What Is Environmental Biotechnology?

Introduction
• Environmental biotechnology is the application
of biotechnology to all aspects of the
environment.
• It is a huge and fast growing field, which
continues to revolutionize the understanding of
basic life sustaining processes in the
environment, by identifying and exploiting the
biomolecules and their utilities to provide clean
technologies, in order to counter environmental
problems.
• The ever increasing temptation to lead an improved
and comfortable life has caused significant damage to
the environment .
• The result of overuse and abuse of the environment by
human made processes are:
• Large scale industrialization
• Release of industrial and domestic wastes to the
environment
• Depletion of natural resources
• Faster rate of consumption of natural resources
• Degradation in the quality of land, air and water
• Scarcity of potable water
• This course is concerned with how biotechnology
can be applied to environmental problems and
issues.
• This will cover existing pollution in terms of the
detection and bioremediation of the
contaminants.
• There is also a need to reduce the production of
pollutants at sources.
• The reduction in the production of industrial
pollutants is known as “clean technology”.
• Every day, we read in the media stories about the CLIMATE
CHANGE, pollution and how we are about to destroy our
world through over exploitation and industrialization.
• There is no doubt that, as a species, living on a planet with
finite resources, we face many problems.
• Solution???????????????
• As a result, we have only one alternative and that is know our
nature (environment and the biological system) to use our
technology and combat any problems that arise from its use.
Environmental biotechnology

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 Environmental biotechnology
• living systems are used to solve environmental problems.
• While the environmental biotechnology has, in the past, been
mainly devoted to waste technology, the subject is beginning
to diversify;
• These improvements include preventing the discharge of
pollutants to the environment, cleaning up contaminated
environments, and generating valuable resources for human
society.
• Environmental biotechnology is essential to society and truly
unique as a technical discipline.

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 Importance of Environmental
Biotechnology
1. Environmental biotechnology is fundamentally rooted in waste, in its
various guises,

 typically being concerned with the remediation of contamination caused by

previous use,
 the impact reduction of current activity or
 the control of pollution.

2. The principal aims of this field are the manufacture of products in

environmentally harmonious ways,

 which allow for the minimization of harmful solids, liquids or gaseous outputs
or the clean-up of the residual effects of earlier human occupation.
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The waste minimization strategy should include the
five R policies.
1. In process - reduction, replacement, reuse and
recovery
2. After process- recycle

7
• Environmental biotechnologists may
– enhance or optimize conditions for existing biological
systems to make their activities happen faster or more
efficiently, or
– they resort to some form of alteration to bring about
the desired outcome.
• The variety of organisms which may play a part in
environmental applications of biotechnology is huge,
ranging from microbes through to trees and all are
utilized on one of the same three fundamental bases –
accept, acclimatize or alter.
• For the vast majority of cases, it is the approach of
accepting and making use of existing species in their
natural, unmodified form, which predominates.
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 The Scope for Use
• There are three key points for environmental
biotechnology interventions, namely -
– in the manufacturing process,
– waste management
– pollution control

• There is a growing realization world over that

biotechnology along with environmental engineering is
going to be the major thrust area in the new millennium.

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• The range of businesses to which environmental
biotechnology has potential relevance is almost limitless.
 The most apparent is with regard to waste.
• All commercial operations generate waste of one form or
another and for many, a proportion of what is produced is
biodegradable.
• With disposal costs rising steadily across the world,
dealing with refuse constitutes an increasingly high
contribution to overheads.
• Thus, there is a clear incentive for all businesses to
identify potentially cost-cutting approaches to waste and
employ them where possible.

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• Changes in legislation throughout Europe, US and
elsewhere, have combined to drive these issues
higher up the political agenda and biological
methods of waste treatment have gained far
greater acceptance.
• For those industries with particularly high biowaste
production, the various available treatment
biotechnologies can offer considerable savings.

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• Manufacturing industries can benefit from the
applications of whole organisms or isolated
biocomponents.
• Compared with conventional chemical processes,
microbes and enzymes typically function at lower
temperatures and pressures.
• The lower energy demands leads to reduced costs,
but also has clear benefits in terms of both the
environment and workplace safety.

12
Likewise,
• biotechnology can be of further commercial
significance by
converting low-cost organic feedstocks into high
value products
enzymatic reactions are more highly specific than
their chemical counterparts,
deriving final substances of high relative purity.

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• manufacturing companies produce wastewaters or
effluents, many of which contain biodegradable
contaminants, in varying degrees.
 traditional permitted discharges to sewer or
watercourses may be adequate for some,
 other industries, particularly those with recalcitrant or
highly concentrated effluents, have found significant
benefits to be gained from using biological treatment
methods themselves on site.
• biotechnology stands as a particularly cost-effective
means of reducing the pollution potential of
wastewater.

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• Likewise, Contaminated land is a growing concern
for the construction industry
 as it seeks to balance the need for more houses
and offices with wider social and environmental
goals.
• The reuse of former industrial sites
– Bioremediation technologies provide a competitive and
sustainable alternative.
• Environmental biotechnology must compete in a
world governed by the best practicable
environmental option (BPEO) and the best available
techniques not entailing excessive cost (BATNEEC).
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• With new developments in treatment technologies
appearing all the time, the list of what can be processed or
remediated by biological means is ever changing.
• The applications for which biotechnological solutions are
sought are also subject to alteration.
• For the biotech sector to keep abreast of these new
demands it may be necessary to examine and possibly
make use of organisms or their derivatives in ways
previously unimagined.
• This is the basis of innovation;
• the inventiveness of an industry is often a good measure of
its adaptability and commercial robustness.

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• Within a very short time, biotechnology has come to play an increasingly important
role in many aspects of everyday life.
• The upsurge of the ‘polluter pays’ principle, increasing pressure to revitalize the
likes of former industrial sites and recent developments within the waste industry
itself have combined to alter the viability of environmental biotechnology radically
in the last few years.
• Once an expensive and largely unfamiliar option, it has now become a realistic
alternative to many established approaches for manufacturing, land remediation,
pollution control and waste management.
• Against a background of burgeoning disposal costs and ever more stringent
legislation and liabilities, the application of biologically engineered solutions seems
certain to continue its growth.
• As a biotechnologists our purpose is a straightforward one:
• i.e. to present a fair reflection of the practical biological approaches currently
employed to address environmental problems, and to provide a working
knowledge of the science that underpins them.

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• Mix ethanol with petrol, MPs tell NOC
By Himalayan News Service
Published: Dec 20, 2007
Issues for Environmental management
• The International issues for attention through
environmental biotechnology include:
• Global warming and Climate change
 Global warming and Climate change
• Major threat to the existing biodiversity and
quality of environment (1990s)
• Some ecosystem might disappear while some
could experience dramatic changes in species
composition.
 Examples

• Increased irregularities in monsoon rains

(India and East Asia)
• Frequent occurrence of warm oceanic
currents
• Drought and forest fires
• Decline in amphibians in the tropical
mountain forest (Pounds et al. 1999)
• Decline in the ground water reserve in the
tropics
 Consequences
• Worldwide increase in energy consumption
• Deforestation
• Methane emission from rice fields and
swampy habitats
• Reduction in the productivity of the tropical
rain forest overtime has been recorded
 Marine pollution
• Attributed to the global increase in ocean
temperature
• Increasing incidence of coral bleaching (after
1989)
• The greatest degree of pollution has been noted
in the coastal oceans that contribute most of the
biomass to the terrestial and oceanic food chain
• nutrient loaded freshwater from land
– cause of changes in the density and diversity in the
coastal life form
• Eutrophication - toxic algal bloom and
sedimentation
• Methane gas production
 Example
• 90% of all the corals died over 1000s of square kilometer
in Indian ocean.(Goreau et al. 2000)
• 1997-1998 El Niño–Southern Oscillation (ENSO) event, in
which all ten reef provinces of the world were affected.
[The 1997–1998 El Niño was regarded as one of the most
powerful El Niño–Southern Oscillation events in recorded
history, resulting in widespread droughts, flooding and
other natural disasters across the globe. It caused an
estimated 16% of the world's reef systems to die, and
temporarily warmed air temperature by 1.5 °C (2.7 °F)
compared to the usual increase of 0.25 °C (0.45 °F) ]
 Air pollution
• Increased incidence of air pollution in last few
decades has been attributed to three major
areas:
• Industrialisation
• Vehicular exhaustion
• Deforestation
Minor
waste deposition and soil erosion
 Most important gases of concern
• Oxides of nitrogen and sulphur
• Volatile carbon compounds
• Greenhouse gases
 Energy crisis
• A global crisis is being felt in the energy sector.
• Crude oil price has gone to an all time high.
• Proportionate decrease in the coal and mineral
reserves
• Decline in forest cover in the developing
countries
• Search to reduce energy consumption through
efficient technology from conventional sources
• Environmental friendly technology to satify
energy demands which is increasing
exponentially.
 Oil spill
• Decline in the forest in the West Asian countries
is due to oil withdrawal and spills in the coastal
soil and ocean
• Accidental releases into the ocean environment
during exploration, removal and transport
• [On July 25, 2024, the Philippine-flagged
industrial fuel tanker MT Terranova[a] (IMO
number: 9092666) capsized and sank in Manila
Bay, off the east coast of Lamao Point, Limay,
Bataan, causing an ongoing oil spill. ]
 Largest oil-tanker spills in history
• Two enormously important oil-tanker spills that took
place in European waters were the Torrey Canyon
disaster off Cornwall, England, in 1967 (119,000 metric
tons of crude oil were spilled) and the Amoco Cadiz
disaster off Brittany, France, in 1978 (223,000 metric
tons of crude oil and ship fuel were spilled).
• In North America the Exxon Valdez oil spill of 1989 in
Prince William Sound, Alaska, caused great ecological
and economic damage, though it ranks well below the
largest oil-tanker spills in history if measured by the
amount of oil spilled (37,000 metric tons).
 Decline and losses of species
• Ecosystem unfriendly events:
– land conversion
– Pollution
– Unsustainable harvesting of natural resources and
– Introduction of exotic species
 Examples
• Land conversion is more intensive in tropical
forests
• Atmospheric nitrogen deposition is more in
northern temperate areas close to cities
• The ultimate causes of species loss are human
population growth, together with the
unsustainable pattern of consumption,
increasing production of wastes and pollution,
urban development
• Decline and extinction of species have emerged as major
environmental issues.
• As per red list by IUCN indicates 24% of mammals and 12%
of bird species are currently regarded as threatened (Hilton
Taylor, 2000)
• The Intergovernmental Science-Policy Platform on
Biodiversity and Ecosystem Services (IPBES) Global
Assessment Report on Biodiversity and Ecosystem Services
is the most comprehensive ever completed. It is the first
intergovernmental Report of its kind and builds on the
landmark Millennium Ecosystem Assessment of 2005,
introducing innovative ways of evaluating evidence.
– The Report finds that around 1 million animal and plant species
are now threatened with extinction, many within decades, more
than ever before in human history.
 Uses of Biotechnology in
Environmental Protection
• Applications of Biotechnology in environmental protection can play
a big role in adopting a sustainable development pathway.
• Biotechnology can immediately help by modifying the solid, liquid
and gaseous waste either by recycling or making new products so
that end product is less harmful to the environment.
• Replacing chemicals with biological materials using biotechnology is
another way to reduce the our harmful impact on the environment.
• five of the many ways in which Biotechnology can help play a role in
environmental protection.
– Bioremediation
– Biosensors
– Biofuels
– Biopesticides
– Bioenzymes
 MICROORGANISMS
IN RELATION TO ENVIRONMENTAL MANAGEMENT

