been developed for the detection and identification of
plant pathogens, each requiring its own protocol, equipment, and expertise. In
addition, some of these techniques permit reliable quantification of the target
pathogen as well, and supply the information required to estimate potential
risks regarding disease development, spread of the inoculum, and economic
Nutrient Film Culture Techniques 13.5

losses. The major challenge at the moment is the development of multiplex

assays that allow accurate detection and quantification of multiple pathogens
in a single assay. In this chapter, we discuss recent advances in molecular plant
pathogen diagnostics that are likely to impact future plant disease-controlling
and, preventing strategies.
Immunological or serological techniques: A first development towards
techniques for molecular pathogen detection was the advent of immunological/
serological or antibody-based detection methods almost 30 years ago. These
techniques were originally developed to detect viruses, as those cannot be
cultured in vitro.
• Immunological or Serological techniques are based on the binding
between diagnostic antibodies and specific antigenic determinants of
the target pathogen.
• Several serological plant pathogen detection methods have been
described, of which, the enzyme-linked immunosorbent assay (ELISA)
is, by far, the most common technique.
• Although, different types of ELISA have been developed, all involve
an enzyme-mediated color change reaction to detect and often, also
quantify antibody binding as a measure for pathogen presence.
• Since its introduction in the late 1970s, ELISA assays have been
routinely used for virus and bacteria detection because of their high-
throughput capacity, the rapid, relatively cheap and simple nature, and
the possibility to quantify the target pathogen.
• A major limitation for the development of serological methods is the
labor-intensive procedure to obtain reliable assays, often due to the
difficulty to generate selective antibodies.
• Although polyclonal antibodies, which recognize multiple epitopes
of the pathogen, have been used successfully for detecting many
viruses, they do not always have the desired degree of specificity and,
importantly, the specificity may vary with each newly-produced batch.
• The accuracy of detection is often improved by using either monoclonal
or recombinant antibodies. Both of these allow the selection of specific
target epitopes to avoid “false positives”.
• However, developing antibodies with the required degree of specificity
is difficult for complex organisms such as bacteria and fungi. In those
cases, it is often hard to find reliable species-specific epitopes that are
ubiquitously shared within a species but not with other species.
Therefore, most antibody-based assays currently available are for the
detection of relatively unsophisticated organisms such as plant viruses, while
those available for the detection of fungi and bacteria are less common.
13.6 Environmental Biotechnology

13.3  MOLECULAR OR NUCLEIC ACID-BASED TECHNIQUES

Before the possibility to amplify nucleic acid sequences existed, the sensitivity
of detection based on those sequences totally relied on the method to translate
their presence into a detectable signal. Since the introduction of amplification
methods for nucleic acids, in particular—the polymerase chain reaction (PCR),
nucleic acid-based methods are increasingly developed for the detection and
identification of plant pathogens. This trend is enhanced by the growing
availability of pathogen sequence data in public databases like GenBank
(http://www.ncbi.nlm.nih.gov/Genbank/) and COGEME (http://www.cogeme.
man.ac.uk/).
A crucial step in the development of nucleic acid-based diagnostic assays
is the selection of sequences that can be employed for pathogen identification.
In general, there are two main approaches that can be used to select target
sequences. The first, and most widespread strategy, involves the use of
ubiquitously conserved genes. The second strategy involves the screening of
random parts of the genome in order to find sequences, harboring the desired
selectivity.
Currently, the nuclear ribosomal DNA (rDNA) operon is the most
commonly used target for bacteria as well as for fungi for a number of reasons.
First, it has been found that this gene provides a powerful means for analyzing
phylogenetic relationships over a wide range of taxonomic levels including,
for example, genus, species, and even below. Apart from this potential, the
large amount of ribosomal sequences in public databases allows to determine
genomic regions that can be used to design selective primers or probes. This is
facilitated even more by the structural nature of this type of gene since it contains
alternating regions with high and low degrees of conservation. This allows to
design primers on sequences that are conserved between species which span
variable domains that can be used for species identification. In addition, the
multiple copies of the gene present in each cell permit a very sensitive detection.
Although rDNA is the main target of many nucleic acid-based analyses,
other targets for detecting fungi include b-tubuline, actin, elongation factor
1 alpha, and mating-type genes.
However, if these genes do not display the desired degree of selectivity, other
regions of the genome need to be assessed. The screening of arbitrary regions
in the genome to find sequences with the required selectivity can be achieved
by several techniques, including RAPD (random amplified polymorphic DNA)
and AFLP (amplified fragment length polymorphism) technology. Diagnostic
markers identified with these approaches can be sequenced and are used
to design specific SCAR (sequence characterized amplified region) primers.
Nevertheless, as these sequences can be derived from anywhere in the genome,
there often is few sequence data available for comparison to multiple other
organisms. Therefore, extensive screening is required to validate the specificity
of the marker.
Nutrient Film Culture Techniques 13.7

Two specific problems can hamper detection of plant pathogens based on

nucleic acid-based techniques because they complicate the identification of
reliable markers.
• First, misclassification of strains is a regularly-occurring phenomenon
in fungal taxonomy. Historically, taxonomists have grouped closely-
related fungi in a single genus or species largely based on similarities of
structural and morphological characteristics. However, especially large
fungal genera and genera containing asexual fungal species are known
to often contain unrelated species. As a result, taxonomic relationships
are not always reflected by the evolutional relationships that are often
revealed using nucleic acid-based techniques. Therefore, finding
selective sequences shared by all members of a species is complicated
for certain species.
• A second problem for molecular detection of certain plant pathogens is
the existence of fungal species that harbor pathogenic as well as non-
pathogenic or, even beneficial strains. This is a known phenomenon for
complex species such as Fusarium oxysporum and Rhizoctonia solani. In
those cases, target sequences should preferably be directly associated
with virulence traits which severely limits the number of sequences.

Nucleic acid-based techniques can be divided into DNA- and

RNA-based technologies
DNA-based techniques: DNA is a highly attractive target for the detection of
plant pathogens in biological samples because it is easier to handle and more
resistant to degradation than RNA. With improved extraction methods (34)
and commercially available extraction kits, highly purified DNA can rather
easily be obtained from complex environmental samples.

13.3.1 Polymerase Chain Reaction (PCR)

Using PCR, millions of copies of specific DNA sequences may be rapidly
synthesized in a thermocyclic process that consists of repetitive cycles of DNA
denaturation, primer annealing, and extension using a thermostable DNA
polymerase. If a DNA sequence unique to a particular organism is determined,
specific PCR primers or probes can be designed that enable determination of
the presence or absence of that sequence, and thus, of the specific organism.
The presence of amplified DNA is traditionally detected by gel electrophoresis,
but alternative detection formats including, colorimetric and fluorimetric
assays, do exist. PCR-based detection methods are very sensitive and can
detect minute quantities of pathogen DNA—even the amount derived from
a single fungal spore. To improve specificity, but sometimes also sensitivity,
PCR products may also be detected using a probe. Other approaches to
increase sensitivity and specificity include the use of immunocapture PCR
(IC-PCR) or nested PCR. IC-PCR utilizes antibodies to isolate the pathogen
13.8 Environmental Biotechnology

from a sample prior to PCR amplification, and has mainly been used to detect
plant pathogenic viruses. Nested PCR involves two consecutive PCR reactions,
the second one using primers that share a sequence within the target DNA
fragment that is amplified in the first reaction. As a result, a specific reaction
products that are generated in the first PCR reaction should not be amplified
in the second reaction.
Many reports describe specific applications of PCR technology in plant
pathology. In addition, increasingly, companies providing diagnostic services
are using PCR to routinely detect and identify plant pathogens.
Quantification of the amount of pathogen DNA, supplying the information
required for disease management decisions and for monitoring the effects of
these decisions has also been pursued using PCR-based methods. Although,
it is relatively easy to quantify the amount of amplicon generated, it is more
difficult to relate this quantity to the initial amount of target DNA present
in a sample. This is caused by the typical non-linear kinetics of template
amplification. Nevertheless, in theory, the exponential nature of PCR allows the
initial amount of DNA to be calculated from the amount of product at any time
point in the reaction. In practice, however, as the reaction proceeds, reagents
become limiting and a plateau level is reached, where the amount of product
is no longer proportional to the original amount of template. Target DNA can
be quantified using competitive PCR, which is based on the co-amplification
of target DNA and a competitor DNA, both with the same primer pair. The
amount of target DNA is subsequently determined on agarose gel by comparing
the relative amounts of target and competitor PCR product. This method has
been used to successfully quantify, for instance, Verticillium wilt pathogens.

13.3.2 Real-time PCR

Especially with respect to quantification purposes, real-time PCR is a
powerful development. This technology differs from conventional PCR by
monitoring products on-line while they accumulate at each reaction cycle in
a closed tube format, without the need of post-reaction processing such as
gel electrophoresis. As a consequence, real-time PCR is generally faster than
conventional PCR, enabling high throughput analyses. In addition, the risk
of post-PCR carry-over contamination of amplicons is eliminated. Real-time
PCR allows accurate template quantification during the exponential phase of
the reaction, before reaction components become limiting. The initial amount
of target DNA can be related to a threshold cycle, defined as the cycle number
at which fluorescence increases above the background level. Target DNA is
quantified using a calibration curve that relates threshold cycles to a specific
amount of template DNA. Typically, DNA amplification is monitored each
cycle based on the emission of fluorescence.
Amplicons can be detected using several chemistries, which can be divided
into amplicon non-specific or amplicon specific methods, using DNA-binding
Nutrient Film Culture Techniques 13.9

Main chemistries for amplicon detection in real-time PCR application

(A) As a DNA-intercalating dye such as SYBR Green® (S) binds to dsDNA, fluorescence
is recorded. (B) Taqman® probes, (C) Molecular Beacons® as well as (D) Scorpion®
primers use a strategy to extinguish fluorescence at certain conditions using a reporter
fluorophore (R) and a fluorogenic quencher (Q). Upon physical separation of both molecules
fluorescence is emitted. (E) The use of FRET probes involves the hybridization of two labeled
oligonucleotides in close proximity. When both probes bind to the target fragment, energy
is transferred from the donor (d) to the acceptor (a) molecule resulting in fluorescence.
dyes and sequence-specific probes, respectively. The use of DNA-intercalating
dyes such as SYBR Green® is a more straightforward and less expensive
approach compared to using probes, but it is also less specific since the dye
binds to all double-stranded DNA (dsDNA) present in the sample. In addition,
the interpretation of results can be disturbed by formation of primer-dimers
or a specific reaction products. It is therefore, crucial to use highly specific
primers and to determine optimal reaction conditions. In addition, melt curve
analysis, at the end of the PCR reaction, allows evaluating the accuracy of
the amplification reaction. In contrast to amplicon non-specific chemistries,
probe-based assays often offer the advantages of increased specificity,
certainly in combination with specific primers, and reducing signals due to
mispriming or primer-dimer formation. Most applications, to date, have used
TaqMan® probes. These probes are single-stranded, short oligonucleotides
which are labeled with a fluorophore and a fluorogenic quencher. Because
of the close proximity of both groups, the fluorescent signal is quenched.
During the annealing phase of each PCR cycle, the probe hybridizes to a
specific region within the target- amplified fragment. The probe is degraded
by 5’ exonuclease activity when the DNA polymerase extends the primer.
Consequently, the fluorophore and the quencher are released independently,
resulting in a fluorescent signal. Variants of this quenching chemistry include,
hairpin-shaped Molecular Beacons® and Scorpion® primers. Whereas,
the loop portion of these molecules contains the probe sequence, the stem,
which is formed by complementary sequences added to both ends of the
probe, holds a fluorophore and a quencher in close proximity. In addition,
Scorpion primers couple the stem-loop-based probe to a PCR primer. Specific
binding of the probe to its target opens the structure, producing a fluorescent
13.10 Environmental Biotechnology

signal. A completely different detection chemistry comprises the use of

fluorescent resonance energy transfer (FRET) probes. With this technology,
two oligonucleotide probes are designed such that they hybridize in very close
proximity to the amplified fragment. Whereas one of the probes contains a
donor fluorophore at its 3’ end, the other probe is labeled at its 5’ end with
an acceptor fluorophore. When both probes properly hybridize to the target
fragment, the energy excited by the donor is transferred to the acceptor
resulting in a fluorescent signal.
Closely related microbial species often, only differ in a single or a few
bases of ubiquitously conserved genes, for instance, the rDNA. The high
degree of specificity of real-time PCR technology allows, independent of the
detection chemistry, the detection of single-nucleotide polymorphisms (SNPs),
meaning that specificity is determined by a single base pair. Therefore, this
technology offers many opportunities in plant pathogen diagnostics. In recent
years, real-time PCR assays have been developed for accurate detection and/or
quantification of specific plant pathogens as well as for monitoring pathogen
infections. Although, not yet used routinely in phytodiagnostics, real-time PCR
has much potential for future applications.
Ligase Chain Reaction (LCR): The ligase chain reaction (LCR) uses two
complementary pairs of oligonucleotides that hybridize in close proximity
on the target fragment. Only when the oligonucleotides correctly hybridize
to the target sequence, the remaining nick between the oligonucleotides is
ligated by a DNA ligase and, a fragment equating to the total sequence of
both oligonucleotides, is generated. Similar as in a PCR reaction, the products
of one reaction serve as templates for subsequent cycles, resulting in an

General principle of the Ligase Chain Reaction (LCR)

Two complementary pairs of adjacent oligonucleotides (pla and plb, p2a, and p2v)
bind to the target sequence. Only if the oligonucleotides bind in close proximity,
DNA ligase seals the nicks and the cycle can be repeated.
Nutrient Film Culture Techniques 13.11

exponential amplification of the desired fragment. To further enhance

sensitivity and, sometimes, also specificity, LCR can also be used following
a PCR pre-amplification. Detection of LCR products can be performed by
polyacrylamide gel electrophoresis. With this technology, SNPs can easily be
differentiated. Although LRC is regularly applied in human disease detection,
it has rarely been reported for detection of plant pathogens.

13.3.3 Rolling Circle Amplification (RCA)

Originally, padlock probes were developed as a new approach for molecular
analysis of complex DNA samples, including analysis of alleles and point
mutations in the human genome. A padlock probe consists of a single-
stranded linear oligonucleotide of about 70–100 nt in length with a target-
complementary region at both ends and a linker segment, in between. The
5’ and 3’ end regions are designed to hybridize next to each other on a target
strand. When properly hybridized to the target sequence, the molecule can be
circularized upon ligation. Because of the need for precise base pairing at the
junction where ligation should take place and the simultaneous hybridization
of two different fragments, padlock probes ensure high specificity.
For sensitive pathogen detection, however, signal amplification is a
prerequisite. One approach for the amplification of padlock probes is a PCR
reaction using primers that hybridize to sequences within the spacer region
of the probe. Another method to amplify padlock probes is rolling circle
amplification (RCA), analogous to replication mechanisms of several viruses
with circular genomes. Two types of RCA have been described: linear and
hyper-ranched RCA. In the first procedure, a primer hybridized at some point
on the circular DNA is extended continuously using a DNA polymerase that
lacks exonuclease activity. As a result, a long linear fragment composed of many
tandem repeats of the complement to the circularized molecule is generated.
In addition, hyperbranched (or cascade) RCA uses a second primer that binds
to each generated RCA repeat. During elongation, the Exonuclease-deficient
DNA polymerase displaces the polymerized strand in front of it. Next, the
displaced strands, which are tandem, repeat with identical sequences to the
original padlock probe, serve again as template for the first primer, resulting
in a cascade of DNA amplification.
As for conventional PCR, detection of amplified products can be achieved
using gel electrophoresis or labeled probes enabling real-time monitoring of the
amplification process. However, although RCA is considered to be one of the
most sensitive amplification methods, the procedure is fairly complicated and
relatively expensive. Therefore, it is important to realize what level of sensitivity
is required for a method to be used for plant pathogen diagnostics. The most
sensitive technique will probably not be required when assessing whether
measures have to be taken in a certain crop to prevent yield losses. Often, such
a decision requires a threshold level to be crossed which can be detected by
many less sensitive techniques. In contrast, sensitivity is very important when
it comes to zero tolerance of quarantine diseases.
13.12 Environmental Biotechnology

General principle of hyperbranched rolling Circle Amplification (RCA)

The 5′ and 3′ ends of a linear padlock probe are designed to hybridize next
to each other on a target strand. When properly hybridized, the molecule is
circularized by ligation. Synthesis of the complementary strand of the circularized
padlock probe is initiated by primer pl. As a strand of linear tandem repeats is
generated, a second primer (p2) hybridizes to each newly generated repeat.
During elongation, the exomclease-deficient DNA polymerase displaces the
polymerized strand in front of it which, in turn, serves as template for the first primer.
All DNA-based methods have in common that they might detect DNA
from dead or non-active organisms as well. Therefore, detection of non-viable
propagules cannot be ruled out. However, DNA from dead cells in soils should
be degraded fairly rapid due to the high microbial activity, suggesting that
interference by DNA from dead cells might be negligible. Nevertheless, the
rate of DNA degradation depends on soil type and moisture content. As DNA
degradation occurs slower in desiccated soils, accurate diagnosis of samples
from dry fields may be biased by detection of dead organisms. However,
since long-lasting soil desiccation generally does not occur in horticultural or
agricultural practice, this should not be a major concern.
To exclude detection of dead organisms, a culturing step on or in a suitable
medium, or even in plants, prior to PCR amplification, could be included. This
technique is referred to as BIO-PCR. Because, only active propagules will be
able to grow, this technique enables selection of viable organisms. In addition,
PCR sensitivity is increased by the culturing step. However, disadvantages are
its labor-intensive and time-consuming nature, and the inability to detect non-
culturable organisms. As an alternative, attempts are made to use DNA-binding
dyes such as ethidium monoazide (EMA) to distinguish viable from non-viable
organisms. Since the membranes of dead cells quickly disintegrate, EMA is able
to selectively enter dead cells where it covalently binds to dsDNA upon light-
exposure. EMA-bound DNA is blocked for PCR amplification, thus enabling
the selective amplification of targets from living organisms. Another alternative
Nutrient Film Culture Techniques 13.13

to prevent detection of dead organisms is the use of RNA as target molecule.

Since RNA is less stable than DNA, RNA will be degraded much faster in dead
organisms. In addition, the stability of DNA can be the cause of persisting
contaminations in diagnosis laboratories where large numbers of samples,
which often contain the same pathogen, are processed. In addition, messenger
RNA (mRNA) is only produced in metabolically-active cells, making mRNA
attractive to selectively detect living microorganisms. However, extraction of
RNA from environmental samples should be a careful procedure.
RNA-based techniques: Whereas DNA-based detection techniques are
increasingly being used to detect and identify pathogenic fungi, bacteria as well
as nematodes, RNA-based techniques are mainly used to detect plant viruses
since most of them have RNA genomes. However, since mRNA may more
accurately reflect metabolicallyactive pathogen material, these techniques can
also be used to selectively detect viable pathogen propagules.
Reverse Transcriptase PCR (RT-PCR): Since PCR can only amplify double-
stranded templates such as DNA, RNA should be converted to DNA (called
complementary DNA or cDNA) prior to use in a PCR-based assay. Typically,
such reverse transcriptase PCR (RT-PCR) consists of an annealing step for
one primer and an extension step to synthesize the complementary or second
strand, followed by a (real-time) PCR reaction. In plant pathology, RT-PCR is
a common strategy to detect plant viruses .
Nucleic Acid Sequence-Based Amplification (NASBA), Transcription
Mediated Amplification (TMA), or Self-Sustained Sequence Replication
(3SR): Nucleic acid sequence-based amplification (NASBA), also known
as transcription mediated amplification (TMA) or self-sustained sequence
replication (3SR), has been used for the direct amplification of RNA. In contrast
to conventional PCR, amplification is carried out in an isothermal process
(avoiding the need for a thermocycler) using three different enzymes, including
a reverse transcriptase, RNase H, and T7 RNA polymerase. Initially, a primer
containing an RNA polymerase promoter sequence at its 5’ end and a target-
specific sequence at its 3’ end is extended by reverse transcription to produce a
cDNA strand. The resulting hybrid is a substrate for RNase H, which degrades
the original RNA strand. Subsequently, a second DNA strand is produced from
a primer designed to bind to the 3’ end of the cDNA, resulting in a dsDNA
molecule that contains the sequence information of the original RNA and
the promoter sequence of the T7 RNA polymerase. In a next step, T7 RNA
polymerase initiates DNA transcription leading to the production of a large
number of antisense RNA molecules. Each antisense RNA molecule is used to
generate new dsDNA molecules based on the same principle, and initiates a
new round of replication.
The amplification products can be visualized using a specific labeled
probe which hybridizes to the RNA amplicons. In addition, amplicons can be
13.14 Environmental Biotechnology

monitored in real-time using a specific detection probe such as a Molecular

Beacon®. This procedure is referred to as AmpliDet RNA and combines the
advantages of both NASBA and real-time PCR.

General principle of Nucleic Acid Sequence-Based Amplification (NASBA)

Upon binding of primer pl that is tailed with a T7 RNA polymerase promoter, reverse
transcriptase (RT) generates a cDNA strand. The result hybrid is a substrate for , which
degrades the original RNA strand. Subsequently, reverse transcriptase generates a
complementary strand to the first cDNA strand using a second primer (p2), resulting in
double-stranded DNA (dsDNAW) with a T7 RNA polymerase promoter. This is a template
for T7 RNA polymerase (T7 pol) that transcribes a large number of antisense RNA
molecules (asRNA) which, in turn, are converted into dsDNA for a next amplification cycle.

13.4 ANTAGONISTIC FUNGI

Fungal antagonists are those fungus species, which act against the harmful
microorganisms and their products.
Trichoderma spp.: Trichoderma viride, T. harzianum and T. virens are
most extensively used fungal antagonists. They are mass-produced using
fermentation technology. They are used for the management of soil-borne
pathogens by seed treatment and soil application.
Aspergillus niger: Aspergillus niger, an ubiquitous fungus is present in all
types of soil with no specific moisture and pH requirements.
It is formulated as wettable powder. It controls a number of devastating
soil-borne pathogens, e.g. Fusarium oxysporum, Macrophomina phaseolina, Pythium
Nutrient Film Culture Techniques 13.15

aphanidermatum, Rhizoctonia solani, and Sclerotinia sclerotiorum, belonging to

different classes of fungi by a single application under different agro-climatic
conditions in different crops
Myrothecium verrucaria: It is a deuteromycete fungus, which produces
cuticle degrading enzymes as well as mycolytic enzymes. It is formulated
either in the form of conidia or unicellular yeast like cells. It is used to control
root-infecting fungus, Sclerotium rolfsii on groundnut.
Trichoderma spp. has been widely used as antagonistic fungal agents against
several pests as well as plant growth enhancers. Faster metabolic rates, anti-
microbial metabolites, and physiological conformation are key factors which
chiefly contribute to antagonism of these fungi. Mycoparasitism, spatial and
nutrient competition, antibiosis by enzymes and secondary metabolites, and
induction of plant defence system are typical biocontrol actions of these fungi.
On the other hand, Trichoderma spp. have also been used in a wide range of
commercial enzyme productions, namely, cellulases, hemicellulases, proteases,
and 1,3-glucanase. Information on the classification of the genus, Trichoderma,
mechanisms of antagonism and role in plant growth promotion has been well
documented.
Trichoderma viride is an antagonistic fungus which prevents the crops from
diseases such as Root rots, Wilts, brown rot, damping off, Charcoal rot and
other soilborne diseases in crops. It is suitable for Sugarcane, Pulses, Oilseeds,
Cotton, Vegetables, Banana, Coconut, Oil palm, Chilies, Lime, Coffee & Tea,
Areca nut & Rubber, Flower crops and Spices.

Mode of disease control

• Trichoderma fungus is well known for disease and nematode control of
crop plants.
• Trichoderma controls diseases by the production of several lytic enzymes
and antibiotics-controlling disease-causing microbes.
• Trichoderma controls nematode infestation by feeding on infective
nematodes.
• These fungi compete with other disease-causing microbes for nutrients
and space.
• These fungi increase the rate of plant growth and development, by
developing more robust roots. These deep roots cause crops, such as
corn, and ornamental plants, to become more resistant to drought.
• Trichoderma also solubilizes phosphates and micronutrients.
• Trichoderma harzianum produces enzymes such as protease which
controls Botrytis cinerea.
• Trichoderma are also helpful in solubilization and sequestration of
inorganic nutrients.
13.16 Environmental Biotechnology

• Induces defense responses in crop plants (Induced resistance)

• Inactivation of the pathogen’s enzymes

13.5 ANTAGONISTIC BACTERIA

Bacterial antagonists are those bacterial species, which act against the harmful
microorganisms and their products. The best example for antagonistic bacteria
is Pseudomonas fluorescens.
Pseudomonas fluorescens: Antagonistic Pseudomonas fluorescens is a bacteria
with high antibiosis potentia. It enters the plant vascular system, and reaches
the various parts of the plant system and acts as systemic bio-control agent
against various fungal and bacterial diseases such as Pythium spp., Phytophtora
spp., Rhizoctonia solani, Fusarium spp., Botrytis cinerea, Sclerotium spp., Sclerotinia
sp. and Ustilogo spp. It is suitable for plants like Tomatoes, Chilly, Cut flowers,
Orchards, Vineyards Ornamentals, Potato, Cucumbers and Eggplant.

Mode of disease control

Pseudomonas produces secondary metabolites, many extracellular hydrolytic
enzymes, which suppress plant disease.
• Antibiotics such as pyrrolnitrin, pyoluteorin, and 2,4-diacetyl-
phloroglucinol inhibit phytopathogen growth. Diseases from
Rhizoctonia solani and Pythium ultimum that affect cotton plants are
inhibited by this strain.
• Pseudomonas fluorescens produces hydrogen cyanide and the
siderophores pyocheline and pyoverdine, which it uses to outcompete
with many pathogenic bacteria and suppress pathogens in the
rhizosphere.
• Pseudomonas fluorescens produce exopolysaccharides which are used
for protection against bacteriophages or dehydration as well as for
defense against the host immune system.
• Pseudomonas fluorescens possess viscosin which is a peptidolipid that
enhances antivirality.

13.6 ANTIFEEDANTS
The possibility of using non-toxic deterrents and repellents as crop protectants
is intuitively attractive. The concept of using insect antifeedants (=feeding
deterrents) gained strength in the 1970s and 1980s with the demonstration of
the potent feeding deterrent effect of azadirachtin and neem seed extracts to a
large number of pest species.
• Indeed, considerable literature, scientific and otherwise, touts neem as
a successful demonstration of the antifeedant concept.
Nutrient Film Culture Techniques 13.17

• In reality, it is the physiological actions of azadirachtin that appear most

reliably linked to field efficacy of neem insecticides; although purely
behavioral effects cannot be ruled out, there is hardly any irrefutable
evidence or documentation of field efficacy based on the antifeedant
effects of neem alone.
• As an academic exercise, the discovery and demonstration of plant
natural products as insect antifeedants has been unquestionably
successful. In addition to the neem triterpenoids, extensive work has
been performed on clerodane diterpenes from the Lamiaceae and
sesquiterpene lactones from the Asteraceae.
• On the other hand, not a single crop protection product, based
unequivocally on feeding or oviposition deterrence, has been
commercialized.
Two main problems face the use of antifeedants in agriculture.
• The first is interspecific variation in response—even closely related
species can differ dramatically in behavioral responses to a substance—
limiting the range of pests affected by a particular antifeedant. Some
substances that deter feeding by one pest can even serve as attractants
or stimulants for other pests.
• The second is the behavioral plasticity in insects—pests can rapidly
habituate to feeding deterrents, rendering them ineffective in a matter
of hours. This has been recently demonstrated not only for pure
substances like azadirachtin, but also for complex mixtures (plant
extracts). Whereas a highly mobile (flying) insect may leave a plant
upon first encountering an antifeedant, a less mobile one (larva) may
remain on the plant long enough for the deterrent response to wane.
Such behavioral changes are important in light of the observation
that some plant substances are initially feeding deterrents but lack
toxicity, if ingested. Azadirachtin is clearly an exception to this rule, as
ingestion leads to deleterious physiological consequences, but many
other compounds or extracts with demonstrated antifeedant effects
lack toxicity when administered topically or via injection.

13.7 INSECTICIDAL ACTIVITIES OF THE COMPOUNDS OF

BOTANICS (BOTANICAL INSECTICDES)
Botanical insecticides are naturally-occurring secondary metabolites
synthesised by plants species, which act on the insect growth and survival.
Natural pesticidal products are available as an alternative to synthetic chemical
formulations, but they are not necessarily less toxic to humans.
Azadirachtin: Azadirachtin is just one of more than 70 limonoids produced
by the neem tree. It is a powerful insect antifeedent and growth regulators.
It is structurally similar to insect hormones called ‘ecdysones,’ which control
13.18 Environmental Biotechnology

the process of metamorphosis as the insects pass from larva to pupa to adult
stage. Azadirachtin occurs in all parts of the neem tree, but is concentrated in
the kernel.
Azadirachtin is used to control whiteflies, aphids, thrips, fungus gnats,
caterpillars, beetles, mushroom flies, mealybugs, leafminers, gypsy moths and
others.