• Microorganisms are very important in the

geochemical cycling of organic and inorganic
nutrients, and the biodegradation and
biotransformation of pollutants in the
environment.
 What makes microorganisms so suited for these tasks ?

• The defining feature of microorganisms is their small sizes.

1. High surface area to volume ratio
• The small sizes of microorganisms result in high surface area
to volume ratio, and this in turn allows for their extensive
interactions with the environment as compared to plant or
animal cells.
• The high surface to volume ratio is responsible for the high
metabolic rates of microorganisms.
 2. Metabolic flexibility & versatility
• Cells from higher plants or animals are relatively
inflexible in their metabolic capabilities.
• Many of their enzyme systems may change
during development, but are less responsive to
changing environmental conditions.
• In microorganisms, extensive adaptability is a
necessity determined by their relatively small
sizes and extensive interactions with the
environment.
• There are several reasons for this adaptability.

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 2.1 Efficient regulation of metabolism through
induction and repression
• Not all the structural genes are expressed all the time.
• The protein molecules are involved in all facets of the
microorganism's activities and processes.
• This requires that the microorganisms be very adaptable.
• Enzymes that are not needed currently are not normally held in
reserve.
• They are usually turned over immediately for other purposes.
• Therefore, many important catabolic enzymes are produced
only if the appropriate substrates are present.
• The regulatory mechanisms such as induction and repression
play a far greater role in microbial cell physiology and are more
easily recognizable than in other higher organisms.
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 2.2 Broad substrate specificity of microbial
enzymes & collective metabolic versatility
• Because of the requirement to be adaptable, many
microbial enzymes are also less specific compared to
those from higher organisms in terms of substrate range,
ie. microbial enzymes in general exhibit broader substrate
specificity than their mammalian counter parts.
• The broad substrate specificity is important in the
biodegradation of many xenobiotic pollutants, as the
microbial enzymes, normally adapted to using natural
substrates, can also catalyse reactions using these novel
compounds, for example, via co-metabolism.
• In fact, without this capability (based on broad substrate
specificity), many xenobiotic compounds would not be
biodegraded at all.
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• Microorganisms collectively hold the monopoly position in the
utilization and metabolism of many unique substrates.

• Moreover, they can collectively metabolize all naturally-

occurring compounds.

• are adaptable from the point of view of being able to survive in

a niche that requires feeding off others' metabolic end products.
In other words, microorganisms have learned to live with others
in a community.

• The collective metabolic flexibility of microorganisms are

important in engineered waste treatment systems.

• a collection of microorganisms can break down the wastes

more readily, provided there are no other toxic materials or
xenobiotics which are not amenable to biodegradation.

• The potential problem with broad substrate specificity is that

some microbial enzymes will attack compounds, eg. by co- 39
metabolism, leading to toxic or dead-end products.
 2.3 Ease of gene transfer & mobilization
• One reason for the metabolic flexibility & versatility of
microorganisms is the ease with which gene transfer occurs,
relative to cells from higher organisms.
• Gene transfer processes include conjugation, transformation,
transduction and transposition.
• Through the acquisition of new genetic elements,
microorganisms can quickly acquire new metabolic capabilities
in new surroundings & environments.
• Similarly, in the absence of selective agents, some
microorganisms can readily lose certain genetic elements. This
can be a concern in using genetically engineered
microorganisms where most of the desired genetic
characteristics are located on plasmids.
• One type of genetic trait that is of concern in environmental
sciences & public health is antibiotic resistance genes. 40
 3. Environmental dispersal of microorganisms
• The small dimension of the microorganisms is of considerable
ecological significance.
• They are found in conditions both extreme and moderate.
Sometimes, species distribution in many extreme locations is
equally diverse as those in soils from temperate climates.
• Because of their low mass, microorganisms are easily carried
by air currents.
• Environmental and nutritional conditions determine which
strains will grow and reproduce. This situation can be exploited
for the isolation of specific microorganisms by enrichment
culture.
• Microorganisms are also carried by water movement to far
away places.
• Microorganisms are also easily transported to far away places
by vectors such as animals, humans, insects, birds, etc.
• The ease with which microorganisms are carried to far
distances is a concern for environmental and public health
reasons when one is considering the use of bioaugmentation 41
(ie. inoculating sites with microorganisms) in bioremediation.
 4. UTILIZATION OF MIXED SUBSTRATES
• Mixed substrate systems are characterized by the presence of
a multiple sources of nutrients that serve a similar physiological
function.
• The term "mixed substrates" encompass all types of nutrients;
such as the carbon sources, energy sources, N-sources, etc.
• Mixed substrates are very relevant in environmental
management.
• The great majority of biological waste treatment processes
involves the metabolism of mixed substrates.
• Also, culture media of many industrial fermentations are
frequently formulated so that they contain complex mixtures of
nutrients.
• In the environment, growth & metabolism of microorganisms
often occurs on low concentrations of diverse mixed
substrates.
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 Patterns of Mixed Substrate Utilization

• Growth of microorganisms on two-substrate

mixtures in batch cultures often result in a
number of different patterns of utilization.
• Substrate utilization may either be sequential or
simultaneous.
• The classic case of mixed substrate utilization is
that of diauxic.
• It was first described by Monod in 1942 on the
utilization of glucose and lactose by Escherichia
coli.

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 Microbial Ecology :

Microbes don’t just live on petri-dishes

 They don’t just make us sick!
 They are an important part of our ecosystem
and we need to understand their ecology and
interactions.
 General Concepts
 Individual cells of the same type multiply to
form populations
 Metabolically related populations constitute
groups called guilds
 Mixtures of different guilds conducting
complementary physiological processes
interact to form microbial communities
 An ecological niche is the role that an organism
plays (or the job it does) in a community Whilst
filling its own niche an organism will interact with
other organisms.
 Microbial communities then interact with
communities of macroorganisms and the
physicochemical environment to define the
entire ecosystem
Population of same species derived from the
cell division
ECOSYSTEM

COMMUNITY

GUILD

POPULATION

INDIVIDUAL
The science of Microbial Ecology has two
broad objectives:

1.To understand the biodiversity of

microorganisms in nature and know
how different guilds interact in
microbial communities

2. To measure the behavior and activities

of microorganisms in natural
environments thereby monitor their
effects on ecosystems
 A few important concepts:

1. Much is known about the activities of only a

small proportion of the microbial world: there
are many microorganisms left to be
discovered and this is a major goal of
microbial ecology.
2. Each type of microorganism has at least one
ecological niche, i.e., a function that it can
successfully compete for in a habitat.
3. Countless microbial niches exist in the
biosphere and are in large part responsible
for the great metabolic diversity and
biodiversity of microorganisms on Earth.
4. Since microbes are small, their habitats are
small, and their ecological niches can also
cover small spatial scales. Despite their small
size, microbes nevertheless affect
ecosystems on greater scales, including
global-level effects, e.g., biogeochemical
cycling of C, N, S needed to sustain life on
Earth.
5. The concept of micro-environment becomes
important in understanding how microbes
actually live and metabolize within their
natural habitats.
6. Such microenvironments commonly
undergo profound changes in physico-
chemical characteristics, resulting in
gradients.
7. Example: respiratory consumption of
oxygen by large populations of aerobic
microbes on biofilms within the human
mouth creates anoxic micro-environments
that allow anaerobic microbes to produce
various volatile fermentation waste
products that cause “bad breath” odor.
 Microbial Habitat
• Can be micro or macroscopic
• Can have several ecological niches based
on varying environmental conditions, for
example:
– Oxygen gradient
– pH gradient
– Salinity gradient
 Interactions among microbial
populations
• Intra-specific = interactions between
members of the same species (or same
population).
• Inter-specific = interactions between
members of different species.
• Competitive exclusion principle = also
known as ‘Gause’s Principle’ states that
no two species can coexist long if they
occupy the same niche.
• Competitive exclusion = the elimination
from an area or habitat of one species by
another through inter-specific competition.
Density dependent population regulation
• population growth rates are impacted by their
own densities, or by the densities of other
species, indicating some sort of species
interaction.
Density independent population regulation
• regulation of population growth rates by factors
not related to population size. Direct effects of
abiotic factors are the most common density
independent regulatory factors.
Allee Principle

• principle stating that both + and -

interactions may occur within a single
population.
Allee Effect
• phenomenon in which populations have
lower or negative growth rates at very
low densities.
 Individual Interactions
• Permit populations to reach optimal size
according to available resources
 Positive interactions
• Cooperative – increase growth rate
Eg.
– Minimum Infectious Dose – Anthrax case
– Degradation of Lignin and Cellulose
– Genetic transfer – Antibiotic resistance,
biodegradation
 Negative Interactions

• Competition
Causes:
– Competition for available resources
– Competition for space
– Accumulation of toxic substances
Eg.
An individual within a population use same substrate
making it not available for other members
Accumulation of lactic acid and fatty acid inhibit the
activity of Lactobacillus
Accumulation of dichloroaniline from 3,4
dichropropionanilide catabolism inhibit Penicillium
piscarium.
Interactions among Populations
• Neutralism -
• Commensalism
• Synergism
• Mutualism
• Competition
• Amensalism
• Predation
• Parasitism
Microbes participate in both positive (+)
and negative (-) interactions:
 Neutralism
• Lack of interaction between two microbial
populations.
• Cannot occur between the organisms having the
same or overlapping functional roles within a
community.
• Occurs between extremely different metabolic
capabilities, spatially distant from one other.
• Eg. Frozen in ice: frozen food products polar
sea, atmosphere, resting stage
 Mutualism
Bidirectional; obligatory relationship,
some reciprocal benefit occurs to both
partners.
Examples:
lichen symbiosis: fungus (mycobiont)
and algae or cyanobacteria phycobiont);
rumen-microbe symbiosis (cow and
anaerobic bacteria),
nitrogen-fixing Rhizobium-legume root
nodule symbiosi
Synergism (protocooperation)

• Bidirectional, non obligatory

• Eg. Cometabolism, degradation of cyclohexane
by Nocardia sps and Pseudomonas sps
 Commensalism
• Unidirectional: non obligatory, one partner
benefits while the other is neither benefited nor
harmed.
Examples:
many interactions in microbial biofilms and
indigenous normal microflora of the intact human
body
 Predation
Predator engulfs, attacks and kills the
prey.
Examples:
density-dependent predation of
bacteria by protozoans in aquatic and
soil habitats.
 Parasitism
Parasite infects host, who is harmed.
Examples;
bacteriophage infecting bacteria,
human infectious diseases.
 Amensalism
Unidirectional release of an inhibitory
compound by one organisms that harms
a susceptible microorganism.
Example:
production of antibiotics or bacteriocins.
 Competition

The demand for a food resource exceeds the

supply: a major cause of restricted
microbial productivity in soil and marine
ecosystems
(limitation of energy-yielding organic carbon).
 Microbial communities
• Microbial populations exhibit various adaptations
for success in diverse communities
• Adaptation as well as population interactions
contribute to the stability of communities
• Communities undergo characteristic
successional changes that may lead to greater
stability
• Stable microbial communities tend to have high
diversities
• The interactions within community are complex
• Model ecosystems and mathematical models
help to understand population dynamics and
Population selection within communities
Microorganisms evolve strategies to successfully
survive and maintain themselves within
communities.
According to Andrews logistic equation for the
growth of a population.
rate of specific population growth = r - (r/K • X)
r = maximal individual growth rate
K = carrying capacity of the habitat
X = population density
This equation describes population growth in the
limited environmental conditions.
• ecological dogma holds that an organism will
optimize either reproductive capacity or
resource conservation, but not both
• in reality organisms exist along a continuum
from pure r-strategy to pure K-strategy
• when population density is low (~ 0),
population increase is maximal (r) (favors
rapid growers)
• when population density approaches
carrying capacity (X = K), the rate of
population growth approaches zero (favors
oligotrophs and resource conservation)
 r-strategy
» high maximal growth rates
» respond rapidly to nutrient inputs
» become numerically dominant at high nutrient
concentrations
» dominate in habitats with abundant resources and
non-crowded conditions
» population sizes fluctuate widely in unstable
environments
» r-strategists – must survive between boom times .
- spores
- cysts
examples of r-strategists
- Gram-negative cells in rhizosphere soils
 30

pop size, [substrate]

25

20

15

10

0
0 5 10 15 20 25 30
time

r-strategist population
nutrients
 K-strategy
• reproduce slowly
• optimized for resource conservation
• grow best under nutrient limitation
• grow well in crowded conditions
• population density remains stable
• a smaller proportion of resources go to
reproduction
examples of K-strategists
» Agrobacterium and Corynebacterium species
that degrade humic materials
» Basidiomycete fungi that degrade cellulose
» Actinomycetes and other antibiotic producers
 30

25
pop. size, [substrate]

20

15

10

0
0 5 10 15 20 25 30
time

K strategists
 Succession

Definitions
• Succession - changes in community structure
over time, i.e. populations in specific niches
being replaced by better adapted populations
• Community Structure - which populations
inhabit which niches
 A general model
– “pioneer” organisms are the first to settle in a virgin habitat,
their only common characteristic is that they have a
dispersal mechanism.
– most pioneer species alter the habitat in such a way as to
encourage further colonization
– lichens may colonize a rocky surface, produce
organics and create new habitat
– the pioneers are succeeded by secondary populations,
which are succeeded by tertiary populations, etc.
– in cases where new niches are created, new populations
can be added making the habitat more diverse
– eventually a stable assemblage is reached called a climax
community
– environmental fluctuations keep communities in dynamic
equilibrium rather than in stasis
• Autogenic succession
– when organisms within the habitat modify the
habitat in such a way that permit new
populations to develop
e.g. removal of oxygen by aerobic respiration that
allows microaerophiles and/or anaerobes to
grow
• Allogenic succession the habitat is modified by
forces other than the resident community
• seasonal changes
• physical/chemical changes
• man-made changes
• Disturbances alter the interactive
equilibrium that has been attained among
the populations in the community,
promoting the accelerated extinction of
some species and facilitating the migration
of new species.
 Autotrophic Succession
• Definitions
P  community primary production rate
R  community respiration rate
• In virgin habitats where inorganic nutrients and sunlight
are available, but organic matter is largely missing
• pioneer organisms are autotrophs
• P/R>1
• organic matter accumulates
• the accumulation of organic matter opens niches for
organotrophs
• P / R ratio approaches 1 (a stable community)
e.g. cyanobacteria or lichens that colonize newly exposed
volcanic rock
– autotrophs produce organic matter
– metabolic activity opens new physical locations for
colonization (weathering)
– autotrophs may trap water, reduce sunlight, etc.
 Heterotrophic Succession
• caused by an episodic input of organic matter
– a tree is uprooted by a storm
– a deer dies in the woods
– a fish is washed up on a beach
• pioneer organisms are organotrophs
• P/R<1
• energy flow is initially high
• eventually stored energy is exhausted
• P / R remains < 1
• community will disappear without an
allocthonous source of organic matter (e.g. gut
and ruminal communities,microbs in
decomposed log)
 Community structure
• Biological communities usually contain a
few species with many individuals and
many species with few individual.
• The less abundant species determine the
species diversity of the community.
• Diversity decreases with high densities of
few species.
• High number signifies successful
competition and dominance by a single
population.
 Symbiosis
Symbiotic relationships are those in which two
or more dissimilar organisms live in close
association; may be endosymbiosis or
ectosymbiosis
Functions of symbiosis
• Protection: Endosymbionts and those
ectosymbionts which live in the body cavities of
animals are protected from adverse
environmental conditions. These habitats
protect the symbionts from desiccation and - in
the case of warm-blooded hosts - from
extremes of temperature.
Provision of a favorable position:
• A symbiotic association may provide one partner
with a position that is favorable with respect to the
supply of nutrients.
• Many marine ciliated protozoa are found only on
the body surfaces of crustacea, where the host's
respiratory and feeding currents assure the
microbe of a constant supply of food.
Provision of recognition devices: Bioluminescence
in many animals often appears to be a recognition
device, promoting schooling, mating, or the
attraction of prey. In some species fish, it is
produced by luminous bacteria living
ectosymbiotically in special glands of the host.
Nutrition: The most common function of symbioses is
to provide nutrients for the partners.
 Plant-Microbe Interactions

• General colonization:
phyllosphere, rhizosphere/rhizoplane
• Specific beneficial associations:
root nodulation, mycorrhizae
• Detrimental pathogenic associations:
crown gall tumorigenesis
 Microbe-Plant Interactions
• Phyllosphere: aerial leaf surface of plants
• Rhizosphere: Narrow zone of soil that is
subjected to the influence of a living root.
• No. micro-organisms in rhizosphere can be 10
- 100 fold greater than in bulk soil
• Bacteria and fungi in the rhizosphere feed on
organic matter released in or from the plant
root
• Rhizoplane: the immediate root epidermal
surface that interfaces the rhizosphere soil
• Rhizobacteria and Mycorrhizal fungi can exist
as symbionts inside plant roots where they
play in important role in nutrient uptake.
 Rhizosphere effect
• Within the rhizosphere , plant roots have a
direct influence on the composition and density
of the soil microbial community known as the
rhizosphere effect.
• R/S ratio - indicates the importance of the root
system to the microbial community.
– R  the number or biomass of microbes in the
rhizoshphere.
– S  the number or biomass of microbes in root-free
soil.
– R/S typically between 5 and 20, can be >100
• In the rhizosphere (relative to root-free
soil)
– abundance of Gram-negative rods is
higher
– abundance of Gram-positive rods and
cocci is lower
• Reflects the influence of plant root
exudates and the selection of
organisms with high growth rates
• Root exudates
– amino acids (proteins)
– keto acids (TCA cycle)
– vitamins (enzyme co-factors)
– sugars (C and energy)
• Roots surrounded by active microbes
produce more exudates than roots in
sterile soil.
• The roots are not just leaky, there is an
interaction with the microbial
community.
Microbial populations are clearly benefited by
the interaction with roots . . . but what does
the plant get?
• One major plant benefit is nitrogen fixation . .
• Nitrogen fixation by free-living bacteria
Azospirillum (microaerophile) & Azotobacter
(aerobe)
– both use root exudates as a source of energy and
cellular carbon
– they can fix up to 40 kg N2 hectare-1 year-1
• Roots surrounded by active microbes produce
more exudates than roots in sterile soil.
Production of Growth Hormones
- the plant supplies exudates to the microbes, and
the microbes provide growth hormone to the
plant
- as the plant develops the amount of exudates
drop off which down-regulates the production of
growth hormone
- Eg. Arthrobacter, Pseudomonas, Agrobacterium
 Other beneficial interactions