13.8 PREDATORS
An entomophagous species that generally consumes more than one prey
individual to complete its development is called a predator.
Cryptolaemus montrouzieri: C. montrouzieri is a voracious feeder of mealybugs
in both larval and adult stage. It is small (about 3–4 mm), dark brown lady
beetle with orange head, larvae have woolly appendages of wax, which makes
them resemble mealy bugs.
It is mass produced on the mealy bugs, Ferrisia virgata or Planococcus sp.
and multiplied on sprouted potatoes or pumpkin fruits.
C. montrouzieri larvae and adults attack citrus and closely related mealy bugs
and some soft scales. Adults predators are released @ 5-10 beetles/vine/tree.
Chrysoperla carnea: They are cosmopolitan in distribution. Adult chrysopids
are medium-sized (7–15 mm), yellowish green to grey in colour with red,
yellow or brown markings. Most of the adults depend upon pollen, nectar and
honeydew and the larval stages are predatory in nature.
These are mass-produced using UV sterilized eggs of Corcyra cephalonica.
The first instar larvae are released in the field @ 1.25 lakh/ha to control sucking
pests and eggs and first instar larvae of lepidopteran insects.
Weed Feeder: Those organisms, which feed on the weeds depleting or
eliminating them, are called Weed Feeder.
Zygogramma bicolorata: This is Chrysomelid beetle used as a biocontrol agent
for the control of the carrot weed, Parthenium hysterophorus. Larvae and adult
beetles feed on the leaves of this weed. The life cycle of this beetle is completed
in 20–43 days. The insect is capable of remaining under soil in diapause from
November to June. Once rain starts, diapause is broken and the beetles come
out of soil and the new cycle is started
 CHAPTER

14 Biotechnology: Industrial Sustainability

uman activities – industrialization, urbanization, agriculture, fishing and

H
aquaculture, forestry and silviculture as well as petroleum and mineral
extraction – have profound impacts on the world’s environment, as well as, on
the quality of life. As a result, there is a growing appreciation that nationally,
regionally and globally, the management and utilization of natural resources
need to be improved and that the amounts of waste and pollution generated
by human activity need to be reduced on a large scale. This will require a
reduction and, if possible, elimination of unsustainable patterns of production
and consumption. As a result, emphasis is growing on industrial sustainability
because this is increasingly recognized as a key means of bringing about such
reduction of environmental impacts and improving quality of life.

14.1  INDUSTRIAL SUSTAINABILITY

The World Commission on Environment and Development has provided
insight on sustainable patterns of production and consumption through its
description of sustainable development:
“Sustainable Development: Strategies and actions that have the objective of
meeting the needs and aspirations of the present without compromising the ability
to meet those of the future”.
This definition of sustainable development can be adapted to provide a
conceptual definition of industrial sustainability:
“Industry is sustainable when it produces goods and services in such a manner as
to meet the needs and aspirations of the present without compromising the ability
of future generations to meet their own needs”.
A closer look shows that industry is sustainable when it is:
• Economically viable (uses natural, financial and human capital to
create value, wealth and profits).
14.2 Environmental Biotechnology

• environmentally compatible (uses cleaner, more eco-efficient products

and processes to prevent pollution, depletion of natural resources as
well as loss of biodiversity and wildlife habitat).
• Socially responsible (behaves in an ethical manner and manages the
various impacts of its production through initiatives such as Responsible
Care).
This “triple bottom line” for industry is captured in a quote from the Shell
Report 2000: “Excellent environmental performance is meaningless if no wealth
is created. Wealth in a destroyed environment is equally senseless. No matter how
wealthy, a society fundamentally lacking in social equity cannot be sustained.”
Moving Toward More Sustainable Industries: Developing sustainable
industries implies constantly assessing and improving industrial performance.
The aim is to uncouple economic growth from environmental degradation
so that industry will be more profitable and, simultaneously, environmental
quality will also improve.
Economic growth provides jobs and income, goods and services and
opportunities to improve the standard of living for an increasing world
population. Environmental protection recognizes the intrinsic value of
nature and living things. It also recognizes the potential of organisms living
in ecosystems to provide insights and the means for developing sustainable
industrial products, processes and production systems. Sustainable
industrial development can be achieved if the three requirements (economic,
environmental and social) outlined above are applied to guide the pathway
and shape the process by which industry and the economy grow.
At a very basic level, sustainable industrial development means doing
more with less – increasing eco-efficiency, that is, decreasing the level of
pollution and at the same time, the amount of energy, material and other inputs
required to produce a given product or service. A major way of accomplishing
this is through cleaner production. Cleaner production involves a paradigm
shift where innovation is used to develop:
• processes and production systems which:
— save costs and are more profitable because they are less wasteful
of materials and energy (resulting in less emissions of greenhouse
gases, persistent organic chemicals and other pollutants).
— enable greater and more efficient utilization of renewable resources
(energy, chemicals and materials), lessening our dependence on non-
renewable resources such as petroleum and reducing associated
greenhouse gas emissions.
• products which are:
— Better performing, more durable and don’t persist after their useful
life.
— Less toxic, more easily recyclable and more biodegradable than their
conventional counterparts.
Biotechnology: Industrial Sustainability 14.3

— Derived as much as possible from renewable resources and

contribute minimally to net greenhouse gas emissions.

14.2 TECHNOLOGY, CLEANER PRODUCTION AND

SUSTAINABILITY
Technological innovation is a key means of achieving cleaner production and
sustainable industrial growth. However, “cleaner” should not be confused
with “sustainable”. Sustainable means, clean enough to meet the needs of
the present without compromising the ability of future generations to meet
their own needs. Making the distinction between “cleaner” and “sustainable”
requires the tools to assess and compare the performance of different
technologies used for industrial production.
Companies that have to take decisions on implementing and improving
production processes can develop these processes on the basis of best available
technologies. Some sources of information already exist on best available
technology, for example, the European Integrated Pollution Prevention and
Control Bureau (http://eippcb.jrc.es) or the UNEP International Cleaner
Production Information Clearinghouse (www.emcentre.com/unepweb/).
Scientifically validated criteria and methods for evaluating the long-
term sustainability of industrial production are still being developed
(see, for example, the Web site of the Canadian National Round Table on
the Environment and the Economy: www.nrtee-trnee.ca). Nevertheless,
it is possible to estimate what is sustainable (“clean enough”) from an
environmental perspective based on the present situation and some simple
assumptions. This helps answer the question:
“If one is to approach environmental sustainability while achieving sustained
economic growth, what should be the environmental performance targets for
technology at the R&D phase today compared to the performance of technology
which is currently the industry standard?”

Eco-efficiency of the economy to keep the environmental footprint constant

To answer this question, it is necessary to determine what environmental
performance will be required of technology in order to keep the environmental
14.4 Environmental Biotechnology

“footprint” (impact) of industrial production at a constant level. Experience

has shown that environmental footprint of industry is directly proportional
to the level of economic activity (that is, if production doubles, then the
environmental footprint doubles), other things being equal. So, as production
increases, so too, must the environmental performance, or “eco-efficiency”,
of technology used if concomitant increases in the environmental footprint of
industrial activities is to be avoided.
What this means is that new technologies, which bring improvements in
production, must also bring improvements in “eco-efficiency”. As is explained
more fully in Fig. 14.1, the lag between the R&D phase of new technologies
and the point at which these become industry standards, means that work
at the R&D stage today needs to target quite significant improvements in
environmental performance.
If the present environmental impact of existing industrial production is not
sustainable, then the environmental performance targets for new technology
to help address this will have to be raised even higher. The following
sections show that advances in technology, especially biotechnology, can help
deliver improvements in environmental performance beyond the factor of
3–4 identified in the example in Fig. 14.1.
The graph sets out a picture of a growing economy over time. The
curve represents two functions. First, it represents the rising environmental
footprint or impact from 4% economic growth without any changes in the
environmental performance of the technology used. And second, the same
curve delineates the factor of eco-efficiency gain required to deliver the same
growth with no impact on the environmental footprint.
So the graph in Fig. 14.1 shows that, in order to bring the environmental
impact back to its original level:
• technologies that are ready to be introduced into the market today
(it takes an average of 25 years for these to become average industry
practice), should have an environmental performance at least three
times better than the current industry average (that is, emissions only
33%, of present).
• technologies at the R&D stage today (it will take an average of 35
years for these to become average industry practice) should have an
environmental performance at least four times better than the current
industry average (that is, emissions only 25%, of present).
The assumptions built into Fig. 14.1 include:
• environmental impacts are directly proportional to level of economic
activity.
• economic growth (the rate of increase in production), for purposes of
this analysis, is set at 4% per year.
• improving the environmental performance (“eco-efficiency”) of
production technology decreases the environmental footprint for a
given level of economic activity.
Biotechnology: Industrial Sustainability 14.5

• in order to be adopted, a technology must provide a significant

net positive value in terms of its economic and/or environmental
performance.
• if newly developed technology is now beginning to be introduced into
industry, it will take an average of 25 years for it to become the average
performance of the industry as a whole.
• technologies at the R&D stage today will take an average of 10 years to
develop to the “market-ready” stage, that is, where it is attractive for
industry to begin adopting them.

14.3 LEARNING FROM NATURE: BIOMIMICRY AND

BIOTECHNOLOGY
It is difficult to achieve a four-fold improvement in environmental performance
through incremental improvements in conventional production technologies.
Improvements of this magnitude usually call for a paradigm shift.
For a growing number of companies, the inspiration for such a paradigm
shift is coming from the products and processes found in natural ecosystems
and the organisms that live in them. Biomimicry is the name coined for this
approach in which industrial production systems imitate nature. Industrial
biotechnology is that set of technologies that come from adapting and
modifying the biological organisms, processes, products, and systems found
in nature for the purpose of producing goods and services.
The organisms, processes, products and systems found in natural
ecosystems have evolved over millions of years to become highly efficient. For
example, all energy in natural ecosystems is renewable and is initially captured
from sunlight through photosynthesis. Also, all bio-organic chemicals and
materials are renewable, biodegradable and recycled. There is no such thing
as “waste” – the by-products of one organism are the nutrients for another.
Most, if not all, metabolic processes are catalyzed by enzymes and are highly
specific and efficient.
Biotechnology has evolved over the last 25–30 years into a set of powerful
tools for developing and optimizing the efficiency of bioprocesses and the
specific characteristics of bioproducts. This increase in efficiency and specificity
has great potential for moving industry along the path to sustainability.
Increased efficiency allows for greater use of renewable resources without
leading to their depletion, degradation of the environment and a negative
impact on quality of life. Biotechnology can become an important tool for
decoupling economic growth from degradation of the environment and
the quality of life. Biotechnology can also enable the design of processes
and products whose performance cannot be achieved using conventional
chemistry or petroleum as feedstock.
Here are some examples of some of the industrial efficiency tools now
coming from the application of biotechnology:
14.6 Environmental Biotechnology

• Enzymes extracted from naturally-occurring micro-organisms, plants

and animals can be used biologically to catalyze chemical reactions with
high efficiency and specificity. Compared to conventional chemical
processes, bio-catalytic processes usually consume less energy, produce
less waste and use less organic solvents (that, then require treatment
and disposal).
• By imitating natural selection and evolution, the performance of
naturally-occurring enzymes can be improved. Enzymes can rapidly
be ‘evolved’ (this technique is called “molecular evolution”) through
mutation or genetic engineering and selected using high-throughput
screening to catalyze specific chemical reactions and to optimize their
performance under certain conditions such as elevated temperature.
• The metabolic pathways of micro-organisms can also be modified by
genetic engineering. The aim is to turn each cell into a highly efficient
“mini reactor” that produces in one step, and at high yield, what would
take an organic chemist a number of steps with much lower yield (this
technique is called “metabolic engineering”).
• A further improvement on metabolic engineering involves engineering
the enzymes in the optimal configuration onto the cell membrane and,
when the cell is ruptured, the cell membrane becomes a bio-catalytic
surface that provides the high efficiency of metabolic engineering
without the energy penalty of keeping the organism alive.
• Plant biomass can be processed and converted by fermentation and
other processes into chemicals, fuels and materials that are renewable
and result in no net emissions of greenhouse gases. Also, these
biologically-derived products (“bio products”) are generally less toxic
and less persistent than their petrochemical counterparts.
• Groups of companies can mimic the co-operative action of organisms in
natural ecosystems by clustering around the processing of a feedstock
such as biomass, so that the by-product of one is the starting material
for another. Also, energy, such as waste heat, can be used efficiently.
This approach is called “industrial ecology”.
• The ability to “evolve” bioprocesses and bio production systems
allows for major improvements in both economic and environmental
performance. This permits a manufacturing facility to increase its
profitability and capacity while maintaining or, even reducing its
environmental footprint.

14.4 BIO-SAFETY
The micro-organisms used for industrial bio-processing or for production
of industrial enzymes are selected to avoid use of pathogenic organisms.
They are subject to stringent environmental regulations in Organization for
Economic Co-operation and Development (OECD) countries. Occupational
Biotechnology: Industrial Sustainability 14.7

health regulations also impose rules on their handling in the workplace and,
after they are used, they are inactivated by sterilization. The resulting organic
material is usually composted. This breaks down the DNA and protein
components and the compost can be used as fertilizer to maintain the level of
organic material in the soil.

Examples of Case Studies

The OECD Task Force on Biotechnology for Sustainable Industrial Development
has recently published a report entitled “The Application of Biotechnology to
Industrial Sustainability”. This report provides case studies of how companies
in a wide range of industrial sectors have used biotechnology to reduce the
cost and environmental impact of their production activities. Summaries of
the case studies are provided below.
Fine Chemicals: Given the cost of developing new bio-processes and bio-
products, it is not surprising that some of the first applications of industrial
biotechnology appear in the pharmaceutical and fine chemicals segment of
the chemical industry, where the value of the products can bear the cost of
technology development. It has long been known that enzymes can catalyze
certain chemical reactions with high efficiency and specificity. Since 1970, Tanabe
Seiyaku (Japan) has used enzymes-derived from certain microorganisms to
produce amino acids. Immobilizing the enzymes on a surface, so they could
be used again and again, led to 40% cost savings. Improving this system of
immobilization of the micro-organisms to optimize the performance of the
enzymes yielded a further 15-fold increase in productivity (i.e., the ratio of
product yield to starting material used), resulting in a major reduction of costs
and waste.
Enzymes usually function in an aqueous solution and this can reduce
the requirement in equivalent conventional chemical processes for organic
solvents, that will later need to be recycled or disposed of by incineration.
Biochemie (Germany/Austria), a subsidiary of Novartis, has developed an
enzyme-catalyzed process for manufacture of the antibiotic cephalosporin.
The efficiency of the enzymes was optimized by genetically modifying
the micro-organisms that produce the enzymes. When compared to the
conventional chemical process, the enzymatic process produces 100 times less
waste solvent to be incinerated and, as a result, the cost of production and the
potential environmental impact of the process are both reduced. Metabolic
engineering is a technique which involves genetically engineering a micro-
organism to contain all the enzyme steps for a series of reactions leading to a
particular product and then uses the cell metabolism to drive the reaction. In
effect, the cell then becomes a highly efficient mini-reactor for synthesizing that
product. Hoffmann La-Roche (Germany) now uses a metabolically engineered
microorganism to produce vitamin B2. This has enabled the company to reduce
a six-step chemical process to one step. As a result, use of non-renewable raw
14.8 Environmental Biotechnology

materials has decreased by 75%, emissions of volatile organic compounds to

air and water have decreased by 50% and operating costs have decreased by
50%. Similarly, DSM (Netherlands) has used a metabolically engineered micro-
organism to reduce the waste produced in the manufacture of cephalexin 3 to
7-fold. This has allowed the company to reduce production costs so that it can
compete effectively in international markets.
Intermediate Chemicals: Other case studies indicate that, once the
underlying biotechnology has been developed and understood, lateral
application can occur in other areas. Thus, biotechnologies developed at
high cost in the pharmaceutical and fine chemicals segment of the chemical
industry can be adapted and applied at lower cost to produce lower value
products, such as intermediate chemicals for synthesis of other chemicals or
plastics.
S-chloropropionic acid is an intermediate chemical used in the synthesis
of certain herbicides. The “S” indicates that the molecule is chiral, that is, one
of two asymmetric isomers (the other isomer is the “R” form). The “S” isomer
is the one that is biologically active. Conventional chemical procedures for
separating chiral molecules are often energy-intensive, or require the use
of additional chemicals which subsequently require disposal. A biological
method for separating chiral molecules involves using a microorganism that
selectively degrades one of the two isomers, leaving the other in essentially
pure form, once it has been isolated. Avecia (United Kingdom) has developed
a bioprocess for producing pure Schloropropionic acid that uses a Pseudomonas
bacterium to selectively degrade the “R” form. Mutation, selection and
adoption of sophisticated means of fermentation resulted in a four-fold increase
in productivity, while use of genetic modification to optimize performance
even further resulted in an additional five-fold increase in productivity. The
bioprocess results not only in lower production costs but also in less waste by-
product that requires treatment and disposal.
Mitsubishi Rayon Company (Japan) produces acrylamide, a chemical
used to produce acrylic polymers. The conventional chemical process for
producing acrylamide from acrylonitrile involves high temperature and
the use of, either a copper catalyst or sulphuric acid. Mitsubishi Rayon has
developed a bioprocess which, instead, uses a naturally-occurring enzyme,
nitrile hydratase, to catalyze the conversion of acrylonitrile into acrylamide.
The performance and yield of this enzyme has been optimized by genetically
engineering the micro-organism which naturally produces the enzyme. The
enzyme-catalyzed process uses 80% less energy, saves costs and yields higher
purity acrylamide than the conventional chemical process.
Polymers: The conventional chemical process for producing certain
polyesters involves the use of either a titanium or tin-based catalyst with
solvents and inorganic acid at high temperature (200°C). Baxenden Chemicals
(United Kingdom) has developed a bioprocess that uses the enzyme lipase
Biotechnology: Industrial Sustainability 14.9

from the yeast Candida antarctica to catalyze the polymerization reaction at

a much lower temperature (60°C). The lipase gene was transferred into
a genetically engineered industrial strain of E. coli bacterium to reduce
the cost of producing the enzyme. The enzyme-catalyzed polymerization
process, when compared with the conventional process, eliminates the use
of organic solvents and inorganic acids and yields energy savings of about
2000 megawatts annually at full industrial scale operation. The polymer from
the bioprocess also has a more uniform polymer chain length. This results in
a melting point over a narrower range of temperature than the conventional
polyester, making it more valuable for use as a hot-melt adhesive. Thus, there
were both environmental and economic benefits from implementing the
enzyme-based bioprocess.
Cargill Dow LLC (United States) has developed polylactic acid (PLA), a
biopolymer that not only involves the use of bioprocesses (developed, using
biotechnology) that are energy and materials-efficient but also utilizes a
renewable agricultural feedstock, corn4. PLA is not only recyclable, but also
biodegradable, and can be composted. It can functionally replace plastics such
as nylon, PET, polyester and polystyrene and life cycle analysis shows that, it
can do so with a net fossil fuel saving of 20-50% and at a cost which reflects
the lower cost of energy and raw material in its manufacture. In the medium-
term, advances in biotechnology will allow PLA to be produced also from the
cellulose found in agricultural and forest by-products. The plastic will then
become a net sink for carbon sequestered from the air by crops and trees.
Cargill-Dow has constructed a plant in Nebraska, USA, that will produce
140,000 tons of PLA annually.
Food Processing: Often, food processing uses large quantities of water
and produces large quantities of organic waste. Biotechnology can help
reduce water usage as well as the production of organic waste. For example,
Pasfrost (Netherlands) has developed a biological treatment system for water
in its vegetable-processing facility that has reduced water use by 50% and led
to significant cost savings. Similarly, Cereol (Germany) has implemented an
enzyme-based system for the degumming of vegetable oil during purification
after extraction. This bioprocess was compared with the conventional
degumming process that used sulphuric acid, phosphoric acid, caustic soda
and large quantities of water. The enzyme system eliminated the need for
treatment with strong acid and base, reduced water use by 92% and waste
sludge by 88% and resulted in an overall cost reduction of 43%.
Fibre Processing: Large quantities of energy, water and chemicals are used
to bleach and treat natural fibres for making textiles and paper. Enzymes can
help reduce some of these input costs and associated environmental impacts.
For example, Windel (Netherlands) uses an enzymatic process to reduce
the energy and time required to wash hydrogen peroxide bleach from textiles
before dyeing. Use of the enzyme made it possible to reduce the temperature
14.10 Environmental Biotechnology

and volume of the second wash from 80 – 95°C to 30 – 40°C, resulting in a

9–14% saving of energy, a 17–18% saving of water and an overall cost saving of
9%. This is very significant in the highly competitive textile industry because
margins are generally quite small.
Domtar (Canada) has begun to use the enzyme xylanase, supplied by
Iogen Corporation (Canada) as an auxiliary brightening agent (this process
is called “bio-bleaching”) for wood pulp in papermaking. The enzyme opens
up the lignin structure of the wood pulp so that it takes 10–15% less chlorine
dioxide to achieve the desired level of brightness. Iogen has reduced the
production cost and improved the performance of xylanase by genetically
engineering the fungus from which it is extracted. The use of xylanase has
helped Domtar reduce the amount of organically-bound chlorine in waste
water by 60% and the cost of bleaching chemicals by 10–15%. Oji Paper (Japan)
has also used xylanase to achieve similar reductions in the requirement for
bleaching chemicals and in levels of organically-bound chlorine in its waste
water. In addition, it produces its own xylanase on-site by fermentation, so its
input costs are reduced even further.
Mining and Metal Refining: Billeton (South Africa) has developed
a bioprocess (“bio-leaching”) to liberate copper from sulphide ore. The
bioprocess uses naturally-occurring bacteria to oxidize the sulphur and
iron present in the ore at ambient temperature. The conventional process
for isolating the copper from the ore involves transporting the mined ore
to a smelter where the impurities are driven-off at high temperature. The
bioleaching process is carried out at the mine site. This saves the cost and
energy required to transport the ore and also eliminates the emission of large
quantities of sulphur oxides, arsenic and other toxic metals into the atmosphere
by the high temperature roasting process. After the copper is extracted from
the acidic leach water, the wastewater is neutralized and toxic substances such
as arsenic are immobilized in a stable form stored at the mine site. The bio-
leaching process can be used to process low-grade ores and arsenic containing
ores that could not be processed effectively by high temperature smelting.
The capital cost requirements of the bio leaching process are 25% less than
for building a smelter. Bio-leaching currently accounts for 20–25% of world
copper production.
Budel Zinc (Netherlands) is a major producer of zinc. The acidic waste
water from its zinc refinery contains zinc and other metals (tin, copper, nickel,
manganese, chromium, lead and iron). The conventional process for treating
this wastewater involves neutralizing it with lime or limestone, which results
in large quantities of gypsum contaminated with heavy metals. Budel has
developed a bioprocess that uses sulphate-reducing bacteria to capture and
recycle zinc and other metals in its wastewater as metal sulphide precipitate.
The metal sulphide precipitate is recycled back into the refinery feedstock.
This process has resulted in a 10- to 40-fold decrease in the concentration of
Biotechnology: Industrial Sustainability 14.11

heavy metals in the refinery wastewater and eliminated the production of

metal-contaminated gypsum which is a hazardous solid waste by-product.
Energy: Examples of biotechnology applications in the energy sector
occur in both the conventional fossil-fuel and the renewable energy segments
of the industry.
Conventional fossil-fuels are usually extracted from deposits buried below
the surface of the earth. Drilling of oil wells requires the use of substances
called drilling fluids or drilling mud. These substances help lubricate the drill
and its pipe as well as hold open the wellbore. Drilling fluids are designed to
deposit a low-permeability layer on the surface of the borehole to limit leakage
of the drilling fluid into the oil-bearing formation and to prevent invasion of
solids into the oil production zones. Once the well is drilled to the desired
depth, the low-permeability layer must be removed in order to maximize oil
production rates. Traditional drilling fluids are muds—dispersions of clay
minerals in water and oil where the clay provides the required viscosity and
the oil provides the lubrication. These muds pose two problems:
(i) the oil used in their formulation can have negative environmental
impacts and requires treatment.
(ii) the strong acid required to remove the low-permeability layer is toxic to
the environment, corrodes equipment and does not uniformly remove
the low-permeability layer.
M-I and British Petroleum Exploration (United Kingdom) are now using
a drilling fluid
containing mixtures of bio-organic polymers such as xanthan gum,
which provides viscosity, and starch or cellulose, which acts as a binder.
The formulation also contains an inert solid called a bridging agent that has
a particle size allowing it to bridge pores in the structure of the rock being
drilled. This formulation is non-toxic and avoids the problems of conventional
drilling muds:
(i) there is no oil or other component which requires treatment before
release into the environment; and,
(ii) the enzymes used in removing the low-permeability layer not only
perform better but also do not corrode equipment or pose environmental
hazard.
Biotechnology has been used to optimize the characteristics of these
enzymes (cellulase, hemicellulase, amylase and pectinase) to work under
the conditions found in a borehole. Although the use of bio-organic drilling
fluid systems is in its early days, it appears in a number of cases that their
performance is satisfactory and permit cost savings of USD 75,000 – 83,000 per
well drilled.
Ethanol is one renewable fuel whose production is increasing rapidly in
response to the need for transportation fuels that produce lower net emissions
of greenhouse gases (GHG). Ethanol is produced by fermentation of sugars
14.12 Environmental Biotechnology

(such as glucose) using brewers’ yeast. The sugar can come from cornstarch.
It takes considerable energy to produce corn, however, so the net reduction in
GHG emissions is around 40–50%, when ethanol from corn is used to replace
gasoline (petrol). If wood cellulose and waste materials are used as the source
of sugar to produce ethanol, the net reduction in GHG emissions is larger,
around 60–70%. Therefore, cellulose-containing materials are, from a GHG
perspective, the material of choice for producing ethanol. However, the lignin in
woody plant material can prevent full conversion of cellulose into fermentable
sugar. Iogen Corporation (Canada) has developed a process utilizing cellulase
enzymes that maximize the conversion of cellulose into fermentable sugar.
The yield and activity of the cellulose enzymes has been optimized using
biotechnology. Iogen is in the scale-up phase of the technology and indications
are that the cost of ethanol produced in this manner will be competitive with
the cost of gasoline produced from oil costing USD 25 per barrel.

Lessons from the Case Studies

It is possible to draw a number of general conclusions from the case studies:
(i) The application of biotechnology in a wide range of industry sectors
(chemicals, plastics, food processing, natural fiber processing, mining
and energy) has invariably led to both economic and environmental
benefits via processes that are less costly and more environmentally
friendly than the conventional processes they replace. In effect, the
application of biotechnology has contributed to an uncoupling of
economic growth from environmental impacts.
(ii) The application of biotechnology to increase the eco-efficiency of
industrial products and processes can provide a basis for moving a
broad range of industries toward more sustainable production. To
achieve this, further development of biotechnology and supporting
technologies will be needed, as well as policies that provide incentives
for achieving more sustainable production.
(iii) The main driving forces for adoption of more efficient bioprocesses
and bio-products are cost savings and improved product quality/
performance. Environmental considerations were (in the case studies,
at least) an important but secondary driving force.
(iv) Successful biotechnology/bioprocess development requires effective
management of technology development by companies and use of
tools that assess both the economic and environmental performance
of technology during its development. There is a need for improved
assessment tools that are easier to use at earlier stages of the technology
development process.
(v) Even large companies may not have, in-house, all the expertise required
to develop more efficient bio-products and bioprocesses. Collaboration
with university and government researchers and other companies is
Biotechnology: Industrial Sustainability 14.13

an important contributing factor for successful introduction of these

products and processes.
(vi) Long lead times are often required for introduction of ‘paradigm shift’
technology into a company; but, development times can be reduced
considerably in subsequent development cycles.
(vii) The application of biotechnology for developing industrial products and
processes is still in its infancy. As awareness builds and the technology
continues to be developed and diffused through different industry
sectors over the next few decades, the economic and environmental
benefits are predicted to grow.
Setting a Path to a Sustainable Future – The Bio-based Economy: The
case studies outlined above show that biotechnology is an effective tool
which provides a means of reconciling the need for economic growth with
the need for environmental protection. The eco-efficiency of industrial bio-
products and bioprocesses can provide a basis for moving a broad range of
industries toward more sustainable production. However, these applications
are occurring as a “thousand points of light”, that is, without a guiding
principle or a strategic orientation. Such a strategic orientation is needed to
avoid investing resources on incremental improvements in the cleanliness of
industrial production systems which may never make it to “clean enough”,
i.e. sustainable. Shifting toward an economy more extensively based on
renewable raw materials—a bio-based economy {The bio-based economy uses
renewable bio-resources (agricultural, forestry and marine) and eco-efficient
processes (including bioprocesses) to produce sustainable bio-products, jobs
and income.}—does provide such an integrating principle. As can be seen in
Table 1, continued use of conventional processes that are not eco-efficient in
combination with non-renewable feed stocks results in continued pollution
and exhaustion of resources.
Choice of Process and Feedstock—Implications for Sustainability
Contentional Processes Cleaner Processes
Non-renewable Status quo-pollution; Extended life of
Feedstock rapid exhaustion of resources— “Postponing
resources the inevitable”
Renewable Feedstock Depletion of renewable Best chance for
(e.g. biomass) resources sustainability
If conventional processes that are not eco-efficient are used in combination
with renewable resources, they may lead to depletion of the renewable resource
as the global economy grows and demand increases. If cleaner production
processes are used on non-renewable resources, they will extend the lifetime of
those resources, but only postpone their inevitable exhaustion. Sustainability
is most likely to be found in utilizing renewable resources through cleaner
14.14 Environmental Biotechnology

processes that are eco-efficient. Developing a sustainable economy more

extensively based on renewable carbon and eco-efficient bioprocesses (a ‘bio-
based economy’) is one of the key strategic challenges for the 21st century.
At present, the global economy depends to a large extent on energy,
chemicals and materials derived from fossil carbon sources, mainly petroleum.
Petroleum provides us with fuels for transportation and heating. It also
yields synthetic chemicals for producing plastics, paints, dyes, adhesives
and a wide range of other useful industrial and consumer products. These
developments have contributed to strong economic growth and employment
and have literally transformed our global society. But this has come at a cost.
The Petrochemical Age has also resulted in massive pollution of air, water and
soil as well as emissions of greenhouse gases responsible for climate change.
Petroleum is also a finite, diminishing resource now subject to strong price
increases and fluctuations. The present level of global energy consumption,
production and industrial growth is ultimately not sustainable because it is
only made possible by continued withdrawals from the stored “bank” of fossil
carbon which is finite and not renewable.
The world was not always dependent on petroleum. A traditional bio-
based economy provided and continues to provide us with food, feed, fiber
and wood. Before the 1920s, many of our industrial products were also
bio-products, such as fuels, chemicals and materials derived from biomass,
primarily wood, and various agricultural crops. Cheap and abundant oil
changed that. However, as seen in the case studies outlined above, advances
in technology, and biotechnology in particular, are making it economically
viable and environmentally attractive to “go back to the future” and begin
supplementing, and eventually perhaps, replacing petroleum with biomass, a
renewable feedstock derived mostly from plants.
Improved understanding of biodiversity, ecology, biology and
biotechnology is making it possible both sustainably to increase biomass
productivity in forestry and agriculture as well as to utilize that biomass and
waste organic materials in a highly efficient and sustainable manner. Without
such advances in science and technology, the move to a bio-based economy
would result in rapid depletion of renewable resources and environmental
degradation. Thus, advances in science and technology are making it possible
to have an economy where industrial development and job creation are not in
opposition to environmental protection and quality of life. Getting there will
be a major challenge, requiring effective tools to assess technology, processes
and products for sustainability and also policies that encourage sustainable
production and consumption.
The life sciences, and in particular, biotechnology, will play a prominent
role in meeting that challenge. For example, the Vision (Vision for Plant/
Crop Based Renewable Resources 2020; www.oit.doe.gov/agriculture/pdfs/
vision2020.pdf) and Technology Roadmap (The Technology Roadmap for
Biotechnology: Industrial Sustainability 14.15