– bacteria may produce substances that inhibit the

growth of competing plants.
– bacteria produce acids that dissolve apatite
(calcium fluorophosphate) releasing phosphorous
– bacteria produce chelating agents that increase the
solubility of iron and manganese
– We tend to think of bacteria as the enemy, most
often that is not true.
– In most cases plants derive benefit from a healthy
microbial community, and participate in maintaining
the community.
N2-fixing symbiosis

• symbiotic relationship with specific legumes

(pod-bearing angiosperms) that develop N2-
fixing root nodules
Legume host Rhizobial cross-inoculation group
many clovers Rhizobium leguminosarum biovar
trifolii
peas, vetch R. leguminosarum biovar viciae
common bean R. leguminosarum biovar phaseoli,
R. etli, R. tropici
soybean Bradyrhizobium japonicum, B.
elkanii, R. fredii
alfalfa Sinorhizobium meliloti
lotus Mesorhizobium loti
Sesbania Azorhizobium caulinodans
 Mycorrhiza: Fungus-plant root symbiosis
• Very common - nearly
universal; roots of ~ 95% of
vascular plants are normally
involved in mycorrhizal
symbiotic associations.
• Ectomycorrhiza -- form a
sheath around the root without
penetration into plant cells;
normal case for many
gymnosperms (e.g., pine)
• Vesicular-Arbuscular
endomycorrhiza – invade plant
root cells; associated with
many angiosperms (e.g., many
agricultural crops).
 Mycorrhizae

– mutualistic associations between fungi

and plant roots
– fungi become integrated into the root
structure
– both partners benefit (not a disease)
 Ectomycorrhizae
– fungi form an external sheath on the root and
extends into intercellular spaces (not inside
individual cells)
– approximately 40 µm thick
– the root association can be up to 40% fungi by
dry weight
– found in most trees in temperate forests
• benefits to the tree include:
» drought resistance
» pathogen resistance
» enhanced nutrient uptake (PO4 and K)
» increased tolerance to pH changes
» increased root growth
• benefits to the fungus includes:
» access to plant exudates
» direct benefit from trees photosynthetic
activity
 Endomycorrhizae
– fungal mycelia penetrate both between cells and
inside individual cells eg. Found in rododendrons,
orchids
– the fungal partner does not fix nitrogen, but does
seem to enhance the uptake of combined
nitrogen
– orchids are pollinated at night and some
mycorrhizal fungi are bioluminescent (insect
attraction?)
– rRNA sequence data place the origin of the
endomycorrhizal fungi at or near the origin of
land plants . . . may indicate a long term co-
evolution.
– benefits to the tree include:
• drought resistance
• pathogen resistance
• enhanced nutrient uptake (combined nitrogen)
• increased tolerance to pH changes
• increased root growth
the most common of all mycorrhizal associations
• wheat, corn, potatoes, beans, soybeans,
tomatoes, strawberries, apples, oranges,
grapes, cotton, tobacco, tea, coffee, cocoa,
sugar cane, rubber
 Plant/Microbe Interactions - Harmful

Disease Symptom
Necrosis (rot) death of plant cells, may appear as localized spots
Canker localized necrosis resulting in lesion, usually on stem
Wilt droopiness due to loss of turgor
Blight loss of foliage
Chlorosis loss of photosynthetic ability, bleaching of chlorophyll
Hypoplasia stunted growth
Hyperplasia excessive growth
Gall tumerous growth
Crown gall tumors on tobacco made in response to
infection by Agrobacterium tumefaciens
 Agrobacterium tumefaciens
– infects fruit trees, sugar beets, etc. and causes
tumor formation
• Agrobacterium enters plant through a cut or
other wound, usually near the soil/stem
interface
• the bacterium transforms plant cells into
tumor cells
– tumor cells grow in the absence of growth
hormone
– tumor cells produce octopine and nopaline
– transformation directed from a large plasmid
carried by the bacterium (Ti plasmid)
• Part of the Ti plasmid is transferred to the plant cell
nucleus.
• The transformed nucleus maintains tumor growth
even in the absence of the bacterium.
• The plasmid consists of two parts:
»the vir genes
»the t-DNA
• vir DNA - codes for the transfer of the t-DNA to the
plant nucleus
– virA - induced by plant phenolic compounds
produced in response to infection, VirA is a
protein kinase that activates the VirG protein
– virG - involved in control of vir gene expression
– virB - sex pilus, involved in transfer of DNA to the
plant cell
• vir DNA - codes for the transfer of the t-DNA to the
plant nucleus
– virD - endonuclease, nicks the Ti plasmid
adjacent to the t-DNA region
– virE - single stranded binding protein, binds to t-
DNA during transfer
 Microbe-Animal Interactions
• Microbes are ingested by animals (especially
in aquatic environments) as a food source.
• Also ingested to act as commensals ...
– help digestion
– synthesize growth factors and vitamins.
– MAINLY act as a barrier against pathogens
• or mutualists ….
– When animals derive all or most nutrients
from compounds that are hard to digest
(e.g. cellulose)- intestinal symbionts
become more specific and mutualistic. (e.g
termites, leaf-cutters).
 Animal/Microbe Interactions - Beneficial
• Grazing on aggregations - aquatic systems
• Gastropods (snails)
• Echinoderms (sea urchins, sea stars, etc.)
• Patellids (limpets)
– have specialized mouth parts for scraping
– good at harvesting biomass from biofilms
e.g. Marine meiofauna secrete slime traits where
bacterial, fungal, algal populations colonize which is
retraced by animals and gaze on the microbial
populations
Many marine, freshwater and soil invertebrates
consume microbes growing on detrital particles
• Earthworms,
• desert snails graze on lichens
 Animal/Microbe Interactions - Beneficial

• Biofilms

– nutrient concentrations are higher at surfaces

– microbes aggregate at surfaces to take advantage
of nutrient availability
– microbial communities breakdown recalcitrant
polymers (cellulose, chitin, etc.)
– grazers benefit by ingestion of microbial biomass
(high N:C ratio) and by ingestion of released
monomers
Animal/Microbe Interactions - Beneficial
 Animal/Microbe Interactions - Beneficial

• Filter feeding-
through gills, tentacles, mucous net
e.g. sponges, sea squirts filter feeders ingest
algae and free swimming microbes
 Animal/Microbe Interactions - Beneficial

• Animal Cultivation of Microbes

– some insects cultivate fungi on plant tissues

– ingest both plant and fungal biomass
– e.g. leaf-cutter ants
– all fungus-growing ants belong to the tribe Attini
– the genus Atta is the best known of the leaf-cutters
– Cultivate fungi Leucocoprinus or Agaricus
Animal/Microbe Interactions - Beneficial

• Leaf-cutter Ants – genus Atta

 Animal/Microbe Interactions - Beneficial

• Leaf-cutter Ants

– leaf sections cut from nearby vegetation by specialized

workers
– leaf sections carried back to the ant nest along “marked”
trails
– very small workers (“minims”) sometimes ride along on leaf
pieces to protect larger workers from parasitic flies.
– processing workers make a mulch from leaf pieces and
inoculate it with fungus
– the fungus degrades the mulch and produces gongylidia
(nutritional bodies), which are easily digested by ants
Animal/Microbe Interactions - Beneficial

• Leaf-cutter Ants
a minim worker on
a leaf fragment

other workers are

tending the garden
 Animal/Microbe Interactions - Beneficial

• Leaf-cutter Ants

– the ant/fungus relationship is mutualistic

– the ant gains nutrition

– the fungus gains shelter and protection from

predation and pathogens

– some fungi are found only in ant gardens

– Eg. Leucocoprinus, Agaricus,Lepiota, other
basidiomycetes
 Animal/Microbe Interactions - Beneficial

• Similar examples

– termites can gain cellulolytic enzymes from

ingested fungi, cultivate species of basidiomycetes
Termitomyces
– wood-boring marine invertebrates were thought to
produce cellulases, now known to harbor bacteria
that produce cellulase and fix nitrogen
– Ambrosia beetles maintain mutualistic
relationships with Monilia, Ceratocystis,
Cladosporium, Penicillium, Cephalosporium and
several other fungi
 Animal/Microbe Interactions - Beneficial

• Intestinal Symbionts - monogastric animals

– the guts of warm blooded animals contain rich

microbial communities (1 gm feces= 1011 microbes)
– the relationship between the host animal and the
microbial community is usually considered
commensal . . .
– but there are benefits to the host.
– Ley et al. (2008), Herbivorous host animals have the
most diverse communities of microorganisms (14
phyla), followed by omnivores(12 phyla), and finally,
by carnivores (six phyla).
 Animal/Microbe Interactions - Beneficial

• Intestinal Symbionts - monogastric animals

– some gut flora produce vitamins required by the host

» E. coli produces vitamin K

– some fermentation products are absorbed by the host animal

– the “normal flora” inhibit the colonization of pathogenic strains

– the walls of the stomach are lined with acid tolerant strains

» lactobacilli
» streptococci
» Helicobacter pylori
 Animal/Microbe Interactions - Beneficial

• Intestinal Symbionts - the human gut

– the large intestine
• strict anaerobes are present at 1010 to 1011 cells/g
» Bacteroides
» Clostridium
» Enterococcus faecalis (fecal strep)

Ley et al. (2008), Firmicutes dominate with 65.7% of the sequences,

followed by the Bacteroidetes (16.3%), Proteobacteria (8.8%),
Actinobacteria (4.7%), and Verrucomicrobia (2.2%). Other groups
occur with less than 1% of the total sequences
 Animal/Microbe Interactions - Beneficial

• Host benefits

– vitamin synthesis – thiamine, riboflavin, pyridoxine, B12,

K
– organic acid production – acetic, propionic, butyric
– glycosidase production - b-glucuronidase, b-
galactosidase, b-glucosidase, a-glucosidase, a-
galactosidase
– One beneficial function of gut microbiota is “colonization
resistance,” which keeps nonnative bacteria from
colonizing the host’s gut.
 Animal/Microbe Interactions - Beneficial

• Importance of intestinal flora

– the gut acts like a chemostat, new growth replaces

continual loss of flora

– antibiotic therapy can inhibit reproduction and

virtually sterilize the gut

– in the absence of normal flora, disease causing

organisms can become established (Bacillus,
Staphylococcus, Proteus, Candida)
 Animal/Microbe Interactions - Beneficial

• Other intestinal symbionts

– birds that eat beeswax maintain bacterial flora that

can degrade the wax, bacteria get required co-factors
from the bird

– aquatic animals carry chitinolytic gut flora, digestible

monomers released from ingested chitin
 Animal/Microbe Interactions - Beneficial

• Ruminal Communities

– in cows the rumen accounts for approximately

15% of the animal’s total weight

– ruminants depend upon microbes that make

cellulases for digestion of grass, leaves, etc.
Animal/Microbe Interactions - Beneficial

• The rumen is a
specialized
esophageal pouch

• it is a stable
environment

• it contains a large and

complex microbial
community
 Animal/Microbe Interactions - Beneficial

• The rumen habitat

– anaerobic
– 30° – 40 ° C
– pH 5.5 – 7.0
– continuous supply of plant material
– continuous removal of toxic metabolites
– 109 – 1010 cells per ml of ruminal fluid
 Animal/Microbe Interactions - Beneficial

• Host benefits

– production of digestible carbohydrate monomers

from complex plant tissue
– production of low molecular weight organic acids
(acetate, propionate, etc.)
– digestible bacterial biomass
– major source of nutrition is by digestion of rumen
microbes in the lower digestive tract
 Animal/Microbe Interactions - Beneficial

• Non-ruminant grazing animals

– e.g. horses, pigs, rabbits

– have an enlarged cecum (pouch at beginning of

large intestine)

– carry cellulolytic community in cecum

 Animal/Microbe Interactions - Beneficial

• Invertebrates with Chemolithotrophs

• Chemolithotrophs are a group of phylogenetically diverse microbes that
can obtain all the energy required for their growth from the oxidation of
inorganic compounds such as hydrogen (H2), hydrogen sulfide (H2S),
and reduced metals
• E.g. The colorless sulfur bacteria, γ-proteobacteria, including the
genera Beggiatoa, Thioploca, Thiothrix, Thiomargarita, Thiomicrospira
– at oceanic rift systems the water is rich in reduced
inorganics (e.g. H2S)
– these “alternative” energy sources support dense
microbial and macrobiological communities
– E.g. Giant tube worm and Clam (molluscs) harbor
chemolithotrophs
 Animal/Microbe Interactions - Beneficial

• Invertebrates with methanotrophics

– bacteria or archaea, can grow aerobically or anaerobically that
metabolize methane as their source of carbon and chemical
energy. Their habitats include wetlands, soils, marshes, rice
paddies, landfills, aquatic systems (lakes, oceans, streams) and
environments where methane is produced
– E.g. Methanosarcina barkeri

– Mussels of family Mytilidae harbor methanotrophic bacteria in

their gills

132
 Symbiotic Light Production
• Some marine invertebrates and fish have
mutualistic relationship with luminescent bacteria.
• Bacteria are contained in special organs, that have
pores to let bacteria in and out.
• Luminescence is used by fish to communicate, for
recognition and to lure prey. Mainly found in deep
sea species. Difficult to study in the lab.
• Four genera responsible for most bacterial
luminescence (Beneckea, Photobacterium, Vibrio,
and Xenorhabdus)
Selected Examples of Interactions between Animals and Microorganisms
Water and wastewater
 water
Key facts

• Over 2 billion people live in water-stressed countries, which is expected to be exacerbated in some
regions as result of climate change and population growth.

• Globally, at least 2 billion people use a drinking water source contaminated with faeces. Microbial
contamination of drinking-water as a result of contamination with faeces poses the greatest risk to
drinking-water safety.

• While the most important chemical risks in drinking water arise from arsenic, fluoride or nitrate,
emerging contaminants such as pharmaceuticals, pesticides, per- and polyfluoroalkyl substances
(PFASs) and microplastics generate public concern.

• Safe and sufficient water facilitates the practice of hygiene, which is a key measure to prevent not
only diarrhoeal diseases, but acute respiratory infections and numerous neglected tropical diseases.

• Microbiologically contaminated drinking water can transmit diseases such as diarrhoea, cholera,
dysentery, typhoid and polio and is estimated to cause 485,000 diarrhoeal deaths each year.

• In 2020, 74% of the global population (5.8 billion people) used a safely managed drinking-water
service – that is, one located on premises, available when needed, and free from contamination.
 Overview
• Safe and readily available water is important for public health, whether it is used for drinking, domestic
use, food production or recreational purposes.

• In 2010, the UN General Assembly explicitly recognized the human right to water and sanitation.

• Everyone has the right to sufficient, continuous, safe, acceptable, physically accessible and affordable
water for personal and domestic use.

• Sustainable Development Goal target 6.1 calls for universal and equitable access to safe and
affordable drinking water.

• The target is tracked with the indicator of “safely managed drinking water services” – drinking water
from an improved water source that is located on premises, available when needed, and free from
faecal and priority chemical contamination.

• Sharp geographic, sociocultural and economic inequalities persist, not only between rural and urban
areas but also in towns and cities where people living in low-income, informal or illegal settlements
usually have less access to improved sources of drinking-water than other residents.

• Climate change, increasing water scarcity, population growth, demographic changes and urbanization
already pose challenges for water supply systems.

• Re-use of wastewater to recover water, nutrients or energy is becoming an important strategy.

• Increasingly countries are using wastewater for irrigation; in developing countries this represents 7%
of irrigated land. While this practice if done inappropriately poses health risks

• safe management of wastewater can yield multiple benefits, including increased food production.

• Improved water supply and sanitation, and better management of water resources, can boost
countries’ economic growth and can contribute greatly to poverty reduction.
 World Health organization (WHO)
• World Health organization (WHO) produces a series of water quality guidelines, including on
drinking-water, safe use of wastewater, and recreational water quality.

• The water quality guidelines are based on managing risks, and since 2004 the Guidelines for
drinking-water quality promote the Framework for safe drinking-water.

• The Framework recommends

– establishment of health-based targets,

– the development and implementation of water safety plans by water suppliers to most effectively identify and
manage risks from catchment to consumer, and

– independent surveillance to ensure that water safety plans are effective and health-based targets are being
met.

• WHO also supports countries to implement the drinking-water quality guidelines through the
development of practical guidance materials and provision of direct country support.

• WHO produces international norms on water quality and human health in the form of guidelines
that are used as the basis for regulation and standard setting world-wide.
– Guidelines for drinking-water quality

– Guidelines for safe recreational water quality: Volume 1 coastal and fresh water

– Guidelines for safe recreational water environments – Volume 2 swimming pools and spas

– International Scheme to Evaluate Household Water Treatment Technologies

 Waste water
Waste water is the used water of a community and
consists of
• domestic waste water, including human excrements
and wash waters.
• water borne wastes from different industrial
establishments.
• agricultural wastes from intensive live stock farming,
where a number of cattle, chickens, etc. are fed in a
small area.
• surface, ground and storm water that enter the
sewage system.
Composition of waste water:
• It consists of approximately 99.5 to 99.9 % water and 0.1-
0.5 % inorganic and organic matter in suspended and
soluble forms.
• The chemical constituents of suspended solids and
soluble materials are quite distinct.
• Lignocellulose, cellulose, proteins, fats and various kinds
of inorganic particulate matter exist in the colloidal state
and constitute suspended solids.
• Sugar, fatty acids, alcohols, amino acids and a number of
inorganic ions constitute soluble materials.
• This small amount of solid matter exist is quite large
when the total quantity of sewage is considered.
Contd.
• The chemical constitutes are extremely important,
because they vary from hour to hour and from day to
day.
• Chemical compounds contributed by domestic wastes
include detergents, antiseptics, pesticides besides
human excrements.
• A variety of organic and inorganic compounds are
added by industrial wastes.
• The kind of wastes depend on the types of industries
such as manufacture of paper, textiles, chemicals,
dairy products, canning, brewing, meat packing,
tannery, oil refinery, mines and metal industries
• Materials that Should Not Be Discharged Into Lakes and Rivers Without
Treatment:
1. Pathogens (in feces)
2. Organics:
a. Non-biodegradable
b. Biodegradable
3. Phosphate –
4. Nitrogen sources (NH4+, NO3-, NO2-)
The main point is that releasing untreated, nutritious materials into the environment
may favor the growth of certain microorganisms, upset the normal balance of
ecosystems, and kill many larger creatures that require oxygen to live, such as fish.
Assay of Waste Treatment

• Liquid Wastes - domestic sewage, industrial wastes

(point sources), and runoff (non-point source)

• natural bodies of water have significant self-cleaning ability

• areas of dense population or industrialization rate of input is higher than rate

of biodegradation
 Biological Oxygen Demand (BOD)

• The Biochemical Oxygen Demand (BOD) assay provides a measure of the

levels of biologically degradable organic material present in water samples.
• The idea is to measure decreases in oxygen concentration following the
oxygen-dependent oxidation of organics by bacteria,
i.e. organics + O2 -----> CO2
• The amount of oxygen consumed (BOD) is difference of the initial and final
concentrations of oxygen in the sample:
[O2]consumed = [O2]initial - [O2]final

e.g. if after 5 days at 20ºC a sample contains 4 mg/L of oxygen, then the
amount consumed is:
[O2]consumed = [O2]initial - [O2]final
= 10 mg/L - 4 mg/L
= 6 mg/L
If 5ml of raw sewage has been diluted to 300 ml
and DO of the diluted sample at the beginning of
the BOD test was 9mg/l and 6.7 mg/l after 5 day
incubation at 20º C, find the BOD of the raw
sewage.
 Efficiency of Wastewater Treatment

To assay the efficiency of wastewater treatment procedure, the results of BOD assays are
compared for water samples taken before and after the treatment.
By definition, the efficiency of treatment is expressed as the percent decrease in BOD (i.e.
the percent decrease in oxygen consumed).