Plant/Crop Based Renewable Resources 2020. Web site: www.oit.doe.gov/

agriculture/pdfs/ag25945.pdf) for Plant/Crop-Based Renewable Resources
2020 provide a view of how this can be conceived, planned and executed
through targeting the development of technologies in the near, medium and
long term for:
• Selecting and developing value-added crop and tree varieties for
conventional and industrial applications.
• High-yield, sustainable crop and tree production.
• Eco-efficient harvesting and processing.
• Sustainable utilization of the resulting products.
• Closing the loop back to the environment to maintain soil organic
content and fertility.
The “bio-based economy” offers hope both for developed and developing
countries. For developed countries, it presents the opportunity to use their
technological capabilities for national energy security to head off major
economic and social disruptions which will be caused by fluctuations in the
availability and price of energy and petrochemicals as the supply of these
finite, non-renewable resources continues to diminish. It will also help them
diversify and grow employment in their rural economies.
For a number of developing countries, it provides the potential to leapfrog
(at least in part) the age of fossil-fuels and petrochemicals to the age of biofuels
and bio chemicals. These are less toxic and more easily biodegradable than
their petrochemical counterparts and can be derived from locally grown
feedstock, leading to local self-sufficiency, an improved economy and a better
quality of life.
However, if we are to see a move to such a future in the 21st century, then,
despite the potential economic, environmental and social benefits, it is not
realistic to assume that a new “green revolution” will sweep spontaneously
over existing industries. Potentially, the move to a bio-based economy could
be at least as big as that caused by the development of the petrochemical age
during the 20th century. But, societal values are different in 2001 from those
of 1901. The transition, therefore, will need to be carefully managed, not least
because it will link such issues as biotechnology and GMOs, preservation
of biodiversity, climate change, globalization, economic growth, sustainable
development and quality of life. The interplay of these issues could pose
complex problems and policy issues for governments, industry and civil
society as they try to optimize economic, environmental and societal benefits,
while enabling and fostering the development of a bio-based economy in
their countries. Visionary thinking is required among stakeholders if we are
to identify proactively the key issues and policy decisions that will have to
be dealt with along the way. Further work on these issues is underway in a
number of countries.
 CHAPTER

15 Ethical Issues in
Environmental Biotechnology

Ethics are the rules or standards that govern the way people behave and their
decisions on the ‘right thing’ to do. It asks basic questions about what is right
and wrong, how we should act towards others and what we should do in
specific situations.
• It is important to note that ethics discussing  biotechnology  and its
applications are not fundamentally different from other situations.
Ethics are practiced by everyone, every day.
• One common feature of ethics is that different people with different
values often disagree on the ‘right thing’ for individuals and society.
One reason for this disagreement is that one thing that benefits some
may not be of benefit to others.
• An example is embryonic stem cell research, which some people see as
having great potential to develop cures for diseases; but others object
to because it involves the destruction of human embryos that have the
potential to become a human being.
• There is no clear right or wrong position in ethics, as a person’s
individual experience and view of the world often guides the way they
make ethical choices.
• For instance, someone who has a strong environmental outlook might
see the use of genetically modified (GM) crops as unnatural. But
someone who has a strong scientific-based view of the world might see
the use of GM crops as a natural extension of traditional crop breeding
technologies.
Many new technologies raise ethical concerns that might not be part
of the worldview held by those who develop the technologies in the first
place. When it comes to developing products for commercial use, the goal is
usually to increase sales and increase profits for shareholders. The decision
for developing products can be seen as good for industry development,
15.2 Environmental Biotechnology

but perhaps not as good for some individuals who do not have products
developed to suit their needs when there is not enough company profit to be
made. Also, in some areas of biotechnology development, the money needed
to fund research projects is out of the range of individuals or small groups and
can only be undertaken by multinational or overseas companies. For some,
this is perceived as acceptable, as it helps local researchers form links with
wealthy larger companies. But others do not think it is not acceptable, as local
research and development leave the community and are then controlled by
international corporations.
• Many people believe that biotechnology products and applications
should respond to, and fulfil community needs. For example, some
products may be of obvious social benefit (such as a drug that treats
cancer), while others may be created by a business by attractive
advertising and skilful marketing (for example, unusual coloured
flowers for the floral industry or fluorescent fish for the pet industry).
• In a world with decreasing resources, where many people go hungry, is
spending research dollars on developing a fluorescent fish an acceptable
thing or not? Your answer will differ depending on your worldview.
When looking at ethical positions, it is important to realise that the ‘right
thing’ for one person may not be right for others and it can be very difficult to
balance these conflicting views.
• There are particular ethical positions that are commonly shared, such as
the view that, it is essential for all biotechnology products to be safe for
humans and the environment (which is why Australia has developed a
sound regulatory system to look at safety). But other ethical positions
are diverse, such as an individual’s rights to do what they want with
their body.
• There are many different ethical ways to view the world and none of
these are inherently right or wrong. There are many approaches, or
frameworks, to ethics. Some of these approaches are listed below:
Action-based (whether or not, actions in a particular circumstance are
ethical):
• Principalism uses benefit-maximising and harm-reducing principles.
• Consequentialism is based on the greatest good for the greatest number.
• Non-consequentialism (deontology) refers to rights and responsibilities.
Agent-based (emphasis on the person rather than the action they perform):
• Virtue-based can acknowledge character traits over consequences.
Situation-based (a broader perspective that takes into account other factors
such as time, place and culture):
Ethical Issues in Environmental Biotechnology 15.3

• Casuistry considers each situation to be completely unique.

• Feminist concentrates on communication, consultation and sensitivity.
• Geocultural focuses on relativity (cultural, special and time-specific
contexts).
Environmental ethics has been called “a triangular affair.” The triangle is
composed of the moral consideration of—
(1) only humans,
(2) animals or
(3) ecosystems.
The first is often called “anthropocentrism” and is atomistic in that
individuals are the focal point. The final is often called “ecocentrism” and is
holistic in, that entire ecosystems, even abiotic components, are considered
in moral deliberations on what is good and right. Between the extremes
is the ethical framework of the animal welfare movement which is non-
anthropocentric in, that the well-being of non-humans is considered but which
is still atomistic in individuals, and not systems, are the unit of consideration.
Ethics of research involving the environment: Gene technology makes
it possible for humans to alter living organisms such as plants, animals,
and bacteria to cater for human needs and wants. Such needs could include
increased crop yields, bigger, leaner, disease-free animals and new drugs or
vaccines. Some biotechnologies are also used for purposes such as veterinary
medicine.  Genetically modified organisms  (GMOs) can even benefit the
environment by cleaning up waste material or converting oil spills into non-
toxic compounds (bioremediation).
Although many scientists and agriculture companies argue that genetically
modified crops require less pesticide and herbicide than other plants, there is
still a great deal of debate about the environmental safety and value of GMOs.
For example, organizations are concerned about the following issues with
regard to genetic engineering:
• Consumer rights and the labelling of GM products
• The ethics of patenting genes and living organisms
• Who benefits from collecting genes from plants, animals, bacteria or
even people?
• Eugenics–the idea that humans can be made perfect
• Human rights
• Animal rights and welfare
• The control of genetic engineering
• Environmental impacts of new organisms
15.4 Environmental Biotechnology

Other examples of concerns about GMOs in the environment include:

• Do we know if they are safe?
• Can they be harmful to humans, animals, or plants?
• Are there other, more environmentally responsible, ways of producing
the effects of genetic modification?
• Do GMOs have advantages over natural organisms and farming
methods?
• Can GMOs independently survive in nature and disturb ecosystems?
• Can GMOs transfer genetic material to other organisms?
• Are we causing harm to animals or plants by genetically modifying
them?
The subject of environmental ethics also raises questions about  genetic
modification for human or environmental benefit.
• Do humans have the right to alter the genetic structure of animals and
plants?
• How do humans see the environment and their connections to it?
• What sort of connections should humans have with other species and
the environment?
• Do humans have a responsibility to protect other species and the
environment?
• Does genetic modification cause suffering for animals or the ecosystem?
• How might releasing GMOs affect the environment in the future?

15.1  RELEASE OF GENETICALLY MODIFIED ORGANISMS (GMOS)

Genetically modified organisms (GMOs), organisms in which genes from
another organism are inserted into the targeted organism’s DNA, have the
potential to both positively and negatively affect the environment and human
health.  Plants can be genetically modified easily because they can be grown
from a single cell or small pieces of tissue.  Thus, one only needs to modify
a single cell to produce an entire genetically modified organism.  Several
methods have been used to insert foreign genes into the target organism. 
• In one method, the target organism takes up a vector with cloned DNA
from a donor organism and incorporates this foreign DNA into its
genome.  
• Another method involves removing the wall of the target cell, allowing
the introduced DNA to easily penetrate. 
• A third method requires using a special gun to inject foreign DNA into
the target cell in hopes that the cell will incorporate the new DNA into
its genome.
Crops have been modified for centuries by humans using selective breeding
techniques, but GMO biotechnology is a more specific and rapid selection
Ethical Issues in Environmental Biotechnology 15.5

process.  For instance, genes from a different species can be incorporated into

the modified crop.  Therefore, GMO technology creates concern over potential
environmental and human health impacts.
Ethical Issues: The use of genetically modified organisms is a practice
still in its infancy.  The long-term effects of this technology are yet to be seen,
and thus, we must proceed with caution, as we develop our practices and
guidelines.

15.1.1  Effects on the Environment

Herbicide Use and Resistance: Effects on the environment are a particular
concern with regard to GMO crops and food production.  One area of
development involves adding the ability to produce pesticides and resistance
to specific herbicides.  These traits are helpful in food production, allowing
farmers to use fewer chemicals, and to grow crops in less-than-ideal
conditions.   However, herbicide use could be increased, which will have a
larger negative effect on the surrounding environment.   Also unintended
hybrid strains of weeds and other plants can develop resistance to these
herbicides through cross-pollination, thus negating the potential benefit of the
herbicide.  
Effects on Untargeted Species: Bt corn, which produces its own pesticide,
is also in use today.   Concerns have been raised regarding adverse effects
on Monarch butterfly populations, which are not the original target of the
pesticide.  Although the pesticide can protect crops against unwanted insects,
they can also have unintentional effects on neutral or even beneficial species.

15.1.2  Effects on Human Health

Allergies: GMO crops could potentially have negative effects on human
health, as well.  When splicing genes between species, there are examples in
which consumers have developed unexpected allergic reactions.  
Long-Term Effects: Because GMO technology has been available for
such a short amount of time, there is relatively little research which has been
conducted on the long-term effects on health.  The greatest danger lies not in
the effects that we have studied, but in those which we cannot anticipate at
this point.
New Proteins: Proteins, which have never been ingested before by
humans, are now part of the foods that people consume every day.   Their
potential effects on the human body are as of yet unknown.
Food Additives: GMOs also present us with possibilities of introducing
additional nutrients into foods, as well as antibiotics and vaccines.   This
availability of technology can provide nutrition and disease-resistance to
those countries that don’t have the means to provide these, otherwise.   The
distribution of these foods is more feasible than mass inoculations for current
diseases.   However, even these possibilities carry with them potential negative
effects such as the creation of antibiotic and vaccine-resistant strains of
15.6 Environmental Biotechnology

diseases. It is imperative that we ensure that environmental issues and human

health are kept at the forefront of development in this field.  It is important
that we not lose sight of the repercussions that could accompany the benefits
if we do not carefully investigate and control development.
The genetic engineering of animals has increased significantly in recent
years, and the use of this technology brings with it ethical issues, some of
which relate to animal welfare — defined by the World Organization for
Animal Health as “the state of the animal…how an animal is coping with the
conditions in which it lives.”
These issues need to be considered by all stakeholders, to ensure that all
parties are aware of the ethical issues at stake and can make a valid contribution
to the current debate regarding the creation and use of genetically engineered
animals. In addition, it is important to try to reflect societal values within
scientific practice and emerging technology, especially, publicly-funded
efforts that aim to provide societal benefits, but that may be deemed ethically
contentious. As a result of the extra challenges that genetically engineered
animals bring, governing bodies have started to develop relevant policies,
often calling for increased vigilance and monitoring of potential animal
welfare impacts. Veterinarians can play an important role in carrying out
such monitoring, especially, in the research-setting, when new genetically
engineered animal strains are being developed.
Several terms are used to describe genetically engineered animals:
genetically modified, genetically altered, genetically manipulated, transgenic,
and biotechnology-derived, amongst others. In the early stages of genetic
engineering, the primary technology used was Trans genesis, literally meaning,
the transfer of genetic material from one organism to another. However,
with advances in the field, new technology emerged that did not necessarily
require trans genesis: recent applications allow for the creation of genetically
engineered animals via the deletion of genes, or the manipulation of genes,
already present. To reflect this progress and to include those animals that are
not strictly transgenic, the umbrella term “genetically engineered” has been
adopted. Hence the scientist offers the following definition of a genetically
engineered animal:
“an animal that has had a change in its nuclear or mitochondrial DNA
(addition, deletion, or substitution of some part of the animal’s genetic
material or insertion of foreign DNA) achieved through a deliberate
human technological intervention.”
Those animals that have undergone induced mutations (for example,
by chemicals or radiation — as distinct from spontaneous mutations that
naturally occur in populations) and cloned animals are also considered to be
genetically engineered due to the direct intervention and planning involved in
creation of these animals.
Ethical Issues in Environmental Biotechnology 15.7

Current context of genetically engineered animals: Genetic engineering

technology has numerous applications involving, companion, wild, and
farm animals, and animal models used in scientific research. The majority
of genetically engineered animals are still in the research phase, rather than
actually in use for their intended applications, or commercially available.
Companion animals: By inserting genes from sea anemone and jellyfish,
zebrafish have been genetically engineered to express fluorescent proteins
— hence the commonly termed “GloFish.” GloFish began to be marketed in
the United States in 2003 as ornamental pet fish; however, their sale sparked
controversial ethical debates in California — the only US state to prohibit the
sale of GloFish as pets.
In addition to the insertion of foreign genes, gene knock-out techniques
are also being used to create designer companion animals. For example, in
the creation of hypoallergenic cats, some companies use genetic engineering
techniques to remove the gene that codes for the major cat allergen. Companion
species have also been derived by cloning. The first cloned cat, “CC,” was
created in 2002.
At the time, the ability to clone mammals was a coveted prize, and after
just a few years, scientists created the first cloned dog, “Snuppy”.
With the exception of a couple of isolated cases, the genetically engineered
pet industry is yet to move forward. However, it remains feasible that
genetically engineered pets could become part of day-to-day life for practicing
veterinarians, and there is evidence that clients have started to enquire about
genetic engineering services, in particular, the cloning of deceased pets.
Wild animals: The primary application of genetic engineering to
wild species involves cloning. This technology could be applied to either
extinct or endangered species; for example, there have been plans to clone
the extinct thylacine and the woolly mammoth point out that, “As many
conservationists are still suspicious of reproductive technologies, it is unlikely
that cloning techniques would be easily accepted. Individuals involved in
field conservation often harbour suspicions that hi-tech approaches, backed
by high profile publicity, would divert funding away from their own efforts.”
However, cloning may prove to be an important tool to be used alongside
other forms of assisted reproduction to help retain genetic diversity in small
populations of endangered species.
Farm animals: There is “an assorted range of agricultural livestock
applications [for genetic engineering] aimed at improving animal productivity;
food quality and disease resistance; and environmental sustainability.”
Productivity of farm animal species can be increased using genetic engineering.
Examples include, transgenic pigs and sheep that have been genetically
altered to express higher levels of growth hormone. Genetically engineered
farm animals can be created to enhance food quality.
15.8 Environmental Biotechnology

Farm species may be genetically engineered to induce disease-resistance

specific examples include, conferring immunity to offspring via antibody
expression in the milk of the mother; disruption of the virus entry mechanism
(which is applicable to diseases such as pseudorabies); resistance to prion
diseases; parasite control (especially, in sheep); and mastitis resistance
(particularly, in cattle). Genetic engineering has also been applied with the
aim of reducing agricultural pollution. Effort has also been made to generate
genetically engineered farm species such as cows, goats, and sheep that
express medically important proteins in their milk.
Research animals: Biomedical applications of genetically engineered
animals are numerous, and include understanding of gene function, modeling
of human disease to either understand disease mechanisms or to aid drug
development, and xenotransplantation.
Through the addition, removal, or alteration of genes, scientists can pinpoint
what a gene does by observing the biological systems that are affected. While
some genetic alterations have no obvious effect, others may produce different
phenotypes that can be used by researchers to understand the function of the
affected genes. Genetic engineering has enabled the creation of human disease
models that were previously unavailable. Animal models of human disease
are valuable resources for understanding how and why a particular disease
develops, and what can be done to halt or reverse the process. As a result,
efforts have focused on developing new genetically engineered animal models
of conditions such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS),
Parkinson’s disease, and cancer. However, as scientists points out: “these
[genetically engineered animal] models do not always accurately reflect the
human condition, and care must be taken to understand the limitation of such
models.”
The use of genetically engineered animals has also become routine
within the pharmaceutical industry, for drug discovery, drug development,
and risk assessment. Nowadays “Transgenic and knock out mouse models
are extremely useful in drug discovery, especially, when defining potential
therapeutic targets for modifying immune and inflammatory responses.
Specific areas for which [genetically engineered animal models] may be
useful are, in screening for drug induced immunotoxicity, genotoxicity, and
carcinogenicity, and in understanding toxicity-related drug metabolizing
enzyme systems.”Perhaps, the most controversial use of genetically engineered
animals in science is to develop the basic research on xenotransplantation —
that is, the transplant of cells, tissues, or whole organs from animal donors into
human recipients. In relation to organ transplants, scientists have developed
a genetically engineered pig, with the aim of reducing rejection of pig organs
by human recipients. This particular application of genetic engineering is
currently at the basic research stage, but it shows great promise in alleviating
the long waiting lists for organ transplants, as the number of people needing
Ethical Issues in Environmental Biotechnology 15.9

transplants currently far outweighs the number of donated organs. However,

as a direct result of public consultation, a moratorium is currently in place
preventing pig organ transplantation from entering a clinical trial phase until
the public is assured that the potential disease transfer from pigs to humans
can be satisfactorily managed.

15.1.3  Environmental Biotechnology - Biosafety Management

Biosafety is associated with the use of genetically modified organisms (GMOs)
and, more generally, with the introduction of non-indigenous species into
natural or managed ecosystems. A relatively new concept in environmental
research, it tempers the adoption of a new technology by carefully considering
its potential effects on human health and the environment.
• Environmental biotechnology has allowed the movement of genetic
material across unrelated species, something impossible with the
traditional breeding methods. This intentional transfer of genetic
material has, in turn, brought biotechnology out from the laboratory to
the field.
• Genetically modified organisms (GMO’s) are organisms whose genetic
material has been artificially modified to change their characteristics
in some way or another. In essence, “genetic modification” or “genetic
engineering” techniques enable scientists to find individual genes
that control particular characteristics, separate them from the original
source, and transfer them directly into the cells of an animal, plant,
bacterium, or virus.
• This technology has many potential applications. These new
opportunities bring along greater public scrutiny and government
regulation. Risk assessment is a common regulatory tool used in the
decision-making process for a proposed commercial release of a GMO
into the environment.
• Environmental applications of microorganisms are wide and varied,
ranging from bio-remediation, bio-pesticides, nitrogen fixation, plant
growth promoter, to bio-control of plant diseases, and other such
agricultural practices.
• The sensible application of recombinant DNA techniques has shown the
potential for genetically improved microorganisms to be used as soil
or seed inoculants. However, when introduced into the environment,
they could have unintended environmental consequences and may
play more pronounced ecological roles than the wild types.
• Genetically improved microorganisms are able to reproduce and
establish themselves as persistent populations and may have subtle and
long-term effects on biological communities and natural ecosystems.
Results of DNA modification may not be limited only to the particular
15.10 Environmental Biotechnology

characteristics of the replaced gene. It is therefore important to ensure

that, when these organisms are released into nature, they do not harm
the environment or human health. Such concerns have led to broader
interests in the theme of risk assessment in the release of GMOs. A
cautious approach is necessary to assess environmental risks which
may occur due to introduction of recombinant organisms in the natural
environment.
Biosafety Guidelines and Regulations: Many countries have formulated
the Biosafety Guidelines for rDNA manipulation with the aims
(i) to minimize the probability of occasional release of GMMs, and
(ii) to ban the deliberate release of such organisms into the environment.
In India, DBT has evolved “the recombinant DNA safety guidelines” to
exercise powers conferred through the Environmental Protection Act,
1986 for the manufacture, use, import, export and storage of hazardous
microorganisms / genetically engineered organisms, cell, etc.
These guidelines are being implemented through the following three
mechanisms:
• the institutional biosafety committees (IBSCs) monitors the research
activity at institutional level,
• the review committee on genetic manipulation (RCGM) functioning in
the DBT which allows the risky research activities in the laboratories,
and
• the genetic engineering approval committee (GEAC) of the Ministry
of Environment and Forest has the power to permit large-scale use of
GMOs at commercial level, and open field trials of transgenic materials
including agricultural crops, industrial products, health care products,
etc.
Genetically modified organisms (GMOs), including GM crops, have been
developed for only a short time. Just like many other major changes in human
history, biotechnology has involved some concerns and mistrust. The issues
concerning environmnetal biotechnology include environmental safety and
biodiversity, human health and food safety, trade and economic impacts,
social and ethical considerations, and much more. Many of the approaches
and policies dealing with these issues are of recent origin, still evolving and
largely unresolved. As a developing country, India considers that it is very
important to have international cooperation and exchange in the regulation
of GMO biosafety. Currently, some of the major challenges facing us are: an
appropriate regulatory approach, a science-based safety assessment, capacity
building, transparency, communication and information exchange. We believe
a good environment is necessary to facilitate the realization of the potential
benefits of modern biotechnology.
Ethical Issues in Environmental Biotechnology 15.11

15.1.4 Environmental Biotechnology & Intellectual Property Rights

(IPRs)
Environmental Biotechnology is a field of technology of growing importance
in which inventions may have a significant effect on our future, particularly
in agriculture, energy and protection of the environment. The science of
environmental biotechnology concerns living organisms, such as plants,
animals and microorganisms, as well as biological material, such as, enzymes,
proteins and plasmids (which are used in “genetic engineering”).
In recent times, scientists have developed processes to modify the genetic
composition of living organisms (genetic engineering). For example, the
modified microorganisms created by Chakrabarty (an inventor in the United
States of America) were able to break down components of oil pollution in
oceans and rivers, and helped to protect environment. The patent on these
microorganisms was the subject of a landmark decision by the United States
Supreme Court, in which modified microorganisms were recognized as
patentable subject matter. The Court noted that the laws of nature, physical
phenomena and abstract ideas were not patentable. The claimed invention,
however, was not directed to an existing natural phenomenon but to new
bacteria with markedly different characteristics from any found in nature.
The invention, therefore, resulted from the inventor’s ingenuity and effort and
could be the subject of a patent. The list of industries using biotechnology has
expanded to include health care, agriculture, food processing, bioremediation,
forestry, enzymes, chemicals, cosmetics, energy, papermaking, electronics,
textiles and mining. This expansion of applications has resulted from
innovations that have led to significant economic activity and development.
Need to protect biotechnological inventions: As in other fields of
technology, there is a need for legal protection in respect of biotechnological
inventions. Such inventions are creations of the human mind just as much as
other inventions, and are generally, the result of substantial research, inventive
effort and investment in sophisticated laboratories. Typically, enterprises
engaged in research, only make investments, if legal protection is available for
the results of their research. As with other inventions and industries, the need
for investment in research and development efforts creates an obvious need
for the protection of biotechnological inventions.
• This need is not only in the interest of inventors and their employers,
but also in the public interest of promoting technological progress.
Modern, flexible intellectual property systems and policies have
contributed to fostering investment needed to establish biotechnology
industries creating tangible products. Flexible intellectual property
policies can play a role in favoring stable legal environments conducive
to public/private partnerships, investment and other economic activity
needed to spread biotechnological innovations to more countries.
• The patenting of biotechnology innovations has been accompanied by
controversy, as has the use of some of these new innovations. Policy
15.12 Environmental Biotechnology

makers of all countries, however, have been careful to avoid extending

patent rights to things, as they exist in nature or to natural phenomena.
A new plant species discovered in the wild, for instance, cannot be
patented and neither can laws of nature. In each country, the laws on
patentability of biotechnological inventions need to be consulted to
learn the availability of patent protection and its scope.
• When considering these issues, one also needs to recognize that legal
regimes other than patent systems are typically relied upon to address
other public interests, such as the environmental or medical safety of
products, efficacy of products, and unfair competition that may occur
in the assertion of patent rights. The confluence of this new technology
with legal and regulatory systems makes biotechnology an evolving
and dynamic component of intellectual property law.
 APPENDIX

Useful Terms and their Meanings of

Environmental Biotechnology

Abatement Debris: Waste resulting from remediation (clean-up) activities.