Percent decrease in BOD = 100 x (BOD before – BOD after)

(BOD before)

e.g. Suppose the BOD is 6mg/L before a treatment and is 2 mg/L after treatment, what
is the percent decrease in BOD?

The percent decrease in BOD is = 100 x (6 mg/L - 2 mg/L)/(6 mg/L) = 66%

Thus a high percentage decrease in BOD indicates that an effective purification has been
achieved.
 Sewage Treatment
• three stages
• Primary - physical removal of solids and particulate matter
• settling tanks
• screens
• skimmers
• Secondary - aerobic or anaerobic degradation by various methods
• Tertiary Treatment - additional treatments to remove non-degraded (potentially
toxic) compounds and microbes.
• chlorophenols
• PCBs
• excess nutrients
• pathogens
1. Primary treatment

• Primary treatment is a strictly physical process

wherein sewage is filtered through screens or grates
to remove large solid materials.
• These solids are incinerated, buried or composted.
• Primary treatment decreases the BOD by 30-40%.
• The liquid is further cleaned up by secondary
treatment.
2. Secondary treatment

• Involves the microbial digestion of organic material

and removal of microorganisms.
• First, the liquid from primary treatment is allowed
to settle for several hours to separate insolubles
("sludge") from the liquid fraction.
• The liquids are treated aerobically by the "activated
sludge" process while the sludge is treated
anaerobically in "sludge digesters".
Types
aerobic or anaerobic degradation by various
methods
1. Fixed Growth – attached organisms
• Trickling filters
• Rotating Biological Contactors (RBCs)

2. Dispersed Growth – suspended organisms

• Activated sludge
• Oxidation ditches/ponds
• Aerated lagoons, stabilization ponds
 suspended growth vs. fixed film biological treatment
• Suspended growth: • Fixed film:
• Biomass well-mixed, in • Biomass layered, attached
suspension • More diverse (snails!), stratified
• Diverse microbiology microbiology
• High energy aeration systems • Frequently relies on draft for aeration
• Process control follows from • Process control is empirically based on
modeling organic and hydraulic loading
• Shocked more easily • Resilient to shock loads
• Biomass recycled • Biomass not typically recycled
• High solids production, easy to • Low solids production, hard to settle
settle (low floc formers)
• High efficiency • Lower efficiency
• DO 2 to 3 mg/L • Higher DO required
• Reliable N & P removal • unreliable N & P removal
Trickling Bed Filters

• waste water is sprayed on a deep bed of porous material

• microbial communities colonies the surface and degrade wastes

• generally works well, but is inhibited by cold temperatures.

• First experimented in England in 1893.
 Construction and Design

• The primary factors that must be considered in the design of trickling filters
include:
• (1) the type of filter media to be used,
• (2) the type and dosing characteristics of the distribution system,and
• (3)the configuration of the underdrain system.
 1. Properties of material used for filter
bed
Material size(mm) surface area(m2/m3)
Slag 50 125
Clinker 62 120
Rounded gravel 25 150

• Depth varies with design-

• ranges from 3-8ft.
• Square shaped new type with plastic medium ranges from 30-40
ft.
 2.Distribution System
• The rotary distributor - reliable and easy to maintain.
• The rotary distributor consists of a hollow vertical center
column carrying two or more radial pipes or arms, each
of which contains a number of nozzles or orifices for
discharging the wastewater onto the bed. All of these
nozzles point in the same direction at right angles to the
arms and the reaction of the discharge through them
causes the arms to revolve.

• The speed of revolution will vary with the flow rate, but it should be in
the range of one revolution in 10 mins or less for a two-arm distributor.
A clearance of 6 to 9 inches should be allowed between the bottom of
the distributor arm and top of the bed.
 3.Underdrain System:

• The underdrain system in trickling filters serves two

purposes:
• to carry the wastewater passing through the filter and the sloughed
solids from the filter to the final clarification process, and
• to provide for ventilation of the filter to maintain aerobic
conditions.

[sloped toward the collection channel at a 1 to 2 percent gradient. Since the underdrains also
provide ventilation for the filter it is desirable that the ventilation openings total at least 20%
of the total floor area]
Micro-organisms

• Common bacteria-
• Achromobacter
• Flavobacterium
• Pseudomonas, Alcaligens
• Sphaerotilus
• Baggiatoa
• Nitrosomonas, Nitrobacter
• Along with fungi, algae and protozoa
 Types of Filters
• Trickling filters are classified as high rate or low rate, based on the organic and
hydraulic loading applied to the unit.
• < 0.40kg BOD5/m3/day – low rate
• High rate-
• depth < 2.5m, continuous loading, effluent recycled, upto 1 kg BOD5/m3/day
Advantages

• Due to its simple design, in actual operation the trickling filter is one
of the most trouble-free types of secondary treatment processes.
• It requires much less operating attention and process control.
• economical and tolerant to changes in wastewater composition.
 Constraints
• efficiency depends upon the biomass in the biofilm.
• Weather Problems - temperature
• Filter Flies are a nuisance to plant personnel and nearby
neighbors.
Other common problems-
• Ponding is normally the result of:(a)excessive organic
loading,(b)use of media which is too small, (c)clogging of
underdrain system, and
(e) trash or debris in filter voids.
• Ponding can cause odors and decrease filter efficiency.
Rotating Biological Contactor
• First experimented in Germany in 1920.
• partially submerged discs are rotated in waste water
• microbial community is constantly aerated
Rotating Biological Contactors
Construction and design

• Material used- polystyrene, polyethylene, polypropylene,

stainless steel, cement, aluminium, glass, rubber, teflon,
wood, wire screen etc.
• Dimension- 2-3 m diameter, 10-20 mm wide,5-8 m length
separated into series of chamber.
• Slime layer on the surface- 1-3mm.
• Disc are partly submerged 40% in the medium
• Slowly rotated at 1-7 rpm
• Hydraulic loading rate- 0.04to 0.06 m3/m2.day
• Organic loading rate- 50 to 60g/m2.d.
Rotating Biological Contactors

Primary
Settling

Secondary
Settling
Sludge
Treatment

Sludge Treatment
Advantages of RBC
• Simple to operate, low maintenance
• Reduced power cost, no sludge recycling.
• Less space
• Effluent quality achieved is as good as after tertiary
treatment.
• Foaming, aerosol, airstripping is reduced
• Filter clogging, ponding, filter flies are eliminated
• Self regulating with respect to cell retention time and
stability of process is a plus point as biomass neatly
attaches to media support.
 Disadvantages with RBC
• Lack of operational control
• Dissolved oxygen may remain limited.
• Odour problem
• Needs covering in cold climates, sun and heavy precipitation.
Aerated Lagoons
• earthwork lagoons are filled with waste water
• large pumps are used to aerate and circulate water
• low tech and low cost
• significant settling of solids and relatively long time for BOD reduction
Parameters:
depth 1-3 m
retention time 7-50 days
BOD removal 70-95%
algal concn 10-100 mg/l
Suspended solid 100-350 mg/l
Aerobic ponds
Facultative ponds
Anaerobic ponds
Septic tank
Septic system schematic
• tank 10 ft from house
• field 100 ft from well or pond
Septic system schematic
 features
• Capacity: 20- 30 gallon / person ...500 gallon
• L= 2B, Depth= 1.5-2 m.
• Liq. Depth= 1.2 m, Air space =12”
• Bottom –sloping towards inlet
• Inlet, outlet ,cover,
• Retention period = 24 hrs
Mechanism:
• sludge reduce volume by anaerobic digestion
• aerobic oxidation of the effluents on subsoil
 Activated Sludge
• Waste water is vigorously oxygenated to promote growth of aerobes,
especially slime forming bacteria such as Zooglea ramigera.
• These organisms oxidize much of the organic material, producing
carbon dioxide.
• In addition, they form a layer of slime called "floc" ("activated sludge")
that traps other organisms and adsorbs soluble organics.
• The floc contains fecal protozoa, enterics, pathogens, viruses etc.
• After the floc is given five to ten hours to settle, it is sent off to a
sludge digestor for further digestion.
• Some of the floc is retained for use as an inoculum to re-populate the
tank for processing the next batch of material.
• The activated sludge process decreases the BOD by 75-90%.
THE PHYSICAL COMPONENTS OF
THE ACTIVATED-SLUDGE PROCESS

• The first is an aeration tank or tanks in which air or

oxygen is introduced into the system to create an
aerobic environment that meets the needs of the
biological community and that keeps the activated
sludge properly mixed
 THE BIOLOGICAL COMPONENT
OF THE ACTIVATED-SLUDGE SYSTEM
• The biological component of the activated sludge
system is comprised of microorganisms.
• Bacteria, fungi, protozoa, and rotifers constitute the
biological mass, of activated sludge.
• In addition, some metazoan, such as nematode
worms, may be present.
• However, the constant agitation in the aeration tanks
and sludge recirculation are deterrents to the growth
of higher organisms.
• The species of microorganism that dominates a
system depends on environmental conditions, process
design, the mode of plant operation, and the
characteristics of the secondary influent wastewater.
• While both heterotrophic and autotrophic bacteria
reside in activated sludge, the former predominate.
• Important genera of heterotrophic bacteria include
Achromobacter, Alcaligenes, Arthrobacter,
Citromonas, Flavobacterium, Pseudomonas, and
Zoogloea.
Modifications of activated sludge

1. Tapered Aeration
2. Step Aeration
3. Contact stabilization
4. Advanced activated sludge processes
Tapered Aeration Activated Sludge -
• Tapered aeration affects only the arrangement of the aeration
devices in the aeration tank.
• In tapered aeration the diffusers or aeration devices are spaced
closer together at the head end of the tank to match the oxygen
demand.
Step feed/aeration activated sludge

• even out the BOD loading to the aeration tank as well as the
air loading along the length of the tank,
• wastewater may flow into the tank at several locations along
the length.
• The BOD is still highest where the waste is injected but the
concentrations are much less than in the conventional
system.
• The air diffusers can then be evenly spaced making the
design much simpler.
Advantage:
• provides flexibility of operation; distributes BOD load to provide
more O2 demand.
Disadvantage:
• more complicated design for process and aeration system
 Contact stabilization
• In this process, wastewater is contacted with the microorganisms for a
much shorter time (1 to 2 hours as compared to 6 to 8 hours for
conventional treatment).
• After settling, the activated sludge is pumped to a re-aeration tank
where the microorganisms metabolize the nutrients they have
extracted from the waste.
Advantage-
• The MLSS concentration in the sludge re-aeration tank is
higher than in the contact tank, the process requires less
aeration than conventional complete mix or plug flow processes.
• Aeration volume requirements are typically 50 percent less
than conventional plug flow.
Disadvantage: operation somewhat more complex than
• conventional complete mix or plug flow processes.
Extended Aeration -

• Use to treat industrial wastewater containing soluble

organics that need longer detention times.
• This is the same as complete mix, with just a longer
aeration.
• Advantage –
• long detention time in the aeration tank; provides
equalization to absorb sudden/temporary shock loads.
• Less sludge is generally produced because some of the
bacteria are digested in the aeration tank.
• One of the simpler modifications to operate.
• Disadvantage: require relatively large aeration tanks
High-purity oxygen activated sludge process

• High-purity oxygen is used instead of air in the

activated sludge process.
• Oxygen is diffused into covered aeration
tanks and is re-circulated.
Advantage:
• requires relatively small aeration tanks;
• adaptable to many types of wastewater
Disadvantage:
• more complex equipment to install, operate and
maintain;
• high peak flows can wash out biomass
 Deep shaft activated sludge reactor:
• A vertical shaft about 120 to 150m deep replaces the primary
clarifiers and aeration basin.
• The shaft is lined with a steel shell and fitted with a concentric pipe to
form an annular reactor.
• Mixed liquor and air are forced down the center of the shaft and
allowed to rise upward through the annulus.
• Flotation is used to separate the biomass; the biomass skimmed off
the flotation tank and recycled back into the reactor.
Advantage:
• increases the amount of dissolved oxygen (25-60 mg/L) in the shaft with
beneficial effects on substrate utilization.
Disadvantage:
• more complex equipment to install, operate and maintain.
Sequencing batch activated sludge reactor:

• The sequencing batch reactor is a fill-and-draw type

reactor system involving a single complete-mix
reactor in which all steps of the activated-sludge
process occur.
• Mixed liquor remains in the reactor during all
cycles, thereby eliminating the need for separate
secondary sedimentation tanks; biomass from
previous cycles is used.
Advantage:
• compact facility; applicable to a variety of plant sizes
Disadvantage:
• high peak flows can disturb operation
Typical operating sequence for sequencing
batch reactor
Activated Sludge Process mechanism

• Organic waste is introduced into a reactor where an aerobic

bacterial culture is maintained in suspension.
• The reactor contents are referred to as the "mixed liquor".
• In the reactor, the bacterial culture carries out the conversion in
general accordance with the stoichiometry shown in the equations
given below ;
The air supply to the aeration basin must be adequate to:
1. Satisfy the BOD of the waste (i.e., to biodegrade the waste)
2. Satisfy the endogenous respiration by the sludge organisms
3. Provide adequate mixing
4. Maintain a minimum dissolved-oxygen concentration of 1 to 2 mg/L
throughout the aeration basin.
If all of the cells can be oxidized completely, the ultimate
BOD of the cells is equal to 1.42 times the concentration of
cells.
Principle of activated sludge process
Continuously Stirred Tank Reactors
(CSTR)

•essentially a stirred vessel without added

support matrix

•aeration and temperature can be

controlled

•less settling of solids.

 fluidized bed reactors
• low density support material is used to colonize the
degrading community

• reactor input lifts and circulates the supporting

material.
• materials for media- sand, carbon, anthracite, glass
calcinated clay etc.
• Size- 0.2-0.3 mm
 Anaerobic secondary treatment
Sludge digestion
• Sludge digestors encourage the growth of anaerobes, including
methanogens.
• Sludge contains a large amount of insoluble organics (20-100 g/L) that
are microbially digested.
• The degradative chemistry is similar to the anaerobic decomposition of
carbon and the biochemistry of the rumen.
• Digestion occurs at 35-37oC, at pH 6.8 for 14-30 days.
• Products include the gases methane and carbon dioxide which are
released into the atmosphere through gas outlets.
• This methane is often recovered and used to power the pumps and
maintain the temperature of the sludge digestor.
• The undigested sludge solids settle and are incinerated or buried.
Sludge digestion decreases the BOD in the remaining liquid fraction by
> 90%.
• anaerobic digestion is helpful in lessening the load of solid waste and
recover of energy
• biomass can be turned into different products, such as hydrogen,
biogas, ethanol, acetone, butanol, organic acids (pyruvate, lactate,
oxalic acid, levulinic acid, citric acid), 2,3-butanediol, 1,4-butanediol,
isobutanol, xylitol, mannitol, and xanthan gum by selecting different
microorganisms in the process of biochemical conversion
• products can synthetize replacements of petroleum-based products
• provides many platforms for the conversion of renewable materials,
fuels, and chemicals
Sketch Diagram of an Anaerobic Digester
THE BIOLOGICAL COMPONENT OF THE SLUDGE DIGESTION SYSTEM
A) Hydrolysis/Liquefaction reactions:

• initial digestion of macromolecules by extracellular enzymes to

soluble organics (glucose, etc.)

• then fermented to form fatty acids (butyric, propionic, lactic, succinic

and acetic acids), ethanol, hydrogen, and carbon dioxide

• Lipids → Fatty Acids

• Polysaccharides → Monosaccharides

• Protein → Amino Acids

• Nucleic Acids → Purines & Pyrimidines

• facultative and obligate anaerobes –

Clostridium, Bacteroides, Peptococcus, Peptostreptococcus, Eubacterium, and
Lactobacillus
B) Acidogenisis (Fermentations):
• fermentation involves the conversion of the sugars, amino acids and fatty acids
to hydrogen, acetate, carbon dioxide, VFAs such as propionic, butyric and acetic
acid, ketones, alcohols and lactic acid by facultative and anaerobic bacteria
• even though a simple substrate such as glucose can be fermented, different
products are produced by the diverse bacterial community fatty acids and
ethanol are then fermented to form acetate, carbon dioxide, hydrogen
C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2
C6H12O6 →2CH3CH2OH + 2CO2
C6H12O6 + 2H2 → 2CH3CH2COOH + 2H2O
• most of the organic matter is converted into readily available substrates for
methanogenic microbes (acetate, hydrogen and carbon dioxide), but a
significant part (approximately 30%) is transformed to short chain fatty acids or
alcohols
• degradable organic matter is removed in this stage
• by-product of amino acids fermentation, ammonia and hydrogen sulphide are
released that can be inhibitory for anaerobic digestion
• bacteria – Syntrophomonas, Syntrophobacter, and Acetobacterium
C) Acetogenesis:

Acetogenesis refers to the synthesis of acetate, which includes the formation of acetate by the
reduction of CO2 and the formation of acetate from organic acids.
• acetogenesis is the conversion of certain fermentation products such as volatile fatty acids
(VFAs) with more than two carbon atoms , alcohols and aromatic fatty acids into acetate and
hydrogen by obligate hydrogen producing bacteria
• most of the acetate is created by hydrogen-producing acetogenic bacteria
• Acetogenesis species can be subdivided into those that are not obligately proton-reducing,
that is, hydrogen-producing species and those that do reduce protons to hydrogen
obligately during acetogenesis.
• The first group is broad, including the Hydrogen-utilizing acetogens i.e. homoacetogens and
species that may direct their metabolisms to proton reduction in the presence of an efficient
hydrogen-removing system.
• Homoacetogenic species are known in the genera Acetobacterium, Acetoanaerobium,
Acetogenium, Butyribacterium, Clostridium, Eubacterium, and Pelobacter.
• In mesophilic sludges, there are approximately 105 homoacetogens per milliliter forming
acetate from H2 + CO2.
• many of the acidogenic species direct their metabolism to acetogenesis.
• obligate proton-reducing acetogens have been described: Syntrophobacter wolinii degrades
propionate, Syntrophomonas wolfei degrades butyrate, and Syntrophus buswellii degrades
benzoate
D) Methanogenesis :

a variety of methane-forming bacteria is required in the anaerobic digestion system,

since no single species can degrade all the available substrates
• approximately 70% of the methane is produced from acetate, while the remaining 30% is
produced from the reduction of carbon dioxide by hydrogen and other electron donors
• according to the type of substrate utilized by the methanogens via methanogenesis is
divided into two main types:
a. hydrogenotrophic methanogenesis: hydrogen and carbon dioxide are converted into
methane according to the reaction:
CO2+4H2→CH4+2H2O
b. acetotrophic or aceticlastic methanogenesis: methane is formed from the conversion of
acetate through the reaction:
CH3COOH →CH4 + CO2
• Bacteria:
methanogenic bacteria; Methanobrevibacter, Methanomicrobium, Methanogenium,
Methanobacterium, Methanococcus, Methanospirillum, methanosarcina

• after 2-4 weeks, the non-degradable material (still called sludge) is settled, then dried and
burned, buried or used for fetlilizer (if it doesn't contain a lot of heavy metals or other
toxic materials
 Advantages of Anaerobic treatment process

• The lower operating cost of the digester makes it commercially viable.