Abiogenesis: Abiogenesis is the study of how life on earth could have
arisen from inanimate matte. It should not be confused with evolution, which
is the study of how groups of living things change over time.
Abiotic stress: The stress caused (e.g., to crop plants) by non-living,
environmental factors such as cold, drought, flooding, salinity, ozone, toxic-
to-that-organism metals (e.g., aluminum, for plants), and ultraviolet-B light.
ABS toxins: ABS toxins are six-component protein complexes secreted by
a number of pathogenic bacteria. All share a similar structure and mechanism
for entering targeted host cells.
Abzyme: See Catalytic antibody.
Acid rain: Rain that is more acidic than normal because raindrops have
dissolved acid gases and/or dust particles from the atmosphere; the principal
gases responsible for increased acidity are oxides of sulfur and nitrogen.
Generally, rain with a pH below about 4.5 is considered environmentally
harmful.
Acidogenesis : Acidogenesis represents the second stage in the four stages
of anaerobic digestion: (1) Hydrolysis: A chemical reaction where particulates
are solubilized and large polymers converted into simpler monomers; (2)
Acidogenesis : A biological reaction where simple monomers are converted
into volatile fatty acids; (3) Acetogenesis : A biological reaction where volatile
fatty acids are converted into acetic acid, carbon dioxide, and hydrogen; and
(4) Methanogenesis : A biological reaction where acetates are converted into
methane and carbon dioxide, while hydrogen is consumed.
Acidophilic autotrophs: Organisms that are able to live solely on sulphides
and in acid conditions.
Activated sludge: Sludge particles produced in raw or settled wastewater
(primary effluent) by the growth of organisms (including zoogleal bacteria) in
A.2 Environmental Biotechnology

aeration tanks in the presence of dissolved oxygen. The term “activated” comes
from the fact that the particles are teeming with bacteria, fungi and protozoa.
Activated sludge is different from primary sludge in that the sludge particles
contain many living organisms which can feed on the incoming wastewater
Active site: The site on the enzyme at which the substrate binds.
Acute toxicity unit: For a given species and a single toxic substance, the 96-
hr median tolerance limit (TLm). For a mixture of toxicants, any combination
of concentrations that would be expected to kill half the individuals of the
same species in 96 hr.
Acute toxicity: Toxicity resulting from exposure to a toxic substance or
stress for a relatively brief period, typically no more than 48–96 hr, but never
more than 10% of the natural lifetime of an organism.
Acute/chronic ratio: The ratio of the concentration or level of a toxic
substance or stress that produces toxic effects after a short period of exposure
to the concentration or level of the same substance or stress that produces toxic
effects after a long period of exposure.
Adaptation: Changes in an organism or population through which they
become more suited for living in the current environment.
Adaptive radiation: The evolution of new species or sub-species to fill
unoccupied ecological niches.
Adherent cells: The cells which grow adhering to cell culture vessel and
are adherent dependent are called adherent cells.
Adverse effect: Any effect that results in functional impairment and/or
pathological lesions that may affect the performance of a whole organism or
that reduces an organism’s ability to respond to an additional challenge.
Aerobe: A microorganism dependent on oxygen for it’s growth.
Aerobic composting: A method of composting organic wastes using
bacteria that need oxygen. This requires that the waste be exposed to air, either
via turning or by forcing air through pipes that pass through the material.
Aerosol: Suspension of small solid particles and/or liquid droplets in a
gas, usually air.
Affinity chromatography: A type of chromatography in which the matrix
contains chemical groups that can selectively bind (ligands) to the molecules
being purified.
Affinity tag: The tagged amino acid sequence which forms a part of the
recombinant protein and acts as an identification tag.
African trypanosomiasis: African trypanosomiasis is a parasitic disease
of people and animals, caused by protozoa of the species Trypanosoma brucei
(which includes Trypanosoma gambiense) and transmitted by the tsetse fly.
Agarose Gel Electrophoresis: Electrophoresis carried out on agarose gel
to separate DNA fragments.
Useful Terms and their Meanings of Environmental Biotechnology A.3

Agricultural Waste: Solid waste generated by the raising of animals or the

production and harvest of crops or trees.
Agrobacterium tumefaciens: A rod-shaped bacterium that causes crown
gall disease by inserting its DNA into plant cells.
Alleles: Alternate forms of a gene or DNA sequence, which occur on either
of two homologous chromosomes in a diploid organism.
Alternative mRNA splicing: The inclusion or exclusion of different exons
to form different mRNA transcripts.
Amino acids: The building blocks or monomeric units of protein composed
of a free amino (NH2) end, a free carboxyl (COOH) end, and a side group (R).
Ampicillin (beta-lactamase): An antibiotic derived from penicillin that
prevents bacterial growth by interfering with cell wall synthesis.
Amplified Fragment Length Polymorphism (AFLP): A sensitive method
for the detection of polymorphism in the genome. It is based on the principle
of RFLP and RAPD.
Amplify: To increase the number of copies of a DNA sequence, in vivo, by
inserting into a cloning vector that replicates within a host cell, or in vitro by
polymerase chain reaction (PCR).
Anaerobe: An organism that lives and reproduces in the absence of
dissolved oxygen, instead, deriving oxygen from the breakdown of complex
substances.
Anaerobic digestion: A method of composting that does not require
oxygen. This composting method produces methane. It is also known as
anaerobic composting. It is a stabilization process, reducing odor, pathogens,
and mass reduction.
Androgenesis: Development of plants from male gametophytes.
Aneuploidy: An abnormal condition of chromosomes, differing from the
usual diploid constitution. This may be due to a loss or gain of chromosomes.
Anneal: The pairing of complementary DNA or RNA sequences, via
hydrogen bonding, to form a double-stranded polynucleotide. Most often
used to describe the binding of a short primer or probe.
Anthrax: Anthrax is an acute disease caused by Bacillus anthracis. It affects
both humans and animals and most forms of the disease are highly lethal.
Antibiotic resistance : Antibiotic resistance is the ability of a microorganism
to withstand the effects of antibiotics. It is a specific type of drug resistance.
Antibiotic resistance evolves via natural selection acting upon random
mutation, but it can also be engineered by applying an evolutionary stress
on a population. Once such a gene is generated, bacteria can then transfer
the genetic information in a horizontal fashion (between individuals) by
plasmid exchange. If a bacterium carries several resistance genes, it is called
multi-resistant or, informally, a superbug. The term antimicrobial resistance is
sometimes used to explicitly encompass organisms other than bacteria.
A.4 Environmental Biotechnology

Antibiotic resistance: The ability of a microorganism to produce a protein

that disables an antibiotic or prevents transport of the antibiotic into the cell.
Antibiotic: A class of natural and synthetic compounds that inhibit the
growth of or kill other microorganisms.
Antibody: An immunoglobulin protein produced by B-lymphocytes of
the immune system that binds to a specific antigen molecule.
Anticodon: A set of three nucleotides in tRNA molecule that are
complementary to a set of three nucleotides (codon) in mRNA.
Antigen: Antigen is the protein or polysaccharide which after
penetration into a cell can stimulate it to produce antibodies—the proteins
(immunoglobulins) which selectively connect with the antigen and inactivate
them (antibodies are produced by lymphocyte cells). The microorganisms,
and specifically their surface structures, e.g. the O-antigens and endotoxins
building the cell wall of gram-negative bacteria, are antigens. Specific antigens
are allergens. The name “antigen” is shortened from the Latin anticorporis
generator that is literally “the producer of antibodies”.
Antigenic determinant: A surface feature of a microorganism or
macromolecule, such as a glycoprotein, that elicits an immune response.
Antigenic switching: The altering of a microorganism’s surface antigens
through genetic rearrangement, to elude detection by the host’s immune
system.
Antimicrobial agent: Any chemical or biological agent that harms the
growth of microorganisms.
Antisense RNA: A complementary RNA sequence that binds to a naturally
occurring (sense) mRNA molecule, thus blocking its translation.
Antisense therapy: The in vivo treatment of a genetic disease by
blocking translation (protein synthesis) with a DNA or RNA sequence that is
complimentary to specific mRNA.
Apoptosis: Programmed cell death
ARS: Autonomously Replicating Sequence
Asexual reproduction: Non-sexual means of reproduction which can
include grafting and budding.
Ash: The noncombustible solid by-products of incineration or other
burning process.
Assisted Reproductive Technology (ART): The manipulations of
reproduction in animals and humans.
ATP: Adenosine Triphosphate
Autoclaving: Sterilization via a pressurized, high-temperature steam
process.
Autoradiography: The process of detection of radioactively labeled
molecules by exposure of an X-ray sensitive film.
Useful Terms and their Meanings of Environmental Biotechnology A.5

Autosome: A chromosome that is not involved in sex determination.

Auxins: A group of plant growth regulators which are involved in cell
elongation, root initiation etc., e.g. Indole acetic acid.
BAC: Bacterial Artificial Chromosome
Bacillus thuringiensis (Bt): A Gram-positive, soil-dwelling bacterium of
the genus Bacillus. Additionally, B. thuringiensis also occurs naturally in the gut
of caterpillars of various types of moths and butterflies, as well as on the dark
surface of plants. B. thuringielnsis was discovered in 1901 in Japan by Ishiwata
and 1911 in Germany by Ernst Berliner, who discovered a disease called
Schlaffsucht in flour moth caterpillars. B. thuringiensis is closely related to B.
cereus, a soil bacterium, and B. anthracis, the cause of anthrax: the three organisms
differ mainly in their plasmids. Like other members of the genus, all three are
aerobes capable of producing endospores. Zakharyan RA et al. first reported
the presence of plasmids in B. thuringiensis and suggested involvement of the
plasmids in endospore/crystal formation. They also described the presence
of large plasmid in the Cry+ variant of B. thuringiensis Upon sporulation, B.
thuringiensis forms crystals of proteinaceous insecticidal dendotoxins (Cry
toxins) which are encoded by cry genes. It was determined that the cry genes
are harbored in the plasmids in most strains of B. thuringiensis. Cry toxins
have specific activities against species of the orders Lepidoptera (moths and
butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), hymenoptera
(wasps, bees, ants and sawflies) and nematodes. Thus, B. thuringiensis serves as
an important reservoir of Cry toxins and cry genes for production of biological
insecticides and insect-resistant genetically modified crops. When insects
ingest toxin crystals the alkaline pH of their digestive tract causes the toxin
to become activated. It becomes inserted into the insect’s gut cell membranes
forming a pore resulting in swelling, cell lysis and eventually killing the insect.
Bacillus: A rod-shaped bacterium.
Backcross: Crossing an organism with one of its parent organisms.
Backyard Composting: The diversion of food scraps and yard trimmings
from the MSW stream through the onsite controlled decomposition of organic
matter by micro-organisms (mainly bacteria and fungi) into a humus-like
product. Backyard composting is excluded from MSW recycling activities and
is considered source reduction because the composted materials never enter
the MSW stream.
Bacterial Artificial Chromosome (BAC): A vector system based on the
F-factor plasmid of E. coli , BAC is used for cloning large (100-300 kb) DNA
segments.
Bacterial oxidation (BIOX): BIOX is a biohydrometallurgical process
developed for precyanidation treatment of refractory gold ores or concentrates.
The bacterial culture is a mixed culture of Thiobacillus ferrooxidans, Thiobacillus
thiooxidans and Leptospirillum ferrooxidans. The bacterial oxidation process
A.6 Environmental Biotechnology

comprises contacting refractory sulfide ROM ore or concentrate with a strain

of the bacterial culture for a suitable treatment period under an optimum
operating environment. The bacteria oxidise the sulfide minerals, thus
liberating the occluded gold for subsequent recovery via cyanidation. Under
controlled continuous plant conditions, the number of bacterial cells and their
activity is optimized to attain the highest rate of sulfide oxidation. The bacteria
require a very acidic environment, a temperature of between 30 and 45°C,
and a steady supply of oxygen and carbon dioxide for optimum growth and
activity. The unusual operating conditions for the bacteria are not favourable
for the growth of most other microbes, thus eliminating the need for sterility
during the bacterial oxidation process. Because organic substances are toxic
to the bacteria, they are non-pathogenic and incapable of causing disease.
The bacteria employed in the process do not, therefore, pose a health risk to
humans or animals. The bacterial oxidation of iron sulfide minerals produces
iron(III) sulfate and sulfuric acid, and in the case of arsenopyrite, arsenic acid
is also produced. The arsenic is removed from the liquor by coprecipitation
with the iron and sulfate in a two-stage neutralization process. This produces
a solid neutralization precipitate containing largely calcium sulfate, basic
iron(III) arsenate and iron(III) hydroxide. The iron(III) arsenate is sufficiently
insoluble and stable to allow the neutralisation product to be safely disposed
of on a slimes dam. The neutralization liquor, purified to contain an acceptable
level of arsenic, can be re-used in the milling, flotation or bacterial oxidation
circuits.
Bacteriocide: A class of antibiotics that kills bacterial cells.
Bacteriocins : Bacteriocins are proteinaceous toxins produced by bacteria
to inhibit the growth of similar or closely related bacterial strain(s). They are
typically considered to be narrow spectrum antibiotics, though this has been
debated They are phenomenologically analogous to yeast and paramecium
killing factors, and are structurally, functionally, and ecologically diverse.
Bacteriocins were first discovered by A. Gratia in 1925. He was involved in
the process of searching for ways to kill bacteria, which also resulted in the
development of antibiotics and the discovery of bacteriophage, all within a
span of a few years. He called his first discovery a colicine because it killed
E. coli. Bacteriocins are categorized in several ways, including producing
strain, common resistance mechanisms, and mechanism of killing. There are
several large categories of bacteriocin which are only phenomenologically
related. These include the bacteriocins from gram-positive bacteria, the
colicins, the microcins, and the bacteriocins from Archaea. The bacteriocins
from E. coli are called colicins. They are the longest studied bacteriocins. They
are a diverse group of bacteriocins and do not include all the bacteriocins
produced by E. coli. For example, the bacteriocins produced by Staphylococcus
warneri, are called as warnerin or warnericin. In fact; one of the oldest known
so-called colicins was called colicin V and is now known as microcin V. It
is much smaller and produced and secreted in a different manner than the
Useful Terms and their Meanings of Environmental Biotechnology A.7

classic colicins. The bacteriocins of lactic acid fermenting bacteria are called
lantibiotics. This naming system is problematic for a number of reasons. First,
naming bacteriocins by what they putatively kill would be more accurate if
their killing spectrum were contiguous with genus or species designations. The
bacteriocins frequently possess spectra that exceed the bounds of their named
taxa and almost never kill the majority of the taxa for which they are named.
Further, the original naming is generally derived not from the sensitive strain
the bacteriocin kills, but instead the organism that produces the bacteriocin.
Bacteriophage (phage or phage particle): A virus that infects bacteria.
Altered forms are used as vectors for cloning DNA.
Bacteriostat: A class of antibiotics that prevents growth of bacterial cells.
Bacterium: A single-celled, microscopic prokaryotic organism: a single
cell organism without a distinct nucleus.
Bacteroidetes : Bacteroidetes is composed of three large classes of bacteria
that are widely distributed in the environment, including in soil, in sediments,
sea water and in the guts of animals. By far, the Bacteroidales class are the
most well-studied, including the genus Bacteroides (an abundant organism in
the feces of warmblooded animals including humans), and Porphyromonas, a
group of organisms inhabiting the human oral cavity. Members of the genus
Bacteroides are opportunistic pathogens. Rarely are members of the other two
classes pathogenic to humans. Researcher Jeffrey Gordon and his colleagues
found that obese humans and mice had intestinal flora (gut flora) with a lower
percentage of Bacteroidetes and relatively more bacteria from the Firmicutes
family. However, they are unsure if Bacteroidetes prevent obesity or if these
intestinal flora are merely preferentially selected by intestinal conditions in
those who are not obese.
Bag-house: A combustion plant emission control device that consists of
an array of fabric filters through which flue gases pass in an incinerator flue.
Particles are trapped and thus prevented from passing into the atmosphere.
Baker’s yeast: The living cells of aerobically grown yeast, Saccharomyces
cerevisiae, used in bread making.
Base Pair (bp): A pair of complementary nitrogenous bases (nucleotides)
in a DNA molecule—adenine-thymine and guanine-cytosine. Also, the unit of
measurement for DNA sequences.
Base ratio: The ratio of A to T, or C to G in a double-stranded DNA.
Basel Convention: An international agreement on the control of trans-
boundary movements of hazardous wastes and their disposal, drawn up in
March 1989 in Basel, Switzerland, with over 100 countries as signatories.
Batch culture: Batch culture is a closed culture system containing limited
amount of nutrients.
Bergmann’s plating technique: The most widely used method for culture
of isolated single plant cells.
A.8 Environmental Biotechnology

beta-DNA: The normal form of DNA found in biological systems, which

exists as a right-handed helix.
beta-Lactamase: Ampicillin resistance gene.
Bio concentration factor (BCF): The biological accumulation factor
associated with direct uptake of a substance from the water in the absence of
any possible intake through the food chain.
Bioaccumulation: It’s studies address the buildup of bioaccumulative
compounds through biomagnification and/or bioconcentration. Bioaccumu-
lation means an increase in the concentration of a chemical in a biological
organism over time, compared to the chemical’s concentration in the
environment. Compounds accumulate in living things any time they are taken
up and stored faster than they are broken down (metabolized) or excreted.
Understanding the dynamic process of bioaccumulation is very important
in protecting human beings and other organisms from the adverse effects of
chemical exposure, and it has become a critical consideration in the regulation
of chemicals.
Bioaugmentation: It is the introduction of a group of natural microbial
strains or a genetically engineered variant to treat contaminated soil or water.
Usually, the steps involve studying the indigenous varieties present in the
location to determine if biostimulation is possible. If the indigenous variety
do not have the metabolic capability to perform the remediation process,
exogenous varieties with such sophisticated pathways are introduced.
Bioaugmentation is commonly used in municipal wastewater treatment to
restart activated sludge bioreactors. At sites where soil and groundwater
are contaminated with chlorinated ethenes, such as tetrachloroethylene
and trichloroethylene, bioaugmentation is used to ensure that the in situ
microorganisms can completely degrade these contaminants to ethylene and
chloride, which are non-toxic. Bioaugmentation is typically only applicable to
bioremediation of chlorinated ethenes, although there are emerging cultures
with the potential to biodegrade other compounds including chloroethanes,
chloromethanes, and MTBE. The first reported application of bioaugmentation
for chlorinated ethenes was at Kelly Air Force Base, TX. Bioaugmentation
is typically performed in conjunction with the addition of electron donor
(biostimulation) to achieve geochemical conditions in groundwater that favor
the growth of the dechlorinating microorganisms in the bioaugmentation
culture.
Bioavailability: The availability of chemicals to degradative micro-
organisms.
Biocenosis: Biocenosis (microbiocenosis) is the association of organisms (of
microorganisms) which reside in a common biotop (the living environment),
between which the various interactions are formed (food web, for example).
The biocenosis along with the biotope creates together the ecological system
called ecosystem.
Useful Terms and their Meanings of Environmental Biotechnology A.9

Biochemical Oxygen Demand (BOD): The oxygen required to meet the

metabolic needs of aerobic organisms in water containing organic compounds.
Biocomposting: It involves combining organic materials under conditions
that enables them to decompose more quickly than they would in nature.
Think about logs and leaves on the ground in a forest. The leaves will break
down and disappear within a year. Logs, of course, will take much longer
to crumble away. Composting involves combining organic materials under
conditions that enables them to decompose more quickly than they would in
nature.
Biodegradable material: Any organic material that can be broken down
by microorganisms into simpler, more stable compounds. Most organic wastes
(e.g., food, paper) are biodegradable.
Biodegradable matter: Organic matter that can be broken down by
bacteria or other microorganisms to more stable forms which will not create a
nuisance or give off foul odors.
Biodegradation: It is nature’s way of recycling wastes, breaking
down organic matter into nutrients that can be used by other organisms.
“Degradation” means decay, and the prefix “bio-” means that the decay is
carried out by a huge assortment of bacteria, fungi, maggots, worms, and other
organisms that eat dead material and recycle it into new forms. In nature, there
is no waste because everything gets recycled. The waste products from one
organism become the food for others, providing nutrients and energy while
breaking down the waste organic matter. Some organic materials will break
down much faster than others, but all will eventually decay. By harnessing
these natural forces of biodegradation, people can reduce wastes and clean
up some types of environmental contaminants. Through composting, we
accelerate natural biodegradation and convert organic wastes to a valuable
resource.
Biodiesel: Biodiesel is a fuel comprised of mono-alkyl esters of long chain
fatty acids derived from biologically produced oils or fats including vegetable
oils, animal fats and microalgal oils. It is a cleaner alternative fuel with
combustion properties very similar to petroleum diesel, most often used as
an additive to improve the lubricity of pure ultra-low sulfur petrodiesel fuel.
Biodiversity: The multitude of different living beings in a particular
ecosystem or on the whole earth.
Bioenergy: In recent decades, efforts were made for evolving were non-
polluting bioenergy sources or energy generation from organic waste or
biomass. These are all eco-friendly solution. Biomass energy supply demand
balances have become a component of energy sector analysis and planning
and assumed greater importance in countries. These are variety of biological
energy sources. Biomass, Biogas, Hydrogen are the example of Bioenergy.
Bio-enrichment: Adding nutrients or oxygen to increase microbial
breakdown of pollutants.
A.10 Environmental Biotechnology

Biofertilizer: To reduce the impact of excess chemical fertilizers in the field

of agriculture the biofertilizer is a potential too; biologically fixed nitrogen is
such a source which can supply an adequate amount of Nitrogen to plants and
other nutrients to some extent. Many free-living and symbiotic bacteria, which
fix atmospheric Nitrogen were used as biofertilizer material as a substitute for
Nitrogen fertilizer. In general, two types of biofertilizers are used: 1. Bacterial
Biofertilizer, 2. Algal Biofertilizer.
Biofilm: A biofilm is a structured community of microorganisms
encapsulated within a self-developed polymeric matrix and adherent to
a living or inert surface. Biofilms are also often characterized by surface
attachment, structural heterogeneity, genetic diversity, complex community
interactions, and an extracellular matrix of polymeric substances.
Biofilteration: The process of removing complex wastes from domestic
and industrial sources by using microorganisms.
Biofuel: The term biofuel is attributed to any alternative fuel that derives
from organic material, such as energy crops (corn, wheat, sugar cane, sugar
beet, cassava, among others), crop residues (e.g. rice straw, rice husk, corn
stover, corn cobs) or waste biomass (for instance, food waste, livestock waste,
paper waste, construction-derived wood residues and others).
Biohazards: The accidents or risks associated with biological materials.
Biohydrometallurgy, biomining, bioleaching: A method of mining and
extracting metals from ores by using microorganisms.
Bioinformatics: Bioinformatics is an interdisciplinary field, which
addresses biological problems using computational techniques. The field is
also often referred to as computational biology. It plays a key role in various
areas such as functional genomics, structural genomics, and proteomics, and
forms a key component in the biotechnology and pharmaceutical sector.
Bioleaching: The use of bacteria to recover valuable metals from ores.
Biolistics: The process of introducing DNA into plants and animal cells,
and organelles by bombardment of DNA-coated pellets under pressure at
high speed. This is also called as ‘microprojectile bombardment’.
Biological accumulation factor: The ratio of the concentration of a
substance in one or more tissues of an aquatic organism to the concentration
of the same substance in the water in which the organism has been living.
Biological warfare (BW): It is the use of pathogens (bacteria, viruses, or
other disease causing agents) as biological weapons (or bioweapons).
Biologics: Agents, such as vaccines, that gives immunity from diseases or
harmful biotic stresses.
Biomarker: It is a biological response to a chemical that gives a measure
of exposure and, sometimes, of toxic effect. Biological markers found in crude
oils and source rock extracts can provide molecular evidence of the correlation
among oils and their sources.
Useful Terms and their Meanings of Environmental Biotechnology A.11

Biomass: The total dry weight of all organisms in a particular sample,

population, or area. (or) The organic mass that can be used as a source of
energy.
Biometry: Application of statistical methods to study biological problems.
Biomining: Biomining is an approach to the extraction of desired minerals
from ores being explored by the mining industry in the past few years.
Microorganisms are used to leach out the minerals, rather than the traditional
methods of extreme heat or toxic chemicals, which have a deleterious effect on
the environment.
Biopesticide: Pest control by biological antagonism appears to be very
useful tool in recent years. Bacterial pesticides are being developed. Heliothis
complex, which lives in close association with plant roots, consists of two major
crop pests-budworm and ball warm. Biological insecticides against both these
insects are being prepared by transfer of a gene from Bacillus thuringiensis.
Biopesticides: The toxic compounds produced by living organisms that
can specifically kill a particular pest species.
Bioprocess technology: A more recent usage to replace fermentation
technology that involves large scale cultivation of microorganisms for
industrial purposes.
Bioreactor: A growth chamber or a vessel for cells or microorganisms. The
cells or cell extracts carry out biological reactions in a bioreactor.
Bioremediation: It is a clean-up technology that uses naturally occurring
microorganisms to degrade hazardous substances into less toxic or non-
toxic compounds. These microorganisms may: Ingest and degrade organic
substances as their food and energy source, degrade organic substances such
as chlorinated solvents or petroleum products, that are hazardous to living
organisms, including humans, and degrade the organic contaminants into
inert products. Because the microorganisms already occur naturally in the
environment, they pose no contamination risk.
Biosensor: Biosensor represents biophysical devices which will detect
the presence and measure the quantities of specific substances in a variety
of environments. These specific substances may include sugars, proteins,
or humas and a variety of toxins in the industrial effluents. In designing a
biosensor, an enzyme or an antibody or even microbial cells are associated
with microchip devices which are used for quantitative estimate of a substance.
Biosorption: The process of microbial cell surface adsorption of metals.
Biosphere: The biosphere is the global sum of all ecosystems. It can also
be called the zone of life on Earth. From the broadest biophysiological point of
view, the biosphere is the global ecological system integrating all living beings
and their relationships, including their interaction with the elements of the
lithosphere, hydrosphere, and atmosphere.
A.12 Environmental Biotechnology

Biostimulation: Addition of specific nutrients to enhance the growth of

naturally occurring microorganisms that convert toxic compounds to non-
toxic compounds.
Biotechnology: The applications of biological principles, organisms and
products to practical purposes.
Biotic stress: Living organisms which can harm plants, such as viruses,
fungi, and bacteria, and harmful insects.
Biotin: A non-radioactive label used for labeling probes, detected through
a cytochemical reaction
Biotransformation: This is a process of biological changes of complex
compound to simpler, toxic to non-toxic, or vice-versa. Several microorganisms
are capable of transforming a variety of compounds found in nature but
generally, with respect to synthetic compounds, they are unable to show any
appropriate action. Biotransfer appears to be one of the major detoxication
methods known so far.
BLAST: Basic Local Alignment Search Tool
BOD: Biochemical Oxygen Demand – the amount of oxygen consumed by
water microorganisms in breaking down the organic matter.
Bottom ash: Relatively coarse, noncombustible, and generally toxic
residue of incineration that accumulates on the grate of a furnace.
Brewer’s Yeast: A strain of yeast usually belonging to Saccharomyces
cerevisiae that is used for the production of beer.
Brewing: Brewing is the production of alcoholic beverages and alcohol
fuel through fermentation.
Broth: Any fluid medium supporting the growth of microorganisms.
Bt. Plants: The plants carrying the toxin producing gene from Bacillus
thuringiensis, and capable of protecting themselves from insect attack.
Bubonic plague: It is the best known manifestation of the bacterial disease
plague, caused by the bacterium Yersinia pestis (formerly known as Pasteurella
pestis).
Bulky waste: Large wastes such as appliances, furniture, and trees and
branches, that cannot be handled by normal MSW processing methods.
Butanol: Butanol is a four-carbon alcohol. It can be produced via clostridial
fermentation.
Callus: A mass of undifferentiated plant tissues formed from plant cells or
tissue cuttings when grown in culture.
Capsid: See Coat protein.
Carcinogen: A substance that induces cancer.
Carcinogens: Carcinogens is the chemical, physical and biological agents
causing cancer (tumour). Most carcinogens are mutagens, because a first step
Useful Terms and their Meanings of Environmental Biotechnology A.13

of the carcinogenesis (process creating a cancer) is the mutation in the gene

controlling cell divisions.
Carcinoma: A malignant tumor derived from epithelial tissue, which
forms the skin and outer cell layers of internal organs.
Casettee mutagenesis: Replacement of a wild type DNA by a synthetic
double-stranded oligonucleotide (a small DNA fragment).
Catalyst: A substance that promotes a chemical reaction by lowering the
activation energy of a chemical reaction, but which itself remains unaltered at
the end of the reaction.
Catalytic antibody (abzyme): An antibody selected for its ability to
catalyze a chemical reaction by binding to and stabilizing the transition state
intermediate.
Catalytic RNA (ribozyme): A natural or synthetic RNA molecule that cuts
an RNA substrate.
Cation: A positively charged ion.
cDNA Library: A library composed of complementary copies of cellular
mRNAs.
cDNA: Complimentary DNA i.e. DNA produced by reverse transcription
from mRNA by the enzyme reverse transcriptase.
Cell culture: The culture of dispersed (or disaggregated) cells obtained
from the original tissue, or from a cell line.
Cell lines: Animal or plant cells that can be cultivated under laboratory
conditions.
Cell: The basic unit by which a landfill is developed. It is the general area
where incoming waste is tipped, spread, compacted, and covered.
Cell-mediated immune response: The activation of the T-lymphocytes of
the immune system in response to a foreign antigen.
Cellular oncogene (proto-oncogene): A normal gene that when mutated
or improperly expressed contributes to the development of cancer.
Cellulose: It is a polymer of glucose. Unlike starch, the glucose monomers
of cellulose are linked together through â-1-4 glycosidic bonds by condensation
resulting in tightly packed and highly crystalline structures that are resistant
to hydrolysis.
Cellulosic ethanol : Cellulosic ethanol is a biofuel produced from wood,
grasses, or the non-edible parts of plants. It is a type of biofuel produced from
lignocellulose, a structural material that comprises much of the mass of plants.
Lignocellulose is composed mainly of cellulose, hemicellulose and lignin.
Corn stover, switchgrass, miscanthus, woodchips and the byproducts of
lawn and tree maintenance are some of the more popular cellulosic materials
for ethanol production. Production of ethanol from lignocellulose has the
advantage of abundant and diverse raw material compared to sources like
A.14 Environmental Biotechnology

corn and cane sugars, but requires a greater amount of processing to make the
sugar monomers available to the microorganisms that are typically used to
produce ethanol by fermentation.
Centers of origin: Usually the location in the world where the oldest
cultivation of a particular crop has been identified.
Central dogma: Francis Crick’s seminal concept that in nature genetic
information generally flows from DNA to RNA to protein.
Centrifugal extractor: A method of solvent extraction that uses the
principle of centrifugal forces.
Centrifugation: Separating molecules by size or density using centrifugal
forces generated by a spinning rotor. G forces of several hundred thousand
times gravity are generated in ultracentrifugation.
Centromere: The central portion of the chromosome to which the spindle
fibers attach during mitotic and meiotic division.
Chemical Precipitation: Precipitation of metals is achieved by the addition
of coagulants such as alum, lime, iron salts and other organic polymers. The
large amount of sludge containing toxic compounds produced during the
process is the main disadvantage.
Chemoautotrophs: Chemoautotrophs generally only use inorganic
energy sources. Most are bacteria or archaea that live in hostile environments
such as deep sea vents and are the primary producers in such ecosystems.
Evolutionary scientists believe that the first organisms to inhabit earth were
chemoautotrophs that produced oxygen as a by-product and later evolved into
both aerobic, animal-like organisms and photosynthetic, plant-like organisms.
Chemoautotrophs generally fall into several groups : methanogens, halophiles,
sulfur reducers, nitrifiers, anammoxbacteria and thermoacidophiles.
Chemocar: A special vehicle for the collection of toxic and hazardous
wastes from residences, shops, and institutions.
Chemotherapy: A treatment for cancers that involves administering
chemicals toxic to malignant cells.
Chemotrophs: Chemotrophs are organisms that obtain energy by the
oxidation of electron donating molecules in their environments. These
molecules can be organic (organotrophs) or inorganic (lithotrophs). The
chemotroph designation is in contrast to phototrophs which utilize solar
energy. Chemotrophs can be either autotrophic or heterotrophic.
Chimera: A recombinant DNA molecule that contains sequences from
different organisms.
Chimeric antibodies: Antibodies in which the individual polypeptide
chains are composed of segments from two different species (usually man and
mouse).
Chloramphenicol: An antibiotic that interferes with protein synthesis.
Useful Terms and their Meanings of Environmental Biotechnology A.15

Chromatid: Each of the two daughter strands of a duplicated chromosome

joined at the centromere during mitosis and meiosis.
Chromatography: An analytical technique dealing with the separation of
closely related compounds from a mixture.
Chromosome walking: It is a technique used to identify the overlapping
sequences of DNA in a chromosome in order to identify a particular locus of
interest.
Chromosome: A single DNA molecule, a tightly coiled strand of DNA,
condensed into a compact structure, in vivo by complexing with accessory
histones or histone-like proteins. Chromosomes exist in pairs in higher
eukaryotes.
Cistron: A DNA sequence that codes for a specific polypeptide; a gene.
Cleaner production : Processes designed to reduce the wastes generated
by production.
Clone: All the individuals derived by asexual reproduction from a single
original individual. In molecular biology, a strain of organism that carries a
particular DNA sequence.
Cloning vector: A plasmid or a phage that carries an inserted foreign DNA
to be introduced into a host cell.
Cloning: The mitotic division of a progenitor cell to give rise to a
population of identical daughter cells or clones.
Coat protein (Capsid): The coating of a protein that encloses the nucleic
acid core of a virus.
Co-disposal: The disposal of different types of waste in one area of
a landfill or dump. For instance, sewage sludges may be disposed of with
regular solid wastes.
Codon: A triplet nucleotide sequence of mRNA coding for an amino acid
in a polypeptide.
Coenzyme (Cofactor): An organic molecule, such as a vitamin, that binds
to an enzyme and is required for its catalytic activity.
Cogeneration: Production of both electricity and steam from one facility,
from the same fuel source.
Coliform index: It is a rating of the purity of water based on a count of
fecal bacteria. Coliform bacteria are microorganisms that primarily originate
in the intestines of warm-blooded animals. By testing for coliforms, especially
the well known E.Coli, which is a thermotolerant coliform, one can determine
if the water has probably been exposed to fecal contamination; that is, whether
it has come in contact with human or animal feces. It is important to know this
because many disease-causing organisms are transferred from human and
animal feces to water, from where they can be ingested by people and infect
them. Water that has been contaminated by feces usually contains pathogenic
A.16 Environmental Biotechnology

bacteria, which can cause disease. Some types of coliforms cause disease, but
the coliform index is primarily used to judge if other types of pathogenic
bacteria are likely to be present in the water. The coliform index is used because
it is difficult to test for pathogenic bacteria directly. There are many different
types of disease-causing bacteria, and they are usually present in low numbers
which do not always show up in tests. Thermotolerant coliforms are present
in higher numbers than individual types of pathogenic bacteria and they can
be tested for relatively easily.
Collection: The process of picking up wastes from residences, businesses,
or a collection point, loading them into a vehicle, and transporting them to a
processing, transfer, or disposal site.
Colony hybridization: A technique that employs nucleic acid probe to
identify a bacterial colony with a vector carrying specific gene (s).
Colony: Colony is the group of cells visible with the naked eye on the
solid medium (e.g. the agar medium) formed by cells originating from the
initial unit—which can be one or more cells. If this is from a single cell then
the colony is the pure strain.
Combustibles: Burnable materials in the waste stream, including paper,
plastics, wood, and food and garden wastes.
Combustion Ash: Residual substance produced during the burning,
combustion or oxidation of waste materials.
Combustion: In context of Municipal Solid Waste Management, the
burning of materials in an incinerator.
Commensalism: The close association of two or more dissimilar organisms
where the association is advantageous to one and doesn’t affect the other(s).
Commercial Waste: Waste generated by businesses, such as office
building, retail and wholesale establishments, and restaurants. Examples
include cardboard, food scraps, office paper, disposable tableware, paper
napkins and yard trimmings.
Commingled Recyclables: A mixture of several recyclable materials.
Commingled: Mixed recyclables that are collected together after having
been separated from mixed Municipal Solid Waste.
Communal collection: A system of collection in which individuals bring
their waste directly to a central point, from which it is collected.
Compactor vehicle: A collection vehicle using high-power mechanical or
hydraulic equipment to reduce the volume of solid waste.
Competence: Ability of a bacterial cell to take in DNA
Competency: An ephemeral state, induced by treatment with cold cations,
during which bacterial cells are capable of up taking foreign DNA.
Complementary DNA or RNA. The matching strand of a DNA or RNA
molecule to which its bases pair.
Useful Terms and their Meanings of Environmental Biotechnology A.17

Complementary Nucleotides: Members of the pairs adenine-thymine,

adenine-uracil, and guanine-cytosine that have the ability to hydrogen bond
to one another.
Composite liner: A liner system for a landfill consisting of an engineered
soil layer and a synthetic sheet of material.
Compost: The material resulting from composting. Compost, also called
humus, is a soil conditioner and in some instances is used as a fertilizer.
Composting Facility: Offsite facility where the organic component of
municipal solid scraps is biologically decomposed under controlled conditions;
an aerobic process in which organic materials are ground or shredded and
then decomposed to humus in windrow piles or in mechanical digesters,
drums or similar enclosures.
Composting: Biological decomposition of solid organic materials by
bacteria, fungi, and other organisms into a soil-like product.
Concatemer: A DNA segment composed of repeated sequences linked
end to end.
Conjugation: The joining of two bacteria cells when genetic material is
transferred from one bacterium to another.
Constitutive promoter. An unregulated promoter that allows for continual
transcription of its associated gene. (See Promoter.)
Construction & Demolition (C&D) Debris: Waste that is generated
during construction, remodeling, repair, or demolition of buildings, bridges,
pavements and other structures. C&D debris includes concrete, asphalt,
lumber, steel girders, steel rods, wiring, dry wall, carpets, window glass, metal
and plastic piping, tree stumps, soil and other miscellaneous items related
to the activities listed, including natural disaster debris. These efforts are
excluded from calculating the MSW recycling rate.
Construction and demolition debris: Waste generated by construction
and demolition of buildings, such as bricks, concrete, drywall, lumber,
miscellaneous metal parts and sheets, packaging materials, etc.
Contigs: These are continuous (contiguous) sequences which have
overlapping regions on either ends.
Contiguous (Contig) Map: The alignment of sequence data from large,
adjacent regions of the genome to produce a continuous nucleotide sequence
across a chromosomal region.
Continuous cell lines: The cell lines that get transformed, and under in
vitro conditions grow continuously, are called Continuous cell lines. These
cells show no contact inhibition and no anchorage dependence.
Controlled dump: A planned landfill that incorporates to some extent
some of the features of a sanitary landfill: siting with respect to hydro-
geological suitability, grading, compaction I some cases, leachate control,
partial gas management, regular (not usually daily) cover, access control, basic
record-keeping, and controlled waste picking.
A.18 Environmental Biotechnology

Copy DNA: See cDNA.

Cosmid: A hybrid vector of plasmid and phage DNA; contains specific
sequence called as cos sites of phage DNA.
Cross-Hybridization: The hydrogen bonding of a single-stranded DNA
sequence that is partially, but not entirely, complementary to a single-stranded
substrate. Often, this involves hybridizing a DNA probe for a specific DNA
sequence to the homologous sequences of different species.
Crossing-Over: The exchange of DNA sequences between chromatids of
homologous chromosomes during meiosis.
Cross-Pollination: Fertilization of a plant from a plant with a different
genetic makeup.
Crumb Rubber: Ground rubber pieces used in rubber or plastic products,
or processed further into reclaimed rubber or asphalt products.
Cryopreservation: Storage and preservation at very low temperatures
(-1960C).
Cryoprotectant: A chemical agent or a compound that can prevent damage
to cells while they are frozen or defrosted.
Culture medium: The nutrients prepared in the form of a fluid (broth) or
solid for the growth of cells/tissues in the laboratory.
Culture: A population of plant or animal cells/microorganisms that are
grown under controlled conditions.
Curbside collection: Collection of compostables, recyclables, or trash at
the edge of a sidewalk in front of a residence or shop.
Curing: Allowing partially composted materials to sit in a pile for a
specified period of time as part of the maturing process in composting.
Cybridization: The process of formation of cybrids.
Cybrids: The cytoplasmic hybrids obtained by the fusion of enucleated
and nucleated protoplasts are called Cybrids.
Cyclic AMP (Cyclic Adenosine Monophosphate): A second messenger
that regulates many intracellular reactions by transducing signals from extra-
cellular growth factors to cellular metabolic pathways.
Cystic fibrosis: A disease affecting lungs and other tissues due to defects
in ion transport. It is caused by the deficiency of CFTR gene.
Cytogenetics: Study that relates the appearance and behavior of
chromosomes to genetic phenomenon.
Cytokines: Various chemicals produced in the body which mediate
immunological responses.
Cytotoxicity: The toxic effects on cells that result in metabolic alterations
including the death of cells.
Useful Terms and their Meanings of Environmental Biotechnology A.19

Dalton: A unit of measurement equal to the mass of a hydrogen atom, 1.67

x 10E-24 gram/L (Avogadro’s number).
Death phase: The final growth phase, during which nutrients have been
depleted and cell number decreases.
Decomposers: Decomposers (or saprotrophs) are organisms that
consume dead or decaying organisms, and, in doing so, carry out the natural
process of decomposition. Like herbivores and predators, decomposers are
heterotrophic, meaning that they use organic substrates to get their energy,
carbon and nutrients for growth and development. Decomposers use
deceased organisms and non-living organic compounds as their food source.
The primary decomposers are bacteria and fungi. Bacteria are the primary
decomposers of dead animals (carrion) and are the primary decomposers
of dead plant matter (litter) in some ecosystems. In soils, active fungal
hyphae and bacteria are much more important in the recycling of nutrients.
Bacteria can also be very important in agricultural fields, because tillage
usually increases the abundance of bacteria relative to fungi. Fungi are the
primary decomposers of litter in many ecosystems. Unlike bacteria, which
are unicellular, most saprotrophic fungi grow as a branching network of
hyphae. While bacteria are restricted to growing and feeding on the exposed
surfaces of organic matter, fungi can use their hyphae to penetrate larger
pieces of organic matter. Additionally, only fungi have evolved the enzymes
necessary to decompose lignin, a chemically complex substance found in
wood. These two factors make fungi the primary decomposers in forests,
where litter has high concentrations of lignin and often occurs in large pieces.
Some animals, like millipedes, woodlice, and various worms are commonly
called decomposers, because such animals consume dead organic matter and
contribute to the process of decomposition. Scientists, however, refer to such
organisms as detritivores. This distinction is made because bacteria and fungi
are capable of digesting many complex chemical molecules that animals are
incapable of digesting. Additionally, bacteria and fungi digest and decompose
organic matter more fully than detritivores, reducing it to inorganic material.
For these reasons, bacteria and fungi play a more fundamental role in the
processes of decomposition and nutrient recycling than animals.
Decomposition: Decomposition refers to the process by which tissues of
dead organisms break down into simpler forms of matter. Such a breakdown
of dead organisms is essential for new growth and development of living
organisms because it recycles the finite chemical constituents and frees up
the limited physical space in the biome. Bodies of living organisms begin to
decompose shortly after death. It is a cascade of processes that go through
distinct phases. It may be categorized in two stages by the types of end
products. The first stage is limited to the production of vapors. The second
stage is characterized by the formation of liquid materials; flesh or plant matter
begin to decompose. The science which studies such decomposition generally
is called taphonomy from the Greek word taphos—which means grave.
A.20 Environmental Biotechnology

Denature: To induce structural alterations that disrupt the biological

activity of a molecule. Often, refers to breaking hydrogen bonds between base
pairs in double-stranded nucleic acid molecules to produce in single-stranded
polynucleotides or altering the secondary and tertiary structure of a protein,
destroying its activity.
Density gradient centrifugation: High-speed centrifugation in which
molecules “float” at a point where their density equals that in a gradient of
cesium chloride or sucrose. (See Centrifugation.)
Diabetes: A disease associated with the absence or reduced levels of
insulin, a hormone essential for the transport of glucose to cells.
Diazotrophs: The microorganisms involved in diazotrophy.
Dideoxynucleotide (didN): A deoxynucleotide that lacks a 3’ hydroxyl
group, and is thus unable to form a 3’-5’ phosphodiester bond necessary for
chain elongation. Dideoxynucleotides are used in DNA sequencing and the
treatment of viral diseases.
Digest: To cut DNA molecules with one or more restriction endonucleases.
Diploid cell: A cell which contains two copies of each chromosome.
Directional cloning: DNA insert and vector molecules are digested with
two different restriction enzymes to create non-complementary sticky ends at
either end of each restriction fragment. This allows the insert to be ligated to
the vector in a specific orientation and prevents the vector from recircularizing.
(See Cloning.)
Disinfection byproducts: Side reactions can occur in water when
chemical oxidants such as chlorine and ozone are used to control potentially
pathogenic microorganisms. These reactions can form low levels of disinfection
byproducts, several of which have been regulated for potential adverse human
health effects.
Disinfection of water: Water systems add disinfectants to destroy
microorganisms that can cause diseases in humans. Primary methods of
disinfection include chlorination, chloramines, chlorine dioxide, ozone, and
ultraviolet light.
Disposal Facilities: Repositories for solid waste including landfills and
combustors intended for permanent containment or destruction of waste
material; excludes transfer stations and composting facilities.
Disposal: The final handling of solid waste, following collection,
processing, or incineration. Disposal most often means placement of wastes
in a dump or a landfill.
Distillation: Distillation is a method of separating chemical compounds
based on their differences in volatility; and volatility is a measure of the speed
at which a chemical compound evaporates.
Diversion rate: The proportion of waste material diverted for recycling,
composting, or reuse and away from landfilling or incineration.
Useful Terms and their Meanings of Environmental Biotechnology A.21

DMSO: Dimethyl sulfoxide

DNA (Deoxyribonucleic acid): An organic acid and polymer composed
of four nitrogenous bases—adenine, thymine, cytosine, and guanine linked
via intervening units of phosphate and the pentose sugar deoxyribose. DNA is
the genetic material of most organisms and usually exists as a double-stranded
molecule in which two anti-parallel strands are held together by hydrogen
bonds between adenine-thymine and cytosine-guanine.
DNA Diagnosis: The use of DNA polymorphisms to detect the presence
of a disease gene.
DNA Fingerprint: The unique pattern of DNA fragments identified by
Southern hybridization (using a probe that binds to a polymorphic region
of DNA) or by polymerase chain reaction (using primers flanking the
polymorphic region).
DNA Fingerprinting: A technique for the identification of individuals
based on the small differences in DNA sequences.
DNA Hybridization: The pairing of two DNA molecules used to detect
the specific sequence in the sample DNA.
DNA Marker: A DNA sequence that exists in two or more readily
identifiable forms (polymorphic forms) which can be used to mark a mal
position on a genome map.
DNA Polymorphism: One of two or more alternate forms (alleles) of
a chromosomal locus that differ in nucleotide sequence or have variable
numbers of repeated nucleotide units.
DNA Probe: A segment of DNA that is tagged with a label (i.e. isotope)
so as to detect a complementary base sequence in the DNA sample after a
hybridization reaction.
DNA Profiling: The term used to describe different methods for the
analysis of DNA to establish the identity of an individual.
DNA Repair: The biochemical processes that correct mutations occurring
due to replication errors or as a consequence of mutagenic agents.
DNA Sequencing: Procedures for determining the nucleotide sequence
of a DNA fragment.
DNAse: Deoxyribonuclease
Dolly: The first mammal (sheep) cloned by Wilmut and Campbell in 1997.
Dominant (-acting) Oncogene: A gene that stimulates cell proliferation
and contributes to oncogenesis when present in a single copy.
Dominant gene: A gene whose phenotype is when it is present in a single
copy.
Dominant: An allele is said to be dominant if it expresses its phenotype
even in the presence of a recessive allele.
A.22 Environmental Biotechnology

Dormancy: A period in which a plant does not grow, awaiting necessary

environmental conditions such as temperature, moisture, nutrient availability.
Double helix: Describes the coiling of the anti-parallel strands of the DNA
molecule, resembling a spiral staircase in which the paired bases form the
steps and the sugar-phosphate backbones form the rails.
Double-stranded complementary DNA (dscDNA): A duplex DNA
molecule copied from a cDNA template.
Drop-Off Center: Method of collection whereby recyclable or compostable
materials are taken by individuals to a collection site and placed in designated
containers. These can be staffed or unstaffed.
Dump: See controlled dump and open dump.
Duplex DNA: Double-stranded DNA.
Ecology: The study of the interactions of organisms with their environment
and with each other.
Ecosystem: The organisms in a plant population and the biotic and abiotic
factors which impact on them.
Edaphic: (i) Of, or pertaining to the soil. (ii) Resulting from, or influenced
by factors inherent in the soil or other substrate, rather than by climatic factors.
Edible vaccines: The vaccines produced in plants which can enter the
body on eating them.
Electrodialysis: In this process, the ionic components (heavy metals)
are separated through the use of semi-permeable ion-selective membranes.
Application of an electrical potential between the two electrodes causes a
migration of cations and anions towards respective electrodes. Because of
the alternate spacing of cation- and anion-permeable membranes, cells of
concentrated and dilute salts are formed. The disadvantage is the formation of
metal hydroxides, which clog the membrane.
Electrophoresis: Electrophoresis is the technique of separation of
macromolecules (nucleic acids, proteins) within the electric field created
between the anode and the cathode. The molecules with the negative charge
migrate to the anode, and molecules with the positive charge—to the cathode.
The separation is achieved due to differences in the speed of the migration of
ions, which in turn depends on their mass, shape and on the charge.
Electroporation: The technique of introducing DNA into cells by inducing
transient pores by electric pulse.
Electrowinning: The final method of extracting the metal, by using an
electrochemical cell.
ELISA: Enzyme Linked Immunosorbent Assay. A technique for the
detection of small quantities of proteins by utilizing antibodies linked to
enzymes, which in turn catalyze the formation of coloured products.
Useful Terms and their Meanings of Environmental Biotechnology A.23

EMBL: European Molecular Biology Laboratory

Embryo rescue: The culture of immature embryos to rescue them from
unripe or hybrid seeds which fail to germinate.
Embryo transfer: The process of implantation of embryos from a donor
animal, or developed by in vitro fertilization into the uterus of a recipient
animal.
Embryonic Stem cells (ES CELLS): The cells of an early embryo that can
give rise to all differentiated cells, including germ cells.
Emissions: Gases released into the atmosphere.
Encapsidation: Process by which a virus’ nucleic acid is enclosed in a
capsid.
End User: Facilities that purchase or secure recovered materials for the
purpose of recycling. Examples include recycling plants and composting
facilities; excludes waste disposal facilities.
Endolith : Endolith is an organism (archaeum, bacterium, fungus, lichen,
alga or amoeba) that lives inside rock, coral, animal shells, or in the pores
between mineral grains of a rock.
Endonuclease. See Nuclease.
Endophyte: An organism that lives inside another.
Endosymbiont : It is any organism that lives within the body or cells of
another organism, i.e. forming an endosymbiosis. Examples are nitrogen-
fixing bacteria (called rhizobia) which live in root nodules on legume roots,
single-celled algae inside reef-building corals, and bacterial endosymbionts
that provide essential nutrients to about 10%-15% of insects. Many instances of
endosymbiosis are obligate, that is either the endosymbiont or the host cannot
survive without the other such as the gut-less marine worms of the genus Riftia,
which get nutrition from their endosymbiotic bacteria. The most common
examples of obligate endosymbiosis are mitochondria and chloroplasts.
However, not all endosymbioses are obligate. Also, some endosymbioses can
be harmful to either of the organisms involved.
Energy recovery: The process of extracting useful energy from waste,
typically from the heat produced by incineration or via methane gas from
landfills.
Entrez: This is an integrated data base retrieval system for obtaining
comprehensive information on a given biological question
Environmental impact assessment (EIA): An evaluation designed to
identify and predict the impact of an action or a project on the environment and
human health and well-being. Can include risk assessment as a component,
along with economic and land use assessment.
Environmental Protection Agency (EPA): The U.S. regulatory agency
for biotechnology of microbes. The major laws under which the agency has
A.24 Environmental Biotechnology

regulatory powers are the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA); and the Toxic Substances Control Act (TSCA).
Environmental risk assessment (EnRA): An evaluation of the interactions
of agents, humans, and ecological resources. Comprised of human health
risk assessment and ecological risk assessment, typically evaluating the
probabilities and magnitudes of harm that could come from environmental
contaminants.
Enzymes: Proteins that control the various steps in all chemical reactions.
Epidemic: An epidemic occurs when new cases of a certain disease occur
in a given human population, during a given period, substantially exceed
what is “expected,” based on recent experience (the number of new cases in
the population during a specified period of time is called the “incidence rate”).
(An epizootic is the analogous circumstance within an animal population.)
In recent usages, the disease is not required to be communicable; examples
include cancer or heart disease. Defining an epidemic can be subjective,
depending in part on what is “expected”. An epidemic may be restricted to one
locale (an outbreak), more general (an “epidemic”) or even global (pandemic).
Because it is based on what is “expected” or thought normal, a few cases of
a very rare disease may be classified as an “epidemic,” while many cases of
a common disease (such as the common cold) would not. Common diseases
that occur at a constant but relatively low rate in the population are said to
be “endemic.” An example of an endemic disease is malaria in some parts
of Africa (for example, Liberia) in which a large portion of the population is
expected to get malaria at some point in their lifetime. The term “epidemic”
is often used in a sense to refer to widespread and growing societal problems,
for example, in discussions of obesity or drug addiction. It can also be used
metaphorically to relate a type of problem like those mentioned above.
Epitopes: The specific antigen determinants located on the antigens.
EPO: Erythropoietin
Escherichia coli: A commensal bacterium inhabiting the human colon that
is widely used in biology, both as a simple model of cell biochemical function
and as a host for molecular cloning experiments.
ESI: Electron Spray Ionization
EST: Expressed Sequence Tag
Esterification: Esterification is a general name for a chemical reaction
between alcohols and acids (carboxylic acids, mineral acids, and acid chlorides)
to form compounds called esters.
Ethidium bromide: A fluorescent dye used to stain DNA and RNA. The
dye fluoresces when exposed to UV light.
Eugenics: The science of improving human stock by selective breeding.
It involves giving better chances for more suitable people in the society to
reproduce than the less suitable people.
Useful Terms and their Meanings of Environmental Biotechnology A.25

Eukaryote: An organism whose cells possess a nucleus and other

membrane-bound vesicles, including all members of the protist, fungi, plant
and animal kingdoms; and excluding viruses, bacteria, and blue-green algae.
Eutrophicaton: Excess growth of algae (in sewage/wastewaters) which
leads to oxygen depletion.
Evolution: The long-term process through which a population of organisms
accumulates genetic changes that enable its members to successfully adapt to
environmental conditions and to better exploit food resources.
Exon: A DNA sequence that is ultimately translated into protein.
Exonuclease: See Nuclease.
Exotoxin: An exotoxin is a toxin excreted by a microorganism, including
bacteria, fungi, algae, and protozoa. An exotoxin can cause damage to the host
by destroying cells or disrupting normal cellular metabolism. They are highly
potent and can cause major damage to the host. Exotoxins may be secreted, or,
similar to endotoxins, may be released during lysis of the cell.
Explant: The whole plants can be regenerated virtually from any plant
referred to as explant.
Exponential phase: This refers to a phase in culture in which the cells
divide at a maximum rate.
Exports: Garbage and recyclables that are transported outside the state or
locality where they originated.
Express: To translate a gene’s message into a molecular product.
Expressed Sequence Tag (EST): A cDNA that is sequenced in order to
gain rapid access to the genes in a genome.
Expression library. (See Library.)
Extremophile: An extremophile is an organism that thrives in and
even may require physically or geochemically extreme conditions that are
detrimental to the majority of life on Earth.
Fecal coliforms: These are facultative-anaerobic, rod-shaped, gram-
negative, non-sporulating bacteria.
Fed-Batch culture: In a Fed-Batch culture, the culture is continuously or
sequentially fed with fresh medium without removing the growing culture.
Fermentation: Fermentation refers to the conversion of sugar to alcohol
using yeast under anaerobic conditions. A more general definition of
fermentation is the chemical conversion of carbohydrates into alcohols or acids.
When fermentation stops prior to complete conversion of sugar to alcohol,
a stuck fermentation is said to have occurred. The science of fermentation
is known as zymology. Fermentation usually implies that the action of the
microorganisms is desirable, and the process is used to produce alcoholic
beverages such as wine, beer, and cider. Fermentation is also employed in
A.26 Environmental Biotechnology

preservation to create lactic acid in sour foods such as pickled cucumbers,

kimchi and yogurt.
Fermenter: A containment system for the cultivation of prokaryotic cells.
Ferrous Metals: Magnetic metals derived from iron (steel). Products
made from ferrous metals include large and small appliances, furniture and
containers and packaging (steel drums and barrels). Examples of recycling
include processing steel cans, strapping and ferrous metals from appliances
into new products.
Filtration: Filtration is the process of removing suspended solids from
water by passing the water through a permeable fabric or porous bed of
material. The most common filtration process employs a granular media (e.g.,
sand, anthracite coal). Filtration is usually a combination of physical and
chemical processes.
Finite Cell Lines: Finite cell lines are those which have a limited life span
and they grow through a limited number of cell generations.
FISH (Fluorescent in situ Hybridization): The method of employing
fluorescent labels for locating markers on chromosomes by detecting the
hybridization positions.
Flanking region: The DNA sequences extending on either side of a specific
locus or gene.
Flaring: The burning of methane emitted from collection pipes at a landfill.
Flavr savr: Transgenic tomato developed by using antisense technology.
Flow cytometry: A method used to sort out cells, organelles or biological
materials by passing through apertures of defined sizes.
Fluidized-bed incinerator: A type of incinerator in which the stoker grate
is replaced by a bed of limestone or sand that can withstand high temperatures.
The heating of the bed and the high air velocities used cause the bed to bubble,
which gives rise to the term fluidized.
Fly ash: The highly toxic particulate matter captured from the flue gas of
an incinerator by the air pollution control system.
Food and Drug Administration (FDA): Agency responsible for regulation
of biotechnology food products. The major laws under which the agency has
regulatory powers include the Food, Drug, and Cosmetic Act; and the Public
Health Service Act.
Food Processing Waste: Food residues produced during agricultural and
industrial operations.
Food Scraps: Uneaten food and food preparation waste from residences
and commercial establishments (grocery stores, restaurants and produce
stands), institutional sources (school cafeterias) and industrial sources
(employee lunchrooms). Excludes food-processing waste from agricultural
Useful Terms and their Meanings of Environmental Biotechnology A.27

and industrial operations. Includes offsite composting but excludes source

reduction activities such as backyard (onsite) composting and use of food
items for human consumption.
Free radicals: Free radicals are labile, extremely reactive forms of
molecules with unpaired electrons, e.g. peroxyl radical O2-. Free radicals form
eg. during UV radiation. Their high oxidative reactivity can cause serious
damage to cellular structures. Antioxidants (e.g. â-carotene, vitamins C, E)
neutralize free radicals.
Fulvic acids: Yellow organic material that remains in solution after
removal of humic acid by acidification.
Fungicide: An agent, such as a chemical, that kills fungi.
Fusion gene: A hybrid gene created by joining portions of two different
genes (to produce a new protein) or by joining a gene to a different promoter
(to alter or regulate gene transcription).
Fusion protein: A protein that is formed by fusion of two polypeptides,
normally coded by separate genes
Fusobacterium : Fusobacterium is a genus of filamentous, anaerobic, Gram-
negative bacteria, similar to Bacteroides. Fusobacterium contribute to several
human diseases, including periodontal diseases, Lemierre’s syndrome, and
topical skin ulcers.
Fusogen: An agent that induces fusion of protoplasts in somatic
hybridization.
Gamete: A haploid sex cell, egg or sperm, that contains a single copy of
each chromosome.
Gametoclonal variations: The variations observed in the regenerated
plants from gametic cells (e.g. anther culture).
Garbage: In everyday usage, refuse, in general. Some MSWM manuals
use garbage to mean “food wastes,” although this usage is not common.
Gasification: Gasification involves a group of processes that turn biomass
into combustible gas by breaking apart the biomass using heat and pressure to
produce a combustible gas, volatiles, char, and ash. The gases can then be used
as a fuel or feedstock chemical.
GEM: Genetically Engineered Microorganism.
Gene Amplification: The presence of multiple genes. Amplification is one
mechanism through which proto-oncogenes are activated in malignant cells.
Gene Bank: A library of genes or clones of an entire genome of a species.
Gene cloning: The process of synthesizing multiple copies of a particular
DNA sequence using a bacteria cell or another organism as a host.
Gene expression: The process of producing a protein from its DNA- and
mRNA-coding sequences.
A.28 Environmental Biotechnology

Gene flow: The exchange of genes between different, but (usually) related
populations.
Gene frequency: The percentage of a given allele in a population of
organisms.
Gene insertion: The addition of one or more copies of a normal gene into
a defective chromosome.
Gene linkage: The hereditary association of genes located on the same
chromosome.
Gene modification: The chemical repair of a gene’s defective DNA
sequence.
Gene pool: The totality of all alleles of all genes of all individuals in a
particular population.
Gene splicing: Combining genes from different organisms into one
organism.
Gene therapy: Treatment of diseases by use of genes or DNA sequences.
Gene translocation: The movement of a gene fragment from one
chromosomal location to another, which often alters or abolishes expression.
Gene: A locus on a chromosome that encodes a specific protein or several
related proteins. It is considered the functional unit of heredity.
Genetic assimilation: Eventual extinction of a natural species as massive
pollen flow occurs from another related species and the older crop becomes
more like the new crop.
Genetic code: The three-letter code that translates nucleic acid sequence
into protein sequence. The relationships between the nucleotide base-pair
triplets of a messenger RNA molecule and the 20 amino acids that are the
building blocks of proteins.
Genetic disease: A disease that has its origin in changes to the genetic
material, DNA. Usually refers to diseases that are inherited in a Mendelian
fashion, although non-inherited forms of cancer also result from DNA
mutation.
Genetic drift: Random variation in gene frequency from one generation
to another.
Genetic engineering: The manipulation of an organism’s genetic
endowment by introducing or eliminating specific genes through modern
molecular biology techniques. A broad definition of genetic engineering also
includes selective breeding and other means of artificial selection.
Genetic library: A collection of clones representing the entire genome of
an organism.
Genetic linkage map: A linear map of the relative positions of genes
along a chromosome. Distances are established by linkage analysis, which
Useful Terms and their Meanings of Environmental Biotechnology A.29

determines the frequency at which two gene loci become separated during
chromosomal recombination.
Genetic maps: Maps giving relative distance and position of one gene
with respect to the other, wherein the distances are based on recombination
values.
Genetic marker: A gene or group of genes used to “mark” or track the
action of microbes.
Genetic Modification (GM): Introduction of isolated genes or pieces of
DNA into another organism. Synonymous terms are gene technology, and
genetic engineering
Genetically Engineered Microorganisms (GEMS): The microorganisms
with genetic modifications are collectively referred to as GEMs.
Genetically Modified (GM) FOODs: The entry of transgenic plants and
animals into the food chain represents GM foods.
Genetically Modified Organisms (GMOS): A term used to represent
organisms that are genetically engineered. It usually describes the transgenic
plants and transgenic animals.
Genotype: The structure of DNA that determines the expression of a trait.
Genome: The total content of DNA represented by the genes contained
in a cell.
Genomic DNA: The DNA of an organism containing the essential genes
of the organism.
Genomic library: A library composed of fragments of genomic DNA.
Genomics: Genomics is the study of the genomes of organisms. The field
includes intensive efforts to determine the entire DNA sequence of organisms
and fine-scale genetic mapping efforts. The field also includes studies of
intragenomic phenomena such as heterosis, epistasis, pleiotropy and other
interactions between loci and alleles within the genome. In contrast, the
investigation of the roles and functions of single genes is a primary focus of
molecular biology and is a common topic of modern medical and biological
research. Research of single genes does not fall into the definition of genomics
unless the aim of this genetic pathway, and functional information analysis
is to elucidate its effect on place in response to the entire genome’s networks.
For the United States Environmental Protection Agency, “the term “genomics”
encompasses a broader scope of scientific inquiry associated technologies than
when genomics was initially considered. A genome is the sum total of all an
individual organism’s genes. Thus, genomics is the study of all the genes of
a cell, or tissue, at the DNA (genotype), mRNA (transcriptome), or protein
(proteome) levels.”
Genomics: The study of the structure and functions of genomes.
Genus: A category including closely related species. Interbreeding
between organisms within the same category can occur.
A.30 Environmental Biotechnology

GEO: Genetically engineered organism.

Geobacter : Geobacter is a genus of Proteobacteria. Geobacter are an
anaerobic respiration bacterial species which have capabilities that may
make them useful in bioremediation. The Geobacter was found to be the first
organism with the ability to oxidize organic compounds and metals, including
iron, radioactive metals and petroleum compounds into environmentally
benign carbon dioxide while using iron oxide or other available metals as
electron acceptor. Geobacter metallireducens was first isolated by Derek Lovley
in 1987 in sand sediment from the Potomac River in Washington D.C. The
first strain was deemed strain GS-15. Geobacter have been found in anaerobic
conditions in soils and aquatic sediment. Research on the potential of the
Geobacter is underway and ongoing. The Geobacter’s ability to consume
oil-based pollutants and radioactive material with carbon dioxide as waste
by-product has already been used in environmental clean-up for underground
petroleum spills and for the precipitation of uranium out of groundwater. The
Geobacter metabolizes the material by creating “pili,” columns with width
of a 3-5 nanometers that act as conduits to pass electrons between the food
material and the Geobacter. This manner of consumption has also led scientists
to theorize that the Geobacter could act as a natural battery.
Geomicrobiology: Geomicrobiology is a subset of the scientific discipline
microbiology. The field of geomicrobiology concerns the role of microbes,
and microbial processes in geological and geochemical processes. The field is
especially important when dealing with microorganisms in aquifers and public
drinking water supplies. Another area of investigation in geomicrobiology
is the study of extremophile organisms—the microorganisms that thrive in
environments normally considered hostile. Such environments may include
extremely hot (hot springs or mid-ocean ridge black smoker) environments,
extremely saline environments, or even space environments such as Martian
soil or comets.
Germ Cell (GERM LINE) Gene Therapy: The repair or replacement of a
defective gene within the gamete-forming tissues, which produces a heritable
change in an organism’s genetic constitution.
Germ cell: Reproductive cell.
Germ Line: Reproductive cells that produce gametes which, in turn, give
rise to sperms and eggs.
Germplasm: Germplasm refers to the sum total of all genes present in a
crop and its related species.
Glucan: Glucan is the anhydrous form of D-glucose as found within a
polysaccharide such as starch or cellulose that has 1 molecule of Water (18 g/
mol) less mass due to a condensation reaction forming the polymer, C6H10O5
GMO: Genetically modified organism. A synonymous term is transgenic
organism.
Useful Terms and their Meanings of Environmental Biotechnology A.31

Golden rice: The genetically engineered rice with provitamin A (beta-

carotene) enrichment.
Gram staining: Gram staining is the method of the differentiation of
microorganisms (mostly bacteria) by staining them with two dyes. Usually,
crystal violet and safranin are used. At first, the cells are stained with the crystal
violet and then are decolorized with alcohol. Those bacteria which do not
decolorize and stay violet are named gram-positive. Those which decolorize
and stain with the second dye (safranin) become pink and are named gram
negative.
GRAS: Generally Regarded as Safe, and is in use in some countries to
represent the safety (no history of causing illness to humans) of foods, drugs,
and other materials. GRAS is also used to represent the host organisms
employed in genetic engineering experiments.
Green biotechnology: Green biotechnology is biotechnology applied to
agricultural processes. An example is the designing of transgenic plants to
grow under specific environmental conditions or in the presence (or absence)
of certain agricultural chemicals. One hope is that green biotechnology might
produce more environmentally friendly solutions than traditional industrial
agriculture. An example of this is the engineering of a plant to express a
pesticide, thereby eliminating the need for external application of pesticides.
An example of this would be Bt corn. Whether or not green biotechnology
products such as this are ultimately more environmentally friendly is a topic
of considerable debate.
Green revolution: Advances in genetics, petrochemicals, and machinery
that culminated in a dramatic increase in crop productivity during the third
quarter of the 20th century.
Greenhouse effect: Trapping of heat from the sun by the atmosphere,
in the same manner as the sun’s heat is trapped by the glass walls and roof
of a greenhouse. The atmosphere, like the glass, is largely transparent to
the sun’s radiation, but it absorbs the longer-wavelength radiation from the
earth’s surface into which the sun’s radiation is converted. The principal gases
responsible for the absorption are carbon dioxide, water vapor, and ozone.
Groundwater: Water beneath the earth’s surface that fills underground
pockets (known as aquifers), supplying wells and springs.
Growth factor: A serum protein that stimulates cell division when it binds
to its cell-surface receptor.
Growth phase (curve): The characteristic periods in the growth of a
bacterial culture, as indicated by the shape of a graph of viable cell number
versus time.
GST: Genomic Sequence Tag
GT-Food: Food produced from GMOs.
A.32 Environmental Biotechnology

Habitat: A habitat (which is Latin word for “it inhabits”) is an ecological

or environmental area that is inhabited by a particular animal or plant
species. It is the natural environment in which an organism lives, or the
physical environment that surrounds (influences and is utilized by) a species
population. The term “species population” is preferred to “organism” because,
while it is possible to describe the habitat of a single black bear, we may not
find any particular or individual bear but the grouping of bears that comprise
a breeding population and occupy a certain biogeographical area. Further,
this habitat could be somewhat different from the habitat of another group
or population of black bears living elsewhere. Thus, it is neither the species
nor the individual for which the term habitat is typically used. A microhabitat
is a physical location that is home to very small creatures, such as woodlice.
Microenvironment is the immediate surroundings and other physical factors
of an individual plant or animal within its habitat.
Half-life: The length of time required for one-half the original radioactive
material to decay to new atoms.
Haploid cell: A cell containing only one set, or half the usual (diploid)
number, of chromosomes.
Hazardous solid waste (HSW): A solid waste that meets any one of the
following four criteria: (1) the waste is specifically listed as a hazardous waste
by regulation, (2) the waste is a mixture containing a hazardous waste, (3) the
waste is derived from the treatment, storage, or disposal of a hazardous waste
(e.g., the ash residue resulting from incineration of a hazardous waste is also
a hazardous waste), or (4) the waste exhibits the characteristics of ignitability,
corrosivity, reactivity, or toxicity.
Hazardous waste: Waste that is reactive, toxic, corrosive, or otherwise
dangerous to living things and/or the environment. Many industrial by-
products are hazardous.
Heavy metals: Metals of high atomic weight and density, such as mercury,
lead, and cadmium, that are toxic to living organisms.
Hela cells: A pure cell line of human cancer cells used for the cultivation
of viruses.
Hemicellulose: Hemicellulose is a highly branched and substituted
polymer comprised mainly of xylose and arabinose, with minor amounts of
galactose and glucose. In the plant cell walls, hemicellulose holds crystalline
microfibers of cellulose in place.
Hemophilia: An X-linked recessive genetic disease, caused by a mutation
in the gene for clotting factor VIII (hemophilia A) or clotting factor IX
(hemophilia B), which leads to abnormal blood clotting.
HEPA: High Efficiency Particulate Air
Herbicide: Any substance that is toxic to plants; usually used to kill
specific unwanted plants.
Useful Terms and their Meanings of Environmental Biotechnology A.33

Heterochromatin: Dark-stained regions of chromosomes thought to be

for the most part genetically inactive.
Heteroduplex: A double-stranded DNA molecule or DNA-RNA hybrid,
where each strand is of a different origin.
Heterogeneous nuclear RNA (hnRNA). The name originally given
to large RNA molecules found in the nucleus, which are now known to be
unedited mRNA transcripts, or pre-mRNAs.
Heterokaryon: A cell in which two or more nuclei of different genetic
make-up are present.
Heterologous: These are gene sequences that are not identical, but show
variable degrees of similarity.
High throughput: Fast rate of sequencing.
Histotypic cultures: The growth and propagation of cells in three
dimensional matrix to high cell density.
Homogenation: Mechanical grinding of cells or tissues.
Homologous chromosomes: Chromosomes that have the same linear
arrangement of genes—a pair of matching chromosomes in a diploid organism.
Homologous recombination: The exchange of DNA fragments between
two DNA molecules or chromatids of paired chromosomes (during crossing
over) at the site of identical nucleotide sequences.
Homozygote: An organism whose genotype is characterized by two
identical alleles of a gene.
Household hazardous waste: Products used in residences, such as paints
and some cleaning compounds, that are toxic to living organisms and/or the
environment.
HPLC: High Performance/Pressure Liquid Chromatography
Human Genome Project (HGP): An international mega project for the
identification of human genome sequences, the genes and their functions.
This project is coordinated by the National Institutes of Health (NIH) and the
Department of Energy (DOE).
Human Growth Hormone (HGH, Somatotrophin): A protein produced
in the pituitary gland that stimulates the liver to produce somatomedins,
which stimulate growth of bone and muscle.
Humic substances: Series of relatively high-molecular-weight, brown-to-
black substances formed by secondary synthetic reactions. The term is generic
in a sense that it describes the coloured material or its fractions obtained on
the basis of solubility characteristics, such as humic acid or fulvic acid.
Humification: Process whereby the carbon of organic residues is
transformed and converted to humic substances by biochemical and chemical
processes.
A.34 Environmental Biotechnology

Humulin: Human insulin used for the treatment of diabetic patients. It

was developed by Eli Liply company and was approved for human use in
1982.
Humus: Total organic compounds in soil exclusive of undecayed plant and
animal tissues, their “partial decomposition” products, and the soil biomass.
The term is often used synonymously with soil organic matter.
Hybrid DNA: DNA composed of sequences from two different organisms,
also called as Recombinant DNA.
Hybrid: The offspring of two parents differing in at least one genetic
characteristic (trait). Also, a heteroduplex DNA or DNA-RNA molecule.
Hybridization: The hydrogen bonding of complementary DNA and/or
RNA sequences to form a duplex molecule.
Hybridoma: A clone of hybrid cells produced by fusion of a myeloma cell
with an antibody-producing cell. Each hybridoma produces only one type of
monoclonal antibody.
Hydrogen bond: A relatively weak bond formed between a hydrogen
atom (which is covalently bound to a nitrogen or oxygen atom) and a nitrogen
or oxygen with an unshared electron pair.
Hydrogenolysis: Hydrogenolysis is the process of cleaving a molecule or
compound with the addition of hydrogen atoms.
Hydrolysis: A reaction in which a molecule of water is added at the site of
cleavage of a molecule into two products.
IEF: Isoelectric Focusing
Immobilized enzymes: An enzyme physically localized in a defined
region enabling it to be reused in a continuous process.
Immortalizing oncogene: A gene that upon transfection enables a primary
cell to grow indefinitely in culture.
Immunoglobulins: The special group of proteins, commonly referred to as
antibodies, produced by B-lymphocytes, and involved in humoral immunity.
Immunology: Immunology is the science that deals with the resistance of
organisms.
Imports: Garbage and recyclables that have been transported to a state or
locality for processing or final disposition, but did not originate in that state.
In situ Hybridisation: The process of annealing a probe in order to screen
a DNA library.
In situ: Refers to performing assays or manipulations with intact tissues.
In vitro GENE BANKS: In vitro gene banks have been made to preserve
the genetic resources by non-conventional methods, i.e. cell and tissue culture
methods.
Useful Terms and their Meanings of Environmental Biotechnology A.35

In vitro: Literally means “in glass” refers to biological activities/reactions

carried out in the test tube rather than the living cell or organism.
In vivo GENE BANK: In vivo gene bank have been made to preserve the
genetic resources by conventional methods e.g. seeds, vegetative propagules
etc.
In vivo GENE THERAPY: The direct delivery of gene(s) to a tissue or an
organ to alleviate genetic disorders.
In vivo: Refers to biological processes that take place within a living
organism or cell.
Incineration: The process of burning solid waste under controlled
conditions to reduce its weight and volume, and often to produce energy.
Incinerator: A furnace for burning solid waste under controlled
circumstances.
Incomplete dominance: A condition where a heterozygous offspring has
a phenotype that is distinctly different from, and intermediate to, the parental
phenotypes.
Indicator bacteria: These are certain species of bacteria used by health
authorities to detect contaminated water. Each gram of human feces contains
approximately 12 billion bacteria; among them may include pathogenic
bacteria, such as Salmonella, associated with gastroenteritis. In addition,
feces may contain pathogenic viruses, protozoa and parasites. If ingested,
these organisms would cause disease. When testing drinking water for
contamination, the variety and often low concentrations of pathogens makes
them difficult to test for individually. Health authorities, therefore, use the
presence of other more abundant and more easily detected fecal bacteria as
indicators of the presence of fecal contamination. Indicator bacteria are not
themselves dangerous to the health but are used to indicate the presence of a
health risk.The most popularized known indicator bacteria are fecal coliforms,
which are found in the intestinal tracts of warm-blooded animals. Another less
commonly used group of indicator organisms are hydrogen sulfide producing
bacteria, which are also found in humans as well as the intestinal tracts of
birds and reptiles—known carriers of Salmonella.
Industrial MSW: Non-hazardous wastes discarded at industrial sites from
packaging and office/administrative sources. Examples of MSW recycling
efforts include cardboard, plastic film, wood pallets, lunchroom wastes
and office paper excludes industrial process wastes from manufacturing
operations.
Industrial Process Waste: Residues and materials produced during
manufacturing operations.
Industrial Sludge: The semi liquid residue remaining after the treatment
of industrial water and wastewater.
A.36 Environmental Biotechnology

Informal sector: The part of an economy that is characterized by private,

usually small-scale, labor-intensive, largely unregulated, and unregistered
manufacturing or provision of services.
Initiation codon: The mRNA sequence AUG, coding for methionine,
which initiates translation of mRNA.
Inorganic waste: Waste composed of material other than plant or animal
matter, such as sand, dust, glass, and many synthetics.
Inositol lipid: A membrane-anchored phospholipid that transduces
hormonal signals by stimulating the release of any of several chemical
messengers.
Insertion mutations: Changes in the base sequence of a DNA molecule
resulting from the random integration of DNA from another source.
Institutional MSW: Waste generated at institutions, such as schools,
libraries, hospitals and prisons. Examples of MSW recycling include cafeteria
and restroom trashcan wastes, office paper, classroom wastes and yard
trimmings.
Insulin: A peptide hormone secreted by the islets of Langerhans of the
pancreas that regulates the level of sugar in the blood.
Integrated solid waste management: Coordinated use of a set of waste
management methods, each of which can play a role in an overall MSVVM
plan.
Interferons: A group of glycoproteins that resist viral infection and
regulate immune responses.
Intergenic regions: DNA sequences located between genes that comprise
a large percentage of the human genome with no known function.
Interleukins: A group of lymphokines important for the function of
immune system.
International NGO: An organization that has an international
headquarters and branches in major world regions, often with the purpose of
undertaking development assistance.
Introgression: Backcrossing of hybrids of two plant populations to
introduce new genes into a wild population.
Intron: A non-coding DNA sequence within a gene that is initially
transcribed into messenger RNA but is later snipped out. See Coding, DNA,
Messenger RNA, Transcription.
Invasiveness: Ability of a plant to spread beyond its introduction site and
become established in new locations where it may have a deleterious effect on
organisms already existing there.
In-vessel composting: Composting in an enclosed vessel or drum with a
controlled internal environment, mechanical mixing, and aeration.
Useful Terms and their Meanings of Environmental Biotechnology A.37

Ion: A charged particle.

Ion-exchange: In this process, metal ions from dilute solutions are
exchanged with ions held by electrostatic forces on the exchange resin. The
disadvantages include: high cost and partial removal of certain ions.
Isotope: One of two or more forms of an element that have the same
number of protons (atomic number) but differing numbers of neutrons (mass
numbers). Radioactive isotopes are commonly used to make DNA probes and
metabolic tracers.
Itinerant waste buyer: A person who moves around the streets buying (or
bartering for) reusable and recyclable materials.
Joining (J) Segment: A small DNA segment that links genes to yield a
functional gene encoding an immunogobulin.
Junk DNA: The intergenic content of DNA is also referred to as junk DNA.
Kanamycin: An antibiotic of the aminoglycoside family that poisons
translation by binding to the ribosomes.
Karyotype: All of the chromosomes in a cell or an individual organism,
visible through a microscope during cell division.
Knock out mouse: A genetically altered mouse lacking the genes for an
entire organ or organ system.
Korarchaeota: These are a group of Archaea that have been found only
in high temperature hydrothermal environments. Analysis of their 16S rRNA
gene sequences suggests that they are a deeply-branching lineage that does
not belong to the main archaeal groups, Crenarchaeota and Euryarchaeota.
Analysis of the genome of one korarchaeote that was enriched from a mixed
culture revealed a number of both Crenarchaeota and Euryarchaeota like features
and supports the hypothesis of a deep-branching ancestry.
Labelling: Attaching radioactive or non-radioactive molecules to specific
substances in order to detect them.
Lag phase: The initial growth phase, during which cell number remains
relatively constant prior to rapid growth.
Land farming: A technique for the bioremediation of hydrocarbon-
contaminated soils.
Landfill gases: Gases arising from the decomposition of organic wastes;
principally methane, carbon dioxide, and hydrogen sulfide. Such gases may
cause explosions at landfills.
Landfilling: The final disposal of solid waste by placing it in a controlled
fashion in a place intended to be permanent. The Source Book uses this term
for both controlled dumps and sanitary landfllls.
Lawn: A uniform and uninterrupted laver of bacterial growth, in which
individual colonies cannot be observed.
A.38 Environmental Biotechnology

Leachate pond: A pond or tank constructed at a landfill to receive

the leachate from the area. Usually, the pond is designed to provide some
treatment of the leachate, by allowing settlement of solids or by aeration to
promote biological processes.
Leachate: Liquid (which may be partly produced by decomposition of
organic matter) that has seeped through a landfill or a compost pile and has
accumulated bacteria and other possibly harmful dissolved or suspended
materials. If uncontrolled, leachate can contaminate both groundwater and
surface water.
Leaching solution: A solution that is used for solubilisation and removal
of metals from an ore by microbial attack.
Lead-Acid Batteries: Batteries used in automobiles, trucks and motorcycles.
They contain plastic, lead (a toxic metal) and sulfuric acid; excludes lead-
acid batteries from large equipment, heavy-duty trucks and tractors, aircraft,
military vehicles and boats.
Legume: A member of the pea family that possesses root nodules-
containing nitrogen-fixing bacteria.
Library: A collection of cells, usually bacteria or yeast, that have been
transformed with recombinant vectors carrying DNA inserts from a single
species. (See cDNA library, Expression library, Genomic library.)
Lift: The completed layer of compacted waste in a cell at a landfill.
Ligand exchange solvent extraction: A method of extracting a metal from
a solution by using ligands.
Ligase (DNA ligase): An enzyme that catalyzes a condensation reaction
that links two DNA molecules via the formation of a phosphodiester bond
between the 3’ hydroxyl and 5’ phosphate of adjacent nucleotides.
Ligate: The process of joining two or more DNA fragments.
Lineage: A chart that traces the flow of genetic information from generation
to generation.
Liner: A protective layer, made of soil and/or synthetic materials, installed
along the bottom and sides of a landfill to prevent or reduce the flow of
leachate into the environment.
Linkage: The frequency of coinheritance of a pair of genes and/or genetic
markers, which provides a measure of their physical proximity to one another
on a chromosome.
Linked Genes/Markers: Genes and/or markers that are so closely
associated on the chromosome that they are coinherited in 80% or more of
cases.
Linker: A short, double-stranded oligonucleotide containing a restriction
endonuclease recognition site, which is ligated to the ends of a DNA fragment.
Lipoplexes: The lipid-DNA complexes also referred to as liposomes.
Useful Terms and their Meanings of Environmental Biotechnology A.39

Liposomes: Membrane-bound vesicles constructed in the laboratory to

transport biological molecules.
Lithotroph: A lithotroph is an organism that uses an inorganic substrate
(usually of mineral origin) to obtain reducing equivalents for use in biosynthesis
(e.g., carbon dioxide fixation) or energy conservation via aerobic or anaerobic
respiration.
Locus (plural = loci): A specific location or site on a chromosome.
Logarithmic phase (log or exponential growth phase): The steepest slope
of the growth curve—the phase of vigorous growth during which cell number
doubles every 20-30 minutes.
Lysis: The destruction of the cell membrane.
Lysogen: A bacterial cell whose chromosome contains integrated viral
DNA.
Lysogenic: A type or phase of the virus life cycle during which the virus
integrates into the host chromosome of the infected cell, often remaining
essentially dormant for some period of time.
Lytic cycle: The replication cycle of bacteria that ultimately results in the
lysis of host cells.
Lytic: A phase of the virus life cycle during which the virus replicates
within the host cell, releasing a new generation of viruses when the infected
cell lyses.
MALDI: Matrix Assisted Laser Desorption /Ionization
Malignant: Having the properties of cancerous growth.
Manual landfill: A landfill in which most operations are carried out
without the use of mechanized equipment.
Mapping: Determining the physical location of a gene or genetic marker
on a chromosome.
Marker gene: A gene which detects insertion of DNA by its inactivation.
Market waste: Primarily organic waste, such as leaves, skins, and unsold
food, discarded at or near food markets.
Mass-burn incinerator: A type of incinerator in which solid waste is
burned without prior sorting or processing.
Material Recovery Facility (MRF): A facility where recyclables are sorted
into specific categories and processed, or transported to processors, for
remanufacturing.
Materials recovery: Obtaining materials that can be reused or recycled.
Medical Waste: Any solid waste generated in the diagnosis, treatment,
or immunization of humans or animals, in research pertaining to humans or
animals, or in the production or testing of biologicals.
A.40 Environmental Biotechnology

Megabase cloning: The cloning of very large DNA fragments.

Meiosis: The reduction division process by which haploid gametes and
spores are formed, consisting of a single duplication of the genetic material
followed by two mitotic divisions.
Membrane filtration: Membrane separation processes use semipermeable
membranes to separate impurities from water. The membranes are selectively
permeable to water and certain solutes. A driving force is used to force the
water to pass through the membrane, leaving the impurities behind as a
concentrate. The amount and type of material removed depends upon the type
of membrane, the type and amount of the driving force and the characteristics
of the water.
Meristem: A localized region of actively dividing cells in plants i.e., tips
of stems and roots.
Messenger RNA (mRNA): The class of RNA molecules that copies the
genetic information from DNA, in the nucleus, and carries it to ribosomes, in
the cytoplasm, where it is translated into protein.
Metabolism: The biochemical processes that sustain a living cell or
organism.
Metagenomics: It is the study of genetic material recovered directly from
environmental samples. Traditional microbiology and microbial genome
sequencing rely upon cultivated clonal cultures. This relatively new field of
genetic research enables studies of organisms that are not easily cultured in
a laboratory as well as, studies of organisms in their natural environment.
Earlier, environmental gene sequencing cloned specific genes (often the 16S
rRNA gene) to produce a profile of diversity in a natural sample. Such work
revealed that the vast majority of microbial diversity had been missed by
cultivation-based methods. Recent studies use “shotgun” Sanger sequencing
or chip-based pyro sequencing to get (mostly) unbiased samples of all genes
from all members of sampled communities.
Metallothionein: A protective protein that binds heavy metals, such as
cadmium and lead.
Methane: An odorless, colorless, flammable, explosive gas, CH, produced
by anaerobically decomposing MSW at landfills.
Methanogenesis: It is the formation of methane by microbes known as
methanogens. Organisms capable of producing methane have been identified
only from the kingdom Archaea a group phylogenetically distinct from both
eukaryotes and bacteria, although many live in close association with
anaerobic bacteria. The production of methane is an important and widespread
form of microbial metabolism. In most environments, it is the final step in
the decomposition of biomass. Recently, some experiments have suggested
that leaf tissues of living plants emit methane. Other research has indicated
that the plants are not actually generating methane; they are just absorbing
Useful Terms and their Meanings of Environmental Biotechnology A.41

methane from the soil and the emitting it through their leaf tissues. There may
still be some unknown mechanism by which plants produce methane, but that
is by no means certain. Methanogenesis in microbes is a form of anaerobic
respiration. Methanogens do not use oxygen to breathe; in fact, oxygen inhibits
the growth of methanogens. The terminal electron acceptor in methanogenesis
is not oxygen, but carbon. The carbon can occur in a small number of organic
compounds, all with low molecular weights.
Methanogens: These are archaea that produce methane as a metabolic
byproduct in anoxic conditions. They are common in wetlands, where they
are responsible for marsh gas, and in the guts of animals such as ruminants
and humans, where they are responsible for the methane content of flatulence.
Microarray: Large number of DNA spots present on a glass slide
representative of the total mRNA of a cell, used for detecting expression
patterns.
Microbial food web: Refers to the combined trophic interactions among
microbes in aquatic environments. These microbes include viruses, bacteria,
algae, heterotrophic protists (such as ciliates and flagellates). In aquatic
environments, microbes constitute the base of the food web. Single celled
photosynthetic organisms such as diatoms and cyanobacteria are generally
the most important primary producers in the open ocean. Many of these
cells, especially cyanobacteria, are too small to be captured and consumed by
small crustaceans and planktonic larvae. Instead, these cells are consumed
by phagotrophic protists which are readily consumed by larger organisms.
Viruses can infect and break open bacterial cells and (to a lesser extent),
planktonic algae (a.k.a phytoplankton). Therefore, viruses in the microbial
food web act to reduce the population of bacteria and, by lysing bacterial cells,
release particulate and dissolved organic carbon (DOC). DOC may also be
released into the environment by algal cells. One of the reasons phytoplankton
release DOC is limited availability of essential nutrient (N22P) essential
nutrients (e.g. nitrogen and phosphorus) are limiting. Therefore, carbon
produced during photosynthesis is not used for the synthesis of proteins (and
subsequent cell growth), but is limited due of a lack of the nutrients necessary
for macromolecules. Excess photosynthate, or DOC is then released, or
exuded. The microbial loop describes a pathway in the microbial food web
where DOC is returned to higher trophic levels via the incorporation into
bacterial biomass.
Microbial mats (biofilms): Layered groups or communities of microbial
populations.
Microenterprise: A synonym for small-scale enterprise: a business, often
family-based or a cooperative that usually employs fewer than ten people and
may operate “informally.”
Microinjection: A means to introduce a solution of DNA, protein, or other
soluble material into a cell using a fine micro capillary pipet.
A.42 Environmental Biotechnology

Micronutrient: An element required by plants and bacteria, in relatively

small amounts, for survival and growth. Micronutrients include: Iron (Fe),
Manganese (Mn), Zinc (Zn), Boron (B), and Molybdenum (Mo).
Micropropagation: This method of tissue culture utilizes the culture of
apical shoots, auxiliary buds, and meristems.
Mining Waste: Residues resulting from the extraction of raw materials
from the earth.
Mitosis: The replication of a cell to form two daughter cells with identical
sets of chromosomes.
Mixed waste: Unsorted materials that have been discarded into the waste
stream.
MoAB/MAb: Monoclonal Antibodies. A specific and single type of
antibody that is produced by hybridoma cells. MAb is directed against a
specific antigenic determinant (epitope).
Modern Biotechnology: Biotechnology, including the use of genetic
modification of the producer cells and other newer procedures.
Modular incinerator: A relatively small type of prefabricated solid waste
combustion unit.
Molecular biology: The study of the biochemical and molecular
interactions within living cells.
Molecular breeding: Breeding assisted by molecular (nucleic acid)
markers is known as molecular breeding.
Molecular cloning: The biological amplification of a specific DNA sequence
through mitotic division of a host cell into which it has been transformed or
transfected.
Molecular genetics: The study of the flow and regulation of genetic
information between DNA, RNA, and protein molecules.
Molecular pharming: Use of transgenic animals to obtain products of
medicinal commercial purposes through recombinant DNA technology
Monellin: A protein found in the fruits of an African plant Discorephyllum
cumminsii which is about 100,000 times sweeter than sucrose.
Monoclonal antibodies: Immunoglobulin molecules of single-epitope
specificity that are secreted by a clone of B cells.
Monoculture: The agricultural practice of cultivating crops consisting of
genetically similar organisms.
Monofill: A landfill intended for one type of waste only.
Monogenic: Controlled by, or associated with, a single gene.
Morphogenesis: The growth and development of an undifferentiated
structure to a differentiated structure or form.
MTCC: Microbial Type Culture Collection
Useful Terms and their Meanings of Environmental Biotechnology A.43

Mulching: The process by which the volume of organic waste is reduced

through shredding or grinding.
Multicellular Tumor spheroids (MCTs): In vitro cellular three-dimensional
proliferating models for the study of tumor cells.
Multi-Locus probe: A probe that hybridizes to a number of different sites
in the genome of an organism.
Municipal Sludge: The semi-liquid residue remaining after the treatment
of municipal water and wastewater.
Municipal solid waste (MSW): All solid waste generated in an area
except industrial and agricultural wastes. Sometimes, includes construction
and demolition debris and other special wastes that may enter the municipal
waste stream. Generally, excludes hazardous wastes except to the extent that
they enter the municipal waste stream. Sometimes, defined to mean all solid
wastes that a city authority accepts responsibility for managing in some way.
Municipal solid waste management (MSWM): Planning and
implementation of systems to handle MSW.
Mushrooms: The fungi belonging to the class Basidiomycetes; some of them
are edible e.g. Agaricus bisporus (button mushroom).
Mutagenesis: The changes in the nucleotides of DNA of an organism by
physical or chemical treatments.
Mutagens: Mutagens are chemical or physical agents causing mutations—
that are the changes in the DNA structure—passed to the next generations
(hereditary). Some pollutants are mutagens, e.g. some aromatic hydrocarbons
and their derivatives (benzo-a-pyrene), and also some fungal toxins (aflatoxins).
Mutation: An alteration in DNA structure or sequence of a gene.
Muteins: The second-generation recombinant therapeutic proteins are
collectively referred to as muteins.
Mycelium: A mass of interwoven thread-like filaments of a fungus or
bacteria.
Mycofiltration: It is the process of using mushroom mycelium mats as
biological filters. The term was coined by mycologist Paul Stamets. Stamets
originally carne up with the technique to control E. coli in the water outflow
from his property. After planting a mushroom bed in the gulch where the
water was leaving, within a year the coliform count had decreased to nearly
undetectable levels. He discovered that the mushroom produced crystalline
entities advancing in front of the growing mycelium, disintegrating when
they encountered E. coli. As they did so, a chemical signal was sent back to
the mycelium that, in tum, generated what appeared to be a customized
macro-crystal which attracted the motile bacteria by thousands, summarily
stunning them. The advancing mycelium then consumed the E. coli, effectively
eliminating them from the environment. Another mushroom, Polyporus
A.44 Environmental Biotechnology

umbellata, has been demonstrated to inhibit Plasmodium falciparum, a parasite

that causes the most dangerous type of malaria infection. One industrial
application of mycofiltration has been to prevent erosion due to water runoff.
Its primary application has been on abandoned logging roads. The approach
here has been to place bark and wood chips onto logging roads, and inoculate
this wood debris with mycelia of native fungal species. As the wood chips
decompose, the mycelial networks develop and they act as filters to prevent
silt-flow. In the process, they also renew top soils, spurring the growth of
native flora and fauna.
Mycoremediation : It is form of bioremediation, comprising the process of
using fungi to return an environment (usually soil) contaminated by pollutants
to a less contaminated state. The term Mycoremediation was coined by Paul
Stamets and refers specifically to the use of fungal mycelia in bioremediation.
One of the primary roles of fungi in the ecosystem is decomposition, which
is performed by the mycelium. The mycelium secretes extracellular enzymes
and acids that break down lignin and cellulose, the two main building blocks
of plant fiber. These are organic compounds composed of long chains of
carbon and hydrogen, structurally similar to many organic pollutants. The
key to mycoremediation is determining the right fungal species to target a
specific pollutant.
Mycorrhizae: Fungi that form symbiotic relationships with roots of more
developed plants.
Myeloma: A tumor cell line derived from a lymphocyte which usually
produces a single type of immunoglobulin.
National Science Foundation (NSF): A non-regulatory agency which has
oversight of biotechnology research activities that the agency funds.
Natural Disaster Debris: Wastes resulting from earthquakes, floods,
hurricanes, tornados, ice storms, natural disasters and other major weather
events; excludes wastes resulting from heavy storms. Natural disaster debris
is classified as C&D debris.
Natural selection: The differential survival and reproduction of organisms
with genetic characteristics that enable them to better utilize environmental
resources.
NCBI : National Centre of Biotechnology Information
NGO: Non-governmental organization. May be used to refer to a range of
organizations from small community groups, through national organizations,
to international ones. Frequently, these are not-for-profit organizations.
Nick translation: A procedure for making a DNA probe in which a DNA
fragment is treated with DNase to produce single-stranded nicks, followed
by incorporation of radioactive nucleotides from the nicked sites by DNA
polymerase I.
Nicked circle (relaxed circle): During extraction of plasmid DNA from
the bacterial cell, one strand of the DNA becomes nicked. This relaxes the
Useful Terms and their Meanings of Environmental Biotechnology A.45

torsional strain needed to maintain super-coiling, producing the familiar form

of plasmid.
Night soil: Human excreta.
NIMBY: “Not In My Back Yard.” An expression of resident opposition to
the siting of a solid waste facility based on the particular location proposed.
Nitrocellulose: A membrane used to immobilize DNA, RNA, or protein,
which can then be probed with a labeled sequence or antibody.
Nitrogen cycle: The nitrogen cycle is the biogeochemical cycle that
describes the transformations of nitrogen and nitrogen-containing compounds
in nature. It is a cycle which includes gaseous components.
Nitrogen fixation: Nitrogen fixation is the process by which nitrogen is
taken from its relatively inert molecular form (N2) in the atmosphere; and
converted into nitrogen compounds (such as ammonia, nitrate and nitrogen
dioxide). This is an essential process for life because fixed nitrogen is needed
to make nucleotides which are needed to make DNA and also to make amino
acids which in turn are needed to produce proteins. Nitrogen fixation is
performed naturally by a number of different prokaryotes, including bacteria,
actinobacteria, and certain types of anaerobic bacteria. Microorganisms that
fix nitrogen are called diazotrophs. Some higher plants, and some animals
(termites), have formed associations (symbioses) with diazotrophs.
Nitrogenous bases: The purines (adenine and guanine) and pyrimidines
(thymine, cytosine, and uracil) that comprise DNA and RNA molecules.
Nitrosomonas europaea: Is a Gram-negative obligate chemolithoautotroph
that can derive all its energy and reductant for growth from the oxidation of
ammonia to nitrite and lives in several places such as soil, sewage, freshwater,
the walls of buildings and on the surface of monuments, especially in polluted
areas where the air contains high levels of nitrogen compounds.
Nodule: The enlargement or swelling on roots of nitrogen-fixing plants.
The nodules contain symbiotic nitrogen-fixing bacteria.
Nonferrous Metals: Nonmagnetic metals such as aluminum, lead and
copper. Products made from nonferrous metals include containers and
packaging such as beverage cans, food and other nonfood cans; nonferrous
metals found in appliances, furniture, electronic equipment; and non-
packaging aluminum products (foil, closures and lids from bimetal cans).
These excludes lead-acid batteries and nonferrous metals from industrial
applications and C&D debris.
Non-hazardous Industrial Process Waste: Waste that is neither MSW nor
considered a hazardous waste under Subtitle C of the Resource Conservation
and Recovery Act, such as certain types of manufacturing wastes and
wastewaters.
Non-target organism: An organism which is affected by an interaction for
which it was not the intended recipient.
A.46 Environmental Biotechnology

Northern hybridization (Northern blotting): A procedure in which RNA

fragments are transferred from an agarose gel to a nitrocellulose filter, where
the RNA is then hybridized to a radioactive probe.
NSF: National Science Foundation.
Nuclease: A class of enzymes that degrades DNA and/or RNA molecules
by cleaving the phosphodiester bonds that link adjacent nucleotides. In
deoxyribonuclease (DNase), the substrate is DNA. In endonuclease, it cleaves
at internal sites in the substrate molecule. Exonuclease progressively cleaves
from the end of the substrate molecule. In ribonuclease (RNase), the substrate
is RNA. In the S1 nuclease, the substrate is single-stranded DNA or RNA.
Nucleic acids: The two nucleic acids, deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA), are made up of long chains of molecules called
nucleotides.
Nuclein: The term used by Friedrich Miescher to describe the nuclear
material, he discovered in 1869, which today is known as DNA.
Nucleocapsid: Nucleocapsid is the complex formed by viral nucleic acid
and capsid, the protein coat that encloses the nucleic acid. Some viruses (e.g.
influenza viruses) are additionally surrounded by a glycoprotein and lipid-
containing membrane called an envelope.
Nucleoid: A term used to represent the DNA containing region of a
prokaryotic cell.
Nucleoside analog: A synthetic molecule that resembles a naturally
occurring nucleoside, but that lacks a bond site needed to link it to an adjacent
nucleotide.
Nucleoside: A building block of DNA and RNA, consisting of a nitrogenous
base linked to a five-carbon sugar.
Nucleotide: A building block of DNA and RNA, consisting of a nitrogenous
base, a five-carbon sugar, and a phosphate group. Together, the nucleotides
form codons, which when strung together form genes, which in turn link to
form chromosomes.
Nucleus: The membrane-bound region of a eukaryotic cell that contains
the chromosomes.
Nylon-eating bacteria: These are a strain of Flavobacterium that is capable
of digesting certain byproducts of nylon-6 manufacture. This strain of
Flavobacterium, Sp. KI72, became popularly known as nylon-eating bacteria,
and the enzymes used to digest the man-made molecules became collectively
known as nylonase.
Obligate anaerobes: These are anaerobic organisms which fail to grow in
the presence of oxygen. Obligate (strict) anaerobes die in presence of oxygen
due to the absence of the enzymes superoxide dismutase and catalase which
would convert the lethal superoxide formed in their cells due to the presence of
Useful Terms and their Meanings of Environmental Biotechnology A.47

oxygen. Instead of oxygen, obligate anaerobes use alternate electron acceptors

for respiration such as sulfate, nitrate, iron, manganese, mercury, and carbon
monoxide. The energy yield of these respiratory processes is less than oxygen
respiration, and not all of these electron acceptors are created equal.
Oligonucleotide-directed mutagenesis: A technique to alter one or more
specific nucleotides in a gene (DNA sequence) so that a protein with specific
amino acid change is produced.
Oncogene: A gene that contributes to cancer formation when mutated or
inappropriately expressed.
Oncogenesis: The progression of cytological, genetic, and cellular changes
that culminate in a malignant tumor.
Oncomouse: The animal model of mouse for cancer which was granted
U.S. patent in 1988, the first animal to be patented.
Open dump: An unplanned “landfill” that incorporates few, if any, of the
characteristics of a controlled landfill. There is typically no leachate control, no
access control, no cover, no management, and many waste pickers.
Open pollination: Pollination by wind, insects, or other natural
mechanisms.
Open Reading Frame (ORF): A long DNA sequence that is uninterrupted
by a stop codon and encodes part or all of a protein.
Operator: A prokaryotic regulatory element that interacts with a repressor
to control the transcription of adjacent structural genes.
Organ culture: The in vitro culture of an organ so as to achieve the
development and/or preservation of the original organ.
Organelle: A cell structure that carries out a specialized function in the
life of a cell.
Organic pump: The uptake of vast quantities of water by plant roots
whereby the water is transpired by the plant into the atmosphere.
Organic waste: Technically, waste containing carbon, including paper,
plastics, wood, food wastes, and yard wastes. In practice, in MSWM, the term
is often used in a more restricted sense to mean material that is more directly
derived from plant or animal sources, and which can generally be decomposed
by microorganisms.
Organogenesis: The process of morphogenesis that finally results in the
formation of organs e.g. shoots, roots.
Origin of replication: The nucleotide sequence at which DNA synthesis
is initiated.
OSHA: Occupational Safety and Health Administration.
Overlapping Reading Frames: Start codons in different reading frames
generate different polypeptides from the same DNA sequence.
A.48 Environmental Biotechnology

Ovum: A female gamete.

Ozone: Ozone is a colorless gas that is extremely unstable and is a strong
oxidizing agent that is capable or reacting with a wide variety of organic and
inorganic solutes in water. Effectiveness of ozone disinfection is a function of
the pH, temperature of water and method for ozone application.
Paleobiology: Is a growing and comparatively new discipline which
combines the methods and findings of the natural science biology with the
methods and findings of the earth science paleontology. It is occasionally
referred to as “geobiology.” Paleobiological research uses biological field
research of current biota and of fossils, millions of years, old to answer
questions about the molecular evolution and the evolutionary history of
life. In this scientific quest, macrofossils, microfossils and trace fossils are
typically analyzed. However, the 21st-century biochemical analysis of DNA
and RNA samples offer much promise, as does the biometric construction of
phylogenetic trees.
Paleontology: The study of the fossil record of past geological periods and
of the phylogenetic relationships between ancient and contemporary plant
and animal species.
Palindromic sequence: A DNA locus whose 5’-to-3’ sequence is identical
on each DNA strand. The sequence is the same when one strand is read left
to right and the other strand is read right to left. Recognition sites of many
restriction enzymes are palindromic.
PAM: Point Accepted Mutation
pAMP: Ampicillin-resistant plasmid developed for the laboratory course.
Parasitism: Is a type of symbiotic relationship between two different
organisms where one organism, the parasite, takes favor from the host,
sometimes for a prolonged time. In general, parasites are much smaller than
their hosts, show a high degree of specialization for their mode of life, and
reproduce more quickly and in greater numbers than their hosts.
Parasitology: Parasitology is the study of parasites, their hosts, and the
relationship between them. As a biological discipline, the scope of parasitology
is not determined by the organism or environment in question, but by their
way of life. This means, it forms a synthesis of other disciplines, and draws
on techniques from fields such as cell biology, bioinformatics, biochemistry,
molecular biology, immunology, genetics, evolution and ecology. The parasitic
mode of life is the most common on the planet, with representatives from all
major taxa, from the simplest unicellular organisms to complex vertebrates.
Every free-living species has its own unique species of parasite, so the number
of parasitic species greatly exceeds the number of free living species. The
study of these diverse organisms means that the subject is often broken up into
simpler, more focused units, which use common techniques, even if they are
not studying the same organisms or diseases. Much research in parasitology
Useful Terms and their Meanings of Environmental Biotechnology A.49

falls somewhere between two or more of these definitions. In general, the study
of prokaryotes fall under the field of bacteriology rather than parasitology.
Patent: A government issued document that provides the holder the
exclusive rights to manufacture, use, or sell an invention for a defined period,
usually 20 years.
Pathogen: An organism capable of causing disease.
pBR322: A derivation of ColE1, one of the first plasmid vectors widely
used.
Pedigree: A diagram mapping the genetic history of a particular family.
PEG: Polyethylene glycol
Persistence: Ability of an organism to remain in a particular setting for a
period of time after it is introduced.
Pesticide: A substance that kills harmful organisms (for example, an
insecticide or fungicide).
PGDF: Platelet-derived growth Factor
Phage ecology: Phage ecology is the study of the interaction of
bacteriophages with their environments. Phage ecology is increasingly an
important component of sessions and symposiums associated with phage
meetings as well as general microbiological meetings.
Phage: A virus infecting bacterium
Phagocytosis: It is the process of taking up small particles (e.g. bacteria)
by some cells of the immune system of the organism (e.g. macrophages).
Phenotype: The observable characteristics of an organism, the expression
of gene alleles (genotype) as an observable physical or biochemical trait.
Pheromone: A hormone-like substance that is secreted into the
environment.
Phosphatase: An enzyme that hydrolyzes esters of phosphoric acid,
removing a phosphate group.
Phosphinothricin (glufosinate): A broad-spectrum herbicide
Phosphodiester bond: A bond in which a phosphate group joins adjacent
carbons through ester linkages. A condensation reaction between adjacent
nucleotides results in a phosphodiester bond between 3’ and 5’ carbons in
DNA and RNA.
Phospholipid: A class of lipid molecules in which a phosphate group is
linked to glycerol and two fatty-acyl groups. A chief component of biological
membranes.
Phosphorylation: The addition of a phosphate group to a compound.
Photoautotrophs or Phototroph: These are organisms (usually plants)
that carry out photosynthesis to acquire energy. Energy from sunlight, carbon
dioxide and water are converted into organic materials to be used in cellular
A.50 Environmental Biotechnology

functions such as biosynthesis and respiration. In an ecological context, they

provide nutrition for all other forms of life (besides other autotrophs such as
chemotrophs). In terrestrial environments, plants are the predominant variety,
while aquatic environments include a range of phototrophic organisms
such as algae (e.g. kelp), other protists (such as euglena) and bacteria (such
as cyanobacteria). One product of this process is starch, which is a storage
or reserve form of carbon, which can be used when light conditions are too
poor to satisfy the immediate needs of the organism. Photosynthetic bacteria
have a substance called bacteriochlorophyll, they live in lakes and pools,
using the hydrogen from hydrogen sulfide awakward construction, for the
chemical process. (The bacteriochlorophyll pigment absorbs light in the
extreme UV and infrared parts of the spectrum, which is outside the range
used by normal chlorophyll). Cyanobacteria live in fresh water, seas, soil and
lichen, and use a plant-like photosynthesis. A photolithotrophic autotroph is an
autotrophic organism that uses light energy, and an inorganic electron source
(eg. H2O, H2, H2S), and CO2 as its carbon source. Examples include plants.
The depth to which sunlight or artificial light can penetrate into water, so that
photosynthesis may occur, is known as the phototrophic zone.
Photochemical smog: A form of air pollution caused by chemical
reactions of oxides of nitrogen and volatile organic compounds in sunshine,
characterized by the formation of elevated concentrations of ozone and of
secondary particles, the latter causing a reduction in visibility.
Photoheterotrophs: These are heterotrophic organisms which use
light for energy, but cannot use carbon dioxide as their sole carbon source.
Consequently, they use organic compounds from the environment to satisfy
their carbon requirements. They use compounds such as carbohydrates, fatty
acids and alcohols as their organic “food”. Examples are purple non-sulfur
bacteria, green non-sulfur bacteria and heliobacteria.
Phyotovolatilization: The use of plants to volatilize pollutants from
polluted soils and water
Physical map: A map showing physical locations on a DNA molecule,
such as restriction sites, and sequence-tagged sites.
Phytoalexins: The secondary metabolites produced in plants in response
to infection.
Phytodegradation: The process where plants are able to metabolically
degrade organic pollutants.
Phytoextraction: The use of plants to extract contaminants from the
environment.
Phytomining: The use of plants to extract metal compounds of high
economic value
Phytoremediation: The use of plants to remediate polluted soil and/or
groundwater.
Useful Terms and their Meanings of Environmental Biotechnology A.51

Phyto stabilization: The use of plants to reduce bioavailability and

migration of contaminants.
pI : Isoelectric point
PITC: Phenyl iso thiocyanate
Plant pathology: It is the scientific study of plant diseases caused by
pathogens (infectious diseases) and environmental conditions (physiological
factors).
Plantlet: Small-rooted shoot or germinated embryo.
Plaque: A clear spot on a lawn of bacteria or cultured cells where cells
have been lysed by viral infection.
Plasmid (p): A circular DNA molecule, capable of autonomous replication,
which typically carries one or more genes encoding antibiotic resistance
proteins. Plasmids can transfer genes between bacteria and are important
tools of transformation for genetic engineers.
Plating Efficiency: The percentage of cells plated which produce cell
colonies.
Pleiotrophy: The effect of a particular gene on several different traits.
Point mutation: A change in a single base pair of a DNA sequence in a
gene.
Pollution: The contamination of soil, water, or the atmosphere by the
discharge of waste or other offensive materials.
Poly(A) polymerase: Catalyzes the addition of adenine residues to the 3’
end of pre-mRNAs to form the poly(A) tail.
Poly(A) tail: A series of A- nucleotides attached to the 3’ end of eukaryotic
mRNA.
Polyacrylamide Gel Electrophoresis (PAGE) : Electrophoresis through
a matrix composed of a synthetic polymer, used to separate proteins, small
DNA, or RNA molecules of up to 1000 nucleotides. Used in DNA sequencing.
Polyclonal antibodies: A mixture of immunoglobulin molecules secreted
against a specific antigen, each recognizing a different epitope.
Polycyclic aromatic hydrocarbons (PAH): A group of chemical substances
comprising two or more fused benzene rings. Some members of the group are
carcinogenic.
Polygenic: Controlled by, or associated with, more than one gene.
Polyhydroxyalkanoates (PHA): Intracellular carbon and energy storage
compounds. They are biodegradable polymers.
Polylinker: A short DNA sequence containing several restriction enzyme
recognition sites that is contained in cloning vectors.
Polymer: A molecule composed of repeated subunits.
A.52 Environmental Biotechnology

Polymerase (DNA): Synthesizes a double-stranded DNA molecule using

a primer and DNA as a template. (See Poly(A) polymerase, Polymerase chain
reaction, RNA polymerase, Taq polymerase.)
Polymerase chain reaction (PCR): Is a technique to amplify a single or
few copies of a piece of DNA across several orders of magnitude, generating
millions or more copies of a particular DNA sequence. The method relies on
thermal cycling, consisting of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA. Primers
(short DNA fragments) containing sequences complementary to the target
region along with a DNA polymerase (after which the method is named)
are key components to enable selective and repeated amplification. As PCR
progresses, the DNA generated is itself used as a template for replication,
setting in motion a chain reaction in which the DNA template is exponentially
amplified. PCR can be extensively modified to perform a wide array of genetic
manipulations.
Polymorphism: The allelic variations in the genomes that results in
different phenotypes.
Polynucleotide: A DNA polymer composed of multiple nucleotides.
Polypeptide (protein): A polymer composed of multiple amino acid units
linked by peptide bonds.
Polyploid: A multiple of the haploid chromosome number that results
from chromosome replication without nuclear division.
Polysaccharide: A polymer composed of multiple units of monosaccharide.
Polyvalent vaccine: A recombinant organism into which has been cloned
antigenic determinants from a number of different disease-causing organisms.
Population: A local group of organisms belonging to the same species and
capable of interbreeding.
Post-consumer materials: Materials that a consumer has finished using,
which the consumer may sell, give away, or discard as wastes.
Primary cell culture: The maintenance of growth of cells dissociated from
the parental tissue in culture medium is known as primary cell culture.
Primary cell: A cell or cell line taken directly from a living organism,
which is not immortalized.
Primary material: A commercial material produced from virgin materials
used for manufacturing basic products. Examples include wood pulp, iron
ore, and silica sand.
Primer: A short sequence of oligonucleotides that hybridizes with template
strand and provides initiation for the nucleic acid synthesis.
Prion: Proteinaceous infectious particle.
Privatization: A general term referring to a range of contracts and other
agreements that transfer the provision of some services or production from
the public sector to private firms or organizations.
Useful Terms and their Meanings of Environmental Biotechnology A.53

Probe: A sequence of DNA or RNA, labeled or marked with a radioactive

isotope, used to detect the presence of complementary nucleotide sequences.
Processing: Preparing MSW materials for subsequent use or management,
using processes such as baling, magnetic separation, crushing, and shredding.
The term is also sometimes used to mean separation of recyclables from mixed
MSW.
Processors: Intermediate operators that handle recyclable materials from
collectors and generators for the purpose of preparing materials for recycling
(material recovery facilities, scrap metal yards, paper dealers, and glass
beneficiation plants). Processors act as intermediaries between collectors and
end users of recovered materials.
Producer responsibility: A system in which a producer of products or
services takes responsibility for the waste that results from the products
or services marketed, by reducing materials used in production, making
repairable or recyclable goods, and/ or reducing packaging.
Prokaryote: A cell lacking a true nucleus; its DNA is usually in one long
strand (E.g. Bacterial Cell).
Promoter: A region of DNA extending 150-300 bp upstream from the
transcription initiation site that contains binding sites for RNA polymerase
and a number of proteins that regulate the rate of transcription of the adjacent
gene.
Pronucleus: Either of the two haploid gamete nuclei just prior to their
fusion in the fertilized ovum.
Protease: An enzyme that cleaves peptide bonds that link amino acids in
protein molecules.
Protein engineering: Production and modification of proteins for
medicinal, industrial, and research purposes.
Protein kinase: An enzyme that adds phosphate groups to a protein
molecule at serine, threonine, or tyrosine residues.
Protein targeting: The process of transport of proteins from one
compartment to other with in a cell. Also called as protein sorting.
Protein: A polymer of amino acids linked via peptide bonds and which
may be composed of two or more polypeptide chains.
Proteinaceous infectious particle (Prion): A proposed pathogen composed
only of protein with no detectable nucleic acid and which is responsible for
Creutzfeldt-Jakob disease and kuru in humans and scrapie in sheep.
Proteolytic: The ability to break down protein molecules.
Proteome: The complete protein complement of cells, tissues, and
organisms is referred to as its proteome.
Proteomics: Large-scale characterization of the entire protein complement
of cells, tissues, and organisms is called proteomics.
A.54 Environmental Biotechnology

PTFE: Poly Tetra Fluoro Ethylene

pUC: A widely used expression plasmid containing a galactosidase gene.
Putrescible: Subject to decomposition or decay. Usually, used in reference
to food wastes and other organic wastes that decay quickly.
Pyrolysis: Chemical decomposition of a substance by heat in the absence
of oxygen, resulting in various hydrocarbon gases and carbon-like residue.
Quorum sensing: It is a type of decision-making process used by
decentralized groups to coordinate behavior. Many species of bacteria use
quorum sensing to coordinate their gene expression according to the local
density of their population. Similarly, some social insects use quorum sensing
to make collective decisions about where to nest. In addition to its function
in biological systems, quorum sensing has several useful applications for
computing and robotics.
R- HuEPO: Recombinant human erythropoietin
Radioactive waste: Waste material containing radioactive elements in
amounts greater than those normally present in the environment. Such waste
is generated in large amounts by nuclear reactors used for production of
electric power or of plutonium for weapons manufacture. Much low-level
waste also results from uranium and phosphate mining and milling, industrial
processes, laboratory research, and discarded materials that were used in
medical diagnosis and therapy.
Raffinate: The aqueous solution that is taken off after solvent extraction.
RAPD: Randomly Amplified Polymorphic DNA—A PCR based method
of DNA profiling. It basically involves the amplification of DNA sequences
using random primers, and use of genetic fingerprints to identify individual
organisms (mostly plants).
RBS: Ribosome Binding site.
Reading frame: A series of triplet codons beginning from a specific
nucleotide. Depending on where one begins, each DNA strand contains three
different reading frames.
Recessive (-acting) Oncogene (anti-oncogene): A single copy of this gene
is sufficient to suppress cell proliferation; the loss of both copies of the gene
contributes to cancer formation.
Recessive gene: Characterized as having a phenotype expressed only
when both copies of the gene are mutated or missing.
Recognition sequence (site): A nucleotide sequence composed typically
of 4, 6, or 8 nucleotides—that is recognized by a restriction endonuclease. Type
II enzymes cut (and their corresponding modification enzymes methylate)
within or very near the recognition sequence.
Recombinant DNA (rDNA) technology: The techniques involved in the
construction, and use of recombinant DNA molecules.
Useful Terms and their Meanings of Environmental Biotechnology A.55

Recombinant DNA: The process of cutting and recombining DNA

fragments from different sources as a means to isolate genes or to alter their
structure and function.
Recombinant Protein: A protein that is produced by the expression of a
cloned gene of a recombinant DNA molecules.
Recombinant: A cell that results from recombination of genes.
Recombination frequency: The frequency at which crossing over occurs
between two chromosomal loci—the probability that two loci will become
unlinked during meiosis.
Recyclables: Those materials recovered from the solid waste stream and
transported to a processor or end user for recycling.
Recycling: The process of transforming materials into raw materials for
manufacturing new products, which may or may not be similar to the original
product.
Red biotechnology: Red biotechnology is applied to medical processes.
Some examples are the designing of organisms to produce antibiotics, and the
engineering of genetic cures through genomic manipulation.
Refuse: A term often used interchangeably with solid waste.
Refuse-derived fuel (RDF): Fuel produced from MSW that has
undergone processing. Processing can include separation of recyclables and
noncombustible materials, shredding, size reduction, and pelletizing.
Regulatory gene: A gene whose protein controls the activity of other
genes or metabolic pathways.
Relaxed plasmid: A plasmid that replicates independently of the main
bacterial chromosome and is present in 10-500 copies per cell.
Renature: The reannealing (hydrogen bonding) of single-stranded DNA
and/or RNA to form a duplex molecule.
Replicon: A chromosomal region containing the DNA sequences necessary
to initiate DNA replication processes.
Repressor: A DNA-binding protein in prokaryotes that blocks gene
transcription by binding to the operator.
Residential Waste: Waste generated by single or multi-family homes
including newspapers, clothing, disposable tableware, food packaging, cans
and bottles, food scraps and yard trimmings. Excludes food wastes and yard
trimmings diverted to backyard composting (onsite).
Residues: The materials remaining after processing, incineration,
composting or recycling activities that have been completed. Usually, disposed
of in a landfill.
Resource recovery: The extraction and utilization of materials and energy
from wastes.
A.56 Environmental Biotechnology

Restriction endonuclease (enzyme): A class of endonucleases that cleaves

DNA after recognizing a specific sequence, such as BamH1 (GGATCC),
EcoRI (GAATTC), and HindIII (AAGCTT). Type I. Cuts nonspecifically a
distance greater than 1000 bp from its recognition sequence and contains
both restriction and methylation activities. Type II. Cuts at or near a short,
and often symmetrical, recognition sequence. A separate enzyme methylates
the same recognition sequence. Type III. Cuts 24-26 bp downstream from a
short, asymmetrical recognition sequence. Requires ATP and contains both
restriction and methylation activities.
Restriction-Fragment-Length Polymorphism (RFLP): Differences
in nucleotide sequence between alleles at a chromosomal locus result in
restriction fragments of varying lengths detected by Southern analysis.
Retro virus: A member of a class of RNA viruses that utilizes the enzyme
reverse transcriptase to reverse copy its genome into a DNA intermediate,
which integrates into the host-cell chromosome. Many naturally occurring
cancers of vertebrate animals are caused by retroviruses.
Reuse: The use of a product more than once in its original form, for the
same or a new purpose. Examples include refilling glass or plastic bottles,
repairing wood pallets, using cardboard or plastic containers for storage and
returning milk containers.
Reverse genetics: Using linkage analysis and polymorphic markers to
isolate a disease gene in the absence of a known metabolic defect, and then
using the DNA sequence of the cloned gene to predict the amino acid sequence
of its encoded protein.
Reverse Osmosis: It is a process in which heavy metals are separated by a
semi-permeable membrane at a pressure greater than osmotic pressure caused
by the dissolved solids in wastewater. The disadvantage of this method is that
it is expensive.
Reverse transcriptase (RNA-dependent DNA polymerase): An enzyme
isolated from retrovirus-infected cells that synthesizes a complementary (c)
DNA strand from an RNA template.
Reverse transcription: The process of synthesis of DNA from RNA.
RFLP: Restriction Fragment Length Polymorphism. A restriction fragment
with variable lengths due to the presence of polymorphic restriction sites at
one or both ends.
Rhizobaceria : These are root-colonizing bacteria that form a symbiotic
relationship with many legumes.
Rhizobia: These are soil bacteria that fix nitrogen (diazotrophy) after
becoming established inside root nodules of legumes (Fabaceae). Rhizobia
require a plant host; they cannot independently fix nitrogen. Morphologically,
they are generally gram-negative, motile, non-sporulating rods. The first
species of rhizobia, R. leguminosarum, was identified in 1889, and all further
Useful Terms and their Meanings of Environmental Biotechnology A.57

species were initially placed in the Rhizobium genus. However, more advanced
methods of analysis have revised this classification, and now, there are many
in other genera. Most research has been done on crop and forage legumes such
as clover, beans, and soy. However, recently more work is occurring on North
American legumes. Although much of the nitrogen is removed when protein-
rich grain or hay is harvested, significant amounts can remain in the soil for
future crops. This is especially important when nitrogen fertilizer is not used,
as in organic rotation schemes or some less-industrialized countries. Nitrogen
is the most commonly deficient nutrient in many soils around the world and
it is the most commonly supplied plant nutrient. Supply of nitrogen through
fertilizers has severe environmental concerns. Nitrogen fixation by Rhizobium
is also beneficial to the environment.
Rhizofiltration: The uptake of contaminants by the roots of plants which
are immersed in water.
Rhizosphere: Zone of soil immediately adjacent to plant roots in which
the kinds, numbers, or activities of microorganisms differ from that of the bulk
soil.
Ribosomal RNA (rRNA): The RNA component of the ribosome.
Ribosome: Cellular organelle that is the site of protein synthesis during
translation.
Ribosome-binding site: The region of an mRNA molecule that binds the
ribosome to initiate translation.
RNA (ribonucleic acid): An organic acid composed of repeating nucleotide
units of adenine, guanine, cytosine, and uracil, whose ribose components are
linked by phosphodiester bonds.
RNA polymerase : Transcribes RNA from a DNA template.
RNA vaccines: RNA molecules which can synthesize antigenic proteins
and offer immunity.
RNAse: Ribonuclease
Root nodule: Specialized structure occurring on roots, especially of
leguminous plants, in which bacteria fix nitrogen and make it available for
the plant.
rRNA: Ribosomal RNA
RT-PCR: Reverse transcriptase Polymerase Chain Reaction
Rubbish: A general term for solid waste. Sometimes used to exclude food
wastes and ashes.
Saccharification: It is the process of hydrolyzing a complex carbohydrate
into a simple soluble fermentable sugar. Starch or oligosaccharides can be
saccharified to produce glucose using glucoamylase enzyme.
Saccharomyces cerevisiae: It is a species of budding yeast. It is perhaps the
most useful yeast owing to its use since ancient times in baking and brewing.
A.58 Environmental Biotechnology

It is believed that it was originally isolated from the skins of grapes. It is one
of the most intensively studied eukaryotic model organisms in molecular
and cell biology, much like Escherichia coli as the model prokaryote. It is the
microorganism behind the most common type of fermentation. Saccharomyces
cerevisiae cells are round to ovoid, 5-10 micrometers in diameter. It reproduces
by a division process known as budding. Many proteins important in human
biology were first discovered by studying their homologs in yeast; these
proteins include cell cycle proteins, signaling proteins, and protein-processing
enzymes.
Salmonella: A genus of rod-shaped, gram-negative bacteria that are a
common cause of food poisoning.
Sanitary landfill: An engineered method of disposing of solid waste on
land, in a manner that meets most of the standard specifications, including
sound silting, extensive site preparation, proper leachate and gas management
and monitoring, compaction, daily and final cover, complete access control,
and record-keeping.
Saprophytic microorganisms: Microbes feeding on organic matter from
dead organisms (as opposed to parasitic microorganisms, feeding on living
organisms and causing diseases). Bacteria and fungi which lead to decay of
organic debris participate in the circulation of matter in nature.
Satellite DNA: Repetitive DNA that forms a satellite band in a density
gradient.
Satellite RNA (viroids): A small, self-splicing RNA molecule that
accompanies several plant viruses, including tobacco ring spot virus.
Scale up : The expansion of laboratory experiments to full-sized industrial
processes.
Scrubber: Emission control device in an incinerator, used primarily to
control acid gases, but also to remove some heavy metals.
SDS-PAGE: Sodium Dodecyl Sulphate - Polyacrylamide gel Electropho-
resis.
Secondary material: A material recovered from post-consumer wastes for
use in place of a primary material in manufacturing a product.
Secondary metabolite: A metabolite that is not required for the growth
and maintenance of cellular functions.
Secure landfill: A disposal facility designed to permanently isolate
wastes from the environment. This entails burial of the wastes in a landfill
that includes clay and/or synthetic liners, leachate collection, gas collection (in
cases where gas is generated), and an impermeable cover.
Selectable marker: A gene whose expression allows one to identify cells
that have been transferred or transfected with a vector containing the marker
gene.
Useful Terms and their Meanings of Environmental Biotechnology A.59

Self-pollination: Pollen of one plant is transferred to the female part of

the same plant or another plant with the same genetic makeup.
Semi-conservative replication: During DNA duplication, each strand of a
parent DNA molecule is a template for the synthesis of its new complementary
strand. Thus, one half of a preexisting DNA molecule is conserved during each
round of replication.
Septage: Sludge removed from a septic tank (a chamber that holds human
excreta).
Septic tanks: Anaerobic digesters of solids of the sewage settled at the
bottom of tanks.
Sequence hypothesis: Francis Crick’s seminal concept that genetic
information exists as a linear DNA code; DNA and protein sequence are
colinear.
Sequence-Tagged Site (STS): A unique (single-copy) DNA sequence used
as a mapping landmark on a chromosome.
Setout container: A box or bucket used for residential waste that is placed
outside for collection.
Sewage sludge: A semi-liquid residue that settles to the bottom of canals
and pipes carrying sewage or industrial wastewaters, or in the bottom of tanks
used in treating wastewaters.
Sewage treatment: It is the process of removing contaminants from
wastewater and household sewage, both runoff (effluents) and domestic. It
includes physical, chemical, and biological processes to remove physical,
chemical and biological contaminants. Its objective is to produce a waste
stream (or treated effluent) and a solid waste or sludge suitable for discharge
or reuse back into the environment.
Sewage: The liquid waste arising mainly from domestic and industrial
sources.
Sexual reproduction: The process where two cells (gametes) fuse to form
one hybrid, fertilized cell.
Shotgun approach: A technique for sequencing of genome in which the
molecules to be sequenced are randomly broken down into fragments, which
are then individually sequenced.
Shuttle vectors: The plasmid vectors that are designed to replicate in two
different hosts e.g. E. coli and Streptomyces sp.
Siderophore: A low molecular weight Fe- chelating protein synthesized
by several soil microorganisms.
Signal peptide: A short sequence of amino acids at the N terminal end of
some proteins that facilitates the protein to cross membrane.
Signal transduction: The biochemical events that conduct the signal of a
hormone or growth factor from the cell exterior, through the cell membrane,
A.60 Environmental Biotechnology

and into the cytoplasm. This involves a number of molecules, including

receptors, proteins, and messengers.
Single Cell Protein (SCP): Cells or protein extracts of microorganisms
produced in large quantities for use as human or animal protein supplement.
Site remediation: Treatment of a contaminated site by removing
contaminated solids or liquids or treating them on-site.
Site-directed mutagenesis: The technique used to produce a specified
mutation at a predetermined position in a DNA molecule.
Sludge: The semi-solid mass produced during the course of sewage/waste
water treatment processes.
Small Nuclear RNA (snRNA). Short RNA transcripts of 100-300 bp that
associate with proteins to form small nuclear ribonucleoprotein particles
(snRNPs), which participate in RNA processing.
SNPs: Single Nucleotide Polymorphisms
Somaclonal variation: The genetic variations found in the cultured plant
cells when compared to a pure breeding strain.
Somatic cell gene therapy: The repair or replacement of a defective gene
within somatic tissue.
Somatic cell: Any body cell as opposed to germ cell. Somatic cell is non-
reproductive and divides by mitosis.
Somatic embryogenesis: Formation of embryos from asexual cells.
Somatotrophin: Human growth hormone.
Source reduction: The design, manufacture, acquisition, and reuse of
materials so as to minimize the quantity and/or toxicity of waste produced.
Source separation: Setting aside of compostable and recyclable materials
from the waste stream before they are collected with other MSW, to facilitate
reuse, recycling, and composting.
Southern hybridization (Southern blotting): A procedure in which DNA
restriction fragments are transferred from an agarose gel to a nitrocellulose
filter, where the denatured DNA is then hybridized to a radioactive probe
(blotting).
Sparger: A device that introduces air into a bioreactor in the form of a fine
stream.
Special wastes: Wastes that are ideally considered to be outside the
MSW stream, but which sometimes enter it and must often be dealt with by
municipal authorities. These include household hazardous waste, medical
waste, construction and demolition debris, war and earthquake debris, oils,
wet batteries, sewage sludge, human excreta, slaughterhouse waste, and
industrial waste.
Species: A classification of related organisms that can freely interbreed.
Useful Terms and their Meanings of Environmental Biotechnology A.61

Spore: A form taken by certain microbes that enables them to exist in a

dormant stage. It is an asexual reproductive cell.
Staphylococcus aureus: Is the most common cause of staph infections. It is
a spherical bacterium, frequently found in the nose and skin of a person.
Stationary phase: The plateau of the growth curve after log growth,
during which cell number remains constant. New cells are produced at the
same rate as older cells die.
Stem cells: A progenitor cell that is capable of dividing continuously
through out the life of an organism.
Sticky end: A protruding, single-stranded nucleotide sequence produced
when a restriction endonuclease cleaves off center in its recognition sequence.
Stirred tank fermenter: A fermentation vessel in which the cells or
microorganisms are mixed by mechanically driven impellers.
Stringency: Reaction conditions — notably temperature, salt, and pH—
that dictate the annealing of single-stranded DNA/DNA, DNA/RNA, and
RNA/RNA hybrids. At high stringency, duplexes form only between strands
with perfect one-to-one complementarity; lower stringency allows annealing
between strands with some degree of mismatch between bases.
Stringent plasmid: A plasmid that only replicates along with the main
bacterial chromosome and is present as a single copy, or at most several copies,
per cell. (See plasmid.)
Structure-functionalism: The scientific tradition that stresses the
relationship between a physical structure and its function, for example, the
related disciplines of anatomy and physiology.
STS: Sequence-tagged site.
Sub-cloning: The process of tranferring a cloned DNA fragment from one
vector to another.
Sub-culturing: Sub-culturing involves removing the growth media,
washing the plate, disassociating the adherent cells, usually enzymatically or
removing by using pipette, and diluting the cell suspension into fresh media.
Subsidy: Direct or indirect payment from government to businesses,
citizens, or institutions to encourage a desired activity.
Sub-unit vaccine: A vaccine composed of a purified antigenic determinant
that is separated from the virulent organism.
Superbug: The first genetically engineered organism (bacterial strain of
Pseudomonas) that was patented. It carries different hydrocarbon-degrading
genes on plasmids.
Super-coiled plasmid: The predominant in vivo form of plasmid, in which
the plasmid is coiled around histone-like proteins. Supporting proteins are
stripped away during extraction from the bacterial cell, causing the plasmid
molecule to supercoil around itself in vitro.
A.62 Environmental Biotechnology

Super-gene: A group of neighboring genes on a chromosome that tend to

be inherited together and are sometimes functionally related.
Supernatant: The soluble liquid & action of a sample after centrifugation
or precipitation of insoluble solids.
Super-ovulation: The process of inducing more ovarian follicles to ripen
and produce more eggs.
Suspension cultures: Cells which do not attach to the surface of the
culture vessel and grow in a suspended manner in the culture medium are
called suspension cultures.
Symbiosis: The term symbiosis commonly describes close and often long-
term interactions between different biological species. The term was first used
in 1879 by the German mycologist Heinrich Anton de Bary, who defined it as
“the living together of unlike organisms”. The definition of symbiosis is in
flux, and the term has been applied to a wide range of biological interactions.
The symbiotic relationship may be categorized as being mutualistic, parasitic,
or commensal in nature. Others define it more narrowly, as only those
relationships from which both organisms benefit, in which case it would be
synonymous with mutualism.
Synapsis: The pairing of homologous chromosome pairs during prophase
of the first meiotic division, when crossing over occurs.
Taq Polymerase: A heat-stable DNA polymerase isolated from the
bacterium Therrnus aquaticus, used in PCR. (See Polymerase.)
TATA box: An adenine- and thymine-rich promoter sequence located 25-
30 bp upstream of a gene, which is the binding site of RNA polymerase.
T-DNA (transfer DNA, tumor-DNA: The transforming region of DNA in
the Ti plasmid of Agrobacterium tumefaciens.
Telomere: The end of a chromosome.
Template: An RNA or single-stranded DNA molecule upon which a
complementary nucleotide strand is synthesized.
Teratogens: Chemical or biological factors causing disturbances in
embryonic development leading sometimes to serious malformations in
embryos. Teratogenic properties can be found in some pesticides, fungal
toxins and viruses (e.g. the rubella virus during early pregnancy).
Termination codon: Any of three mRNA sequences (UGA, UAG, UAA)
that do not code for an amino acid, and thus, signal the end of protein synthesis.
Also known as stop codon.
Terminator Region: A DNA sequence that signals the end of transcription.
Tetracycline: An antibiotic that interferes with protein synthesis in
prokaryotes.
Thaumatin: A protein extracted from berries which is about 3000 times
sweeter than sucrose.
Useful Terms and their Meanings of Environmental Biotechnology A.63

Thiobacillus ferroxidans: Micro-organisms that can get all their energy

from oxidising Fe2+ to Fe3+ and are able to live solely on sulphides and in acid
conditions.
Thymidine kinase (tk): An enzyme that allows a cell to utilize an alternate
metabolic pathway for incorporating thymidine into DNA. Used as a selectable
marker to identify transfected eukaryotic cells.
Ti (tumor-inducing) PLASMID: A giant plasmid of Agrobacterium
tumefaciens that is responsible for tumor formation in infected plants. Ti
plasmids are used as vectors to introduce foreign DNA into plant cells.
TIGR: The Institute of Genomic Research
Tipping fee: A fee for unloading or dumping waste at a landfill, transfer
station, incinerator, or recycling facility.
Tipping floor: Unloading area for vehicles that are delivering MSW to a
transfer station or incinerator.
Tissue culture: A process where individual cells, or tissues of plants or
animals, are grown artificially.
Tissue engineering: The application of the principles of engineering to
cell culture for the construction of functional anatomical units.
T-Lymphocytes (T cells): The lymphocytes that are dependent on the
thymus for their differentiation, and are involved in cell-mediated immune
response.
Tm: Melting temperature
Totipotent: A term used to describe a cell that is not committed to a
single developmental pathway, and thus it is capable of forming all types of
differential cells.
Traditional (old) biotechnology: The age-old practices for the preparation
of foods and beverages, based on the natural capabilities of microorganisms.
Trans-capsidation: The partial or full coating of the nucleic acid of one
virus with a coat protein of a differing virus.
Transcription: The process of creating a complementary RNA copy of
DNA.
Transduction: The transfer of DNA sequences from one bacterium to
another via lysogenic infection by a bacteriophage (transducing phage).
Transfection: The uptake and expression of a foreign DNA sequence by
cultured eukaryotic cells.
Transfer point: A designated point, often at the edge of a neighborhood,
where sma collection vehicles transfer waste to larger vehicles for transport to
disposal sites.
Transfer station: A major facility at which MSW from collection vehicles
is consolidated into loads that are transported by larger trucks or other means
to more distant final disposal facilities, typically landfills.
A.64 Environmental Biotechnology

Transfer: The act of moving waste from a collection vehicle to a larger

transport vehicle.
Transformant: In prokaryotes, a cell that has been genetically altered
through the uptake of foreign DNA. In higher eukaryotes, a cultured cell that
has acquired a malignant phenotype.
Transformation efficiency: The number of bacterial cells that uptake and
express plasmid DNA divided by the mass of plasmid used (in transformants/
microgram).
Transformation: In prokaryotes, the natural or induced uptake and
expression of a foreign DNA sequence—typically a recombinant plasmid in
experimental systems. In higher eukaryotes, the conversion of cultured cells
to a malignant phenotype—typically through infection by a tumor virus or
transfection with an oncogene.
Transforming oncogene: A gene that upon transfection converts a
previously immortalized cell to the malignant phenotype.
Transgenic animal: Genetically engineered animal or offspring of
genetically engineered animals. The transgenic animal usually contains
material from at lease one unrelated organism, such as from a virus, plant, or
other animal (See Transgenic).
Transgenic plant: Genetically engineered plant or offspring of genetically
engineered plants. The transgenic plant usually contains material from at least
one unrelated organisms, such as from a virus, animal, or other plant. (See
Transgenic).
Transgenic: An organism in which a foreign DNA gene (a transgene) is
incorporated into its genome early in development. The transgene is present
in both somatic and germ cells, is expressed in one or more tissues, and is
inherited by offspring in a Mendelian fashion.
Transition-state intermediate: In a chemical reaction, an unstable and
high-energy configuration assumed by reactants on the way to making
products. Enzymes are thought to bind and stabilize the transition state, thus
lowering the energy of activation needed to drive the reaction to completion.
Translation: The process of converting the genetic information of an
mRNA on ribosomes into a polypeptide. Transfer RNA molecules carry the
appropriate amino acids to the ribosome, where they are joined by peptide
bonds.
Translocation: The movement or reciprocal exchange of large-
chromosomal segments, typically between two different chromosomes.
Transposition: The movement of a DNA segment within the genome of
an organism.
Transposon (transposable, or movable genetic element): A relatively
small DNA segment that has the ability to move from one chromosomal
position to another.
Useful Terms and their Meanings of Environmental Biotechnology A.65

tRNA (transfer RNA): The class of small RNA molecules that transfer
amino acids to the ribosome during protein synthesis. See Transfer RNA.
Trypsin: A proteolytic enzyme that hydrolyzes peptide bonds on the
carboxyl side of the amino acids arginine and lysine.
TSCA: The Toxic Substances Control Act
Tumor virus: A virus capable of transforming a cell to a malignant
phenotype.
Ultrafiltration: They are pressure-driven membrane operations that use
porous membranes for the removal of heavy metals. The main disadvantage
of this process is the generation of sludge.
Ultraviolet (UV): UV light is electromagnetic radiation with a wavelength
shorter than that of visible light, but longer than X-rays, in the range
10 nm to 400 nm, and energies from 3 eV to 124 eV. It is so named because
the spectrum consists of electromagnetic waves with frequencies higher than
those that humans identify as the color violet. UV light is found in sunlight
and is emitted by electric arcs and specialized lights such as black lights.
As an ionizing radiation, it can cause chemical reactions, and causes many
substances to glow or fluoresce. Most people are aware of the effects of UV
through the painful condition of sunburn, but the UV spectrum has many
other effects, both beneficial and damaging, on human health. The discovery
of UV radiation was intimately associated with the observation that silver
salts darken when exposed to sunlight. In 1801, the German physicist Johann
Wilhelm Ritter made the hallmark observation that invisible rays just beyond
the violet end of the visible spectrum were especially effective at darkening
silver chloride-soaked paper. He called them “de-oxidizing rays” to emphasize
their chemical reactivity and to distinguish them from “heat rays” at the other
end of the visible spectrum. The simpler term “chemical rays” was adopted
shortly thereafter, and it remained popular throughout the 19th century. The
terms chemical and heat rays were eventually dropped in favor of ultraviolet
and infrared radiation, respectively.
Ultraviolet light: Ultraviolet light is electromagnetic energy that is located
in the electromagnetic spectrum at wavelengths between those of X-rays
and visible light. UV light, that is effective is destroying microbial entities,
is located in the 200- to 310-nm range of the energy spectrum. Most typical
applications of UV at water treatment plants apply UV light in the wavelength
range of 250 to 270 nm.
Vaccine: A preparation of dead or weakened pathogen, or of derived
antigenic determinants, that is used to induce formation of antibodies or
immunity against the pathogen.
Vaccinia: The cowpox virus used to vaccinate against smallpox and,
experimentally, as a carrier of genes for antigenic determinants cloned from
other disease organisms.
A.66 Environmental Biotechnology

Variable Surface Glycoprotein (VSG): One, of a battery of antigenic

determinants expressed by a microorganism, to elude immune detection.
Variation: Differences in the frequency of genes and traits among
individual organisms within a population.
Vector: A vehicle for carrying cloned DNA.
Vectors: Organisms that carry disease causing pathogens. At landfills,
rodents, flies, and birds are the main vectors that spread pathogens beyond
the landfill site.
Vegetative propagation: The asexual propagation of plants from the
detached parts of the plants.
Vermiculture: Worm culture.
Viral oncogene: A viral gene that contributes to malignancies in vertebrate
hosts.
Virgin materials: Any basic material for industrial processes that has
not previously been used, for example, wood-pulp trees, iron ore, crude oil,
bauxite.
Viroid: A plant pathogen that consists of a naked RNA molecule of
approximately 250-350 nucleotides, whose extensive base pairing results in a
nearly correct double helix. (See Satellite RNA.)
Virulence: Is the degree of pathogenicity of an organism, or in other
words, the relative ability of a pathogen to cause disease. The word virulent,
which is the adjective for virulence, derives from the Latin word virulentus,
which means “full of poison.” From an ecological point of view, virulence can
be defined as the host’s parasite-induced loss of fitness.
Virus: An infectious particle composed of a protein capsule and a nucleic
acid core, which is dependent on a host organism for replication. A double-
stranded DNA copy of an RNA virus genome that is integrated into the host
chromosome during lysogenic infection.
VSG: Variable surface glycoprotein
Waste characterization study: An analysis of samples from a waste stream
to determine its composition.
Waste collector: A person employed by a local authority or a private firm
to collect waste from residences, businesses, and community bins.
Waste dealer: A middleman who buys recyclable materials from waste
generators and itinerant buyers and sells them, after sorting and some
processing, to wholesale brokers or recycling industries.
Waste Generation: The amount (weight or volume) of materials and
products that enter the waste stream before recycling, composting, landfilling
or combustion takes place.
Useful Terms and their Meanings of Environmental Biotechnology A.67

Waste management hierarchy: A ranking of waste management

operations according to their environmental or energy benefits. The purpose
of the waste management hierarchy is to make waste management practices as
environmentally sound as possible.
Waste picker:A person who picks out recyclables from mixed waste
wherever it may be temporarily accessible or disposed of.
Waste reduction: All means of reducing the amount of waste that is
produced initially and that must be collected by solid waste authorities. This
ranges from legislation and product design to local programs designed to
keep recyclables and compostables out of the final waste stream.
Waste Stream: The total flow of solid waste from homes, businesses,
institutions and manufacturing plants that must be recycled, incinerated or
disposed of in landfills; or any segment thereof, such as the “residential waste
stream” or the “recyclable waste stream.”
Waste-to-energy (WTE) plant: A facility that uses solid waste materials
(processed or raw) to produce energy. WTE plants include incinerators that
produce steam for district heating or industrial use, or that generate electricity;
they also include facilities that convert landfill gas to electricity.
Waste-To-Energy Facility/Combustor: A facility where recovered MSW
is converted into a usable form of energy, usually through combustion. SC
identifies waste-to-energy activities (not incineration) as recycling.
Wastewater: The used water from households and industrial plants. Storm
runoff and infiltration water may also be included in the wastewater.
Water table: Level below the earth’s surface at which the ground becomes
saturated with water.
Weathering: Disintegration and chemical decomposition of rocks by slow
reaction with air and water at or near the earth’s surface.
Weed: An undesirable plant.
Weediness: Unwanted effects of a plant.
Wetland: An area that is regularly wet or flooded and has a water table
that stands at or above the land surface for at least part of the year.
White biotechnology: White biotechnology, also known as grey
biotechnology, is biotechnology applied to industrial processes. An example
is the designing of an organism to produce a useful chemical. White
biotechnology tends to consume less in resources than traditional processes
used to produce industrial goods.
Wild type: An organism as found in nature; the organism before it is
genetically engineered.
Windrow: An elongated pile of aerobically composting materials that
are turned periodically to expose the materials to oxygen and to control the
temperature to promote biodegradation.
A.68 Environmental Biotechnology

Worin castings: The material produced from the digestive tracts of

worms as they live in earth or compost piles. The castings are rich in nitrates,
potassium, phosphorous, calcium, and magnesium.
Working face: The length and width of the row in which waste is being
deposited at a landfill. Also known as the tipping face.
Worm culture: A relatively cool, aerobic composting process that uses
worms and microorganisms. Also known as vermiculture.
Xenobiotic: Compound foreign to biological systems. Often refers to
human-made compounds that are resistant or recalcitrant to biodegradation
and decomposition.
Xerophiles: These are extremophilic organisms that can grow and
reproduce in conditions with a low availability of water, also known as water
activity.
Xerophyte: Is a plant which is able to survive in an environment with
little available water or moisture, usually in environments where potential
evapotranspiration exceeds precipitation for all or part of the growing season.
X-Linked disease: A genetic disease caused by a mutation on the X
chromosome. In X-linked recessive conditions, a normal female “carrier”
passes on the mutated X chromosome to an affected son.
X-RAY crystallography: The diffraction pattern of X-rays passing through
a pure crystal of a substance.
YAC: Yeast Artificial Chromosome
Yard waste: Leaves, grass clippings, prunings, and other natural organic
matter discarded from yards and gardens.
Yoghurt: Is a dairy product produced by bacterial fermentation of milk.
Fermentation of the milk sugar (lactose) produces lactic acid, which acts on
milk protein to give yoghurt its texture and its characteristic tang.
Z-DNA: A region of DNA that is “flipped” into a left-handed helix,
characterized by alternating purines and pyrimidines, and which may be the
target of a DNA-binding protein.
Zoogleal film: A complex population of organisms that form a “slime
growth” on trickling-filter media and break down the organic matter in
wastewater.
Zygote: The fertilized egg formed by the fusion of two gametes.
Parts Per Million (ppm): Indicates the number of units of an element or
compound contained in a million units of soil.
2-DE: 2-Dimensional Gel Electrophoresis.
 Index

A Conservation of biodiversity, 1.34

Cryopreservation, 11.3
Agricultural productivity, 1.5
Cytotoxicity, 8.24
Algae-biotechnology, 9.1
Ammensalism, 2.18 D
Animal biotechnology, 11.1
Decomposition, 1.21
Antagonism, 2.16 DNA hybridization, 3.35
Apiculture
E
B Ecosystem, 1.2
Beneficial microorganisms, 2.36 Ecosystem modeling, 1.13
Biochemical oxygen demand, 3.12 Ectotrophic mycorrhiza, 2.21
Biodegradation, 3.12 Effective microorganisms, 5.1
Biofertilizers, 1.2 Endotrophic mycorrhiza, 2.21
Biofuels, 9.3 Environmental biotechnology, 1.1
Biological reprocessing, 7.10 Environmental microbiology, 2.1
Environmental monitoring, 1.2
Biomedical waste, 7.2
Environmental pollution, 1.2
Biomimicry, 14.5
Environmental protection agency, 1.4
Bioremediation, 1.2
Esterification, A.24
Bioresource technology
Eugenics, 15.3
Bio-safety, 14.6
Eutrophicaton, A.25
Biosafety management, 15.9
Biotransformation, 1.2 F
Fecal coliforms, A.25
C Fermentation, 1.5
Cellulose decomposition, 2.11 Food processing waste, A.26
Cellulosic ethanol, A.10
Chemoautotrophs, 3.2 G
Coliform index, A.15 Genetically engineered microbes, 1.2
Commensalisms, 2.17 Geomicrobiology, A.30
Composting , 1.17 Green biotechnology, A.31
I.2 Index

H O
Hazardous solid waste, 14.11 Oil spillage, 1.6
Hazardous waste, 4.25 Organic matter, 1.14
Human activities , 1.2 Organic pollutants, 3.22
Humus decomposition, 2.12
Hydraulic control, 6.2 P
Parasitism, 2.16
I
Pest management, 2.31
Indicator organisms, 4.22
Phosphorus cycle, 2.52
Industrial sludge, A.35
Photoheterotrophs, A.50
Industrial solid waste , 7.2
Phytoextraction, 6.2
Industrial sustainability , 14.1
Phytoremediaiton, 6.5
Intellectual property rights, 15.11
Phytostabilization 6.6
L Phytostimulation , 6.4
Lignin decomposition, 2.11 Phytotransformation , 6.3
Plant biotechnology, 10.1
M Pollutants, 1.2
Membrane filtration, 7.26 Polymerase chain reaction, 3.35
Metagenomics, A.40 Predation, 2.16
Metal accumulator plant species, 6.8
Proto-cooperation, 2.16
Metal excluders, 6.8
Putrefaction, 5.13
Metal indicators,6.8
Metal transformations, 1.12 Q
Methanogenesis, A.1
Quorum sensing, A.54
Microalgae, 1.20
Microbial ecology, 2.33 R
Microbial mats (biofilms), A.41
Reclaimed water, 7.29
Municipal solid waste, 1.19
Municipal waste reduction, 7.17 Reverse osmosis, 3.24

Mutualism, 2.16 Rhizodegradation, 6.4

Mycofiltration, A.43 Rhizofiltration, 6.2
Mycoremediation, A.44 Rhizosphere, 1.23
Risk assessment, 1.22
N
Nature, 1.1 S
Nitrogen cycle, 2.14 Sericulture
Nucleic acid-based techniques, 3.33 Silk synthesis
Index I.3

Soil fertility, 1.11 T

Soil microorganisms, 1.22 Treated wastewater, 7.29
Soil phytoedaphon, 2.6
Soil quality, 5.2 W
Solid waste disposal , 7.1 Wastewater management, 7.7

Sulphur cycle, 2.51 Wastewater treatment, 3.21

Water purification , 4.22
Sustainable agriculture, 1.9
Waterweeds , 7.25