• Sludge occupies less volume and is easier to dry.

• Reduce production of landfill gas, which when damaged leads to an outburst of

methane (major greenhouse gas)

• Methane produced in the digestor can be used as biogas, an alternative source of

energy.

• It reduces the energy footprint of conventional wastewater treatment technology.

• It has reduced the use of chemical fertilizer as the digestate (the content of the
reactor after completion of digestion) can
be used as fertilizer.
 Disadvantages of Anaerobic treatment process

• Slow process
• Anaerobes are more sensitive to toxic compounds than aerobes
• Products are more odoursous, corrosive and less stable
• Narrow pH range
• Need for alkalinity addition to overcome the acidity induced by CO2 production.
 Applications
• Treatment of organic wastewaters of distillery, brewery, pulp and paper
manufacturing and petrochemical industries.
• Eg.
• Anaerobic suspended growth process
• anaerobic attached growth
• Fluidized bed attached growth
• Upflow anaerobic sludge blanket reactor (UASB)
Anaerobic suspended growth process

Three types-
1. Complete mix process
2. Anaerobic contact process
3. Anaerobic sequencing batch reactor
Complete mix process

• Contents of tank are intimately mixed with incoming wastewater.

• Hydraulic retention and solid retention time (SRT) are equal.
• 15-30 days
• Useful for high concentration of organic solids
Anaerobic contact process

• Biomass is separated and returned to the complete mix or contact

reactor so that the solid retention time is longer than the hydraulic
retention time.
 Anaerobic sequencing batch reactor (ASBR)
• It is a suspended growth type.
• Four stages-
• Feeding
• Reacting
• Settling (30 min.)
• Decanting
 Upward flow anaerobic sludge blanket reactor (UASB)

• Consists of cylindrical or rectangular tank of 4-5 m of liquid depth.

• Upward flow at the velocity of 0.15-0.3 mm/sec.
• Retention time 8-10 hrs.
• Takes 30-90 days for acclimatization.
• Biogas is collected at the top
• Scum removal every 2-3 years
• The tank is emptied once in 5-7 years to improve the efficiency
• pH –neutral to slightly alkaline
 Anaerobic attached growth process

1. Upflow packed bed attached growth reactor-

• Packing material can be variable as in trickling filter
( may be 50-70% of total depth)
• 2-8m diameter
• Depth-3-13 m
• Media is submerged with water
• Microbes are loosely held in the void space of packing material
 Anaerobic attached growth process
2. Upflow attached growth anaerobic expanded bed reactor
(AEBR)-
• Fine media as sand (0.2-0.5 mm) is used
3. attached growth anaerobic fluidized bed reactor (FBR)-
• Principle similar to AEBR
• 0.6-0.8 mm diameter- sand/ activated carbon/ adsorbing media
• Sand undergoes 100% expansion and behaves as fluid.
 Tertiary Treatment
• Tertiary treatment methods purify the wastewater further until
it is reusable /drinkable.
• The chemical and physical methods used are -
• activated charcoal
• removes organic and chlorinated compounds
• chemical precipitation
• precipitation of phosphate with Ca or Fe to reduce the
likelihood of blooms
• stripping nitrogen as NH3 at high pH to reduce the
likelihood of blooms
• disinfection
• removal of pathogenic microbes by chlorination or UV
treatment
Tertiary Treatment

• (at home)
• physical filtration - for the removal of cysts
• ion exchange - for the removal of chlorine and metals
• boiling - for removal of pathogenic microbes
Non-biodegradable organics:

(i) Activated charcoal binds non-specifically to many of the dissolved

organics and remaining particulates, removing them from the water.
(ii) Chlorine (Cl2) oxidizes and precipitates organics that can be removed
after they settle.
 Phosphate:
Selective precipitation removes phosphate from solution as insoluble
salts. These precipitates, which also carry along most remaining
microorganisms, are allowed to settle and are then removed.
(i) Calcium:
• It is usually added in the form of lime Ca(OH)2. It reacts with
the natural alkalinity in the wastewater to produce calcium
carbonate, which is primarily responsible for enhancing SS removal.
As the pH value of the wastewater increases beyond about 10,
excess calcium ions will then react with the phosphate, to precipitate
in hydroxylapatite (The lime dose required can be approximated at
1.5 times the alkalinity as CaCO3)
• Addition of calcium chloride (CaCl2) - leads to the formation of
calcium phosphate [Ca3(PO4)2], that comes out of solution.
(ii) Iron:
• Addition of ferric chloride (FeCl3) leads to the formation of ferric
phosphate (FePO4), that comes out of solution.
• Ferric chloride or sulphate and ferrous sulphate also know as
copperas, are all widely used for phosphorous removal
iii) Aluminium:
Alum or hydrated aluminium sulphate is widely used precipitating
phosphates and aluminium phosphates (AlPO4).
 Biological processes
• several biological suspended growth process configurations have been used to accomplish
biological phosphorous removal.
• The principal advantages of biological phosphorous removal are reduced chemical costs and
less sludge production as compared to chemical precipitation.

Anaerobic/Aerobic EBPR Process

• Phosphorus accumulating organisms (PAO) are encouraged to grow and consume

phosphorous. Eg. Candidatus Accumulibacter phosphatis (Accumulibacter)
• Under anaerobic conditions, PAO assimilate fermentation products (i.e. volatile fatty acids)
into storage products within the cells with the concomitant release of phosphorous from
stored polyphosphates. Using energy available from stored polyphosphates, the PAO
assimilate acetate and produce intracellular polyhydroxybutyrate (PHB) storage products.
• In the aerobic zone: Stored PHB is metabolized, providing energy from oxidation and carbon
for new cell growth. The energy released from PHB oxidation is used to form polyphosphate
bonds in cell storage. PHB utilization also enhances cell growth and this new biomass with
high polyphosphate storage accounts for phosphorous removal.
Nitrogen:

• Treatment with the strong base

(i.e. NaOH), converts the ammonium ions (NH4+) to ammonia gas
(NH3) that bubbles out of solution.
NH4+ + OH- -----> NH3 gas + H2O
 Nitrate
• Nitrate is considered one of the essential minerals in agriculture.
• It plays an important role in nitrification and denitrification processes that takes place in
nitrogen cycle.
• It possesses the characteristics of high water solubility.
• Its high concentration is regarded as the most common contaminant in groundwater
• Nitrate is also one of the elements for stimulating eutrophication.
• The forms of nitrogen that affect water are ammonium, nitrate and nitrite.
• Nitrate present in the water when enters human body easily reduced to nitrite with the help
of bacterial action in the stomach and into forms of carcinogenic N-nitrosamine compounds.
• Additionally, increased nitrate concentration reports gastric cancer and congenital disabilities.
• Nitrates in drinking water easily form nitrosamine by reacting with amine chemically or
enzymatically, resulting in cancer and methemoglobinemia in newborn infants
• Increased nitrate concentration leads to low dissolved oxygen content, very low light
transmission, reduction of biodiversity, and increased production of phytoplankton blooms by
the activity of increased phosphate concentration.
• The intermediate product formed from nitrate is dinitrogen monoxide, which possesses more
potential in causing global warming
removal techniques
• Ion exchange chromatography, reverse osmosis and electrodialysis
Others:
biological methods, chemical methods, adsorption methods, and
nanotechnology
 Biological Nitrogen Removal Process

• The main source of nitrogen in sewage is ammonium.

• Nitrification is a biological process oxidizing ammonium to nitrite and then nitrite to
nitrate.
• Bacteria responsible for the nitrification process are mostly autotrophs, while there are
some heterotrophs as well.
• Autotroph bacteria utilize carbon dioxide as carbon source and gain their required
energy from inorganic compounds, while heterotrophs gain their energy and carbon
from organic compounds.
• The nitrification process consists of two steps.
• Firstly, ammonia oxidizing bacteria (AOB) oxidize ammonium to nitrite.
• Secondly, nitrite-oxidizing bacteria (NOB) oxidize nitrite to nitrate
Nitrifying bacteria oxidize NH3 or NH4- in 2 steps to NO2- and then NO3-.

NH3 + O2 -------> NO2- + energy -------(Nitrosomonas)

NO2- + O2 ------> NO3- + energy ------- (Nitrobacter)

• Nitrification can occur in secondary biological treatment systems under conditions of low
organic loading and warm temperature (20 to 35oC )

103
Denitrification

• Nitrification is followed by denitrification in order to remove nitrogen from waste-

water.
• In the denitrification process, nitrate is reduced to gaseous nitrogen, and
consequently N2 is released to the atmosphere.
• Denitrification process requires anoxic conditions

• Bacteria responsible in the denitrification process are heterotrophs which include

Thiobacillus, Micrococcus, Serratia, and Pseudomonas-reducing nitrate and/or nitrite in
the absence of dissolved oxygen.
• Pseudomonas are the most common denitrifiers which can utilize various energy
sources.

104
Dendrogram based on 16S rDNA sequences of ammonia-oxidizing bacteria
(Koops and Pommerening-Röser 2001)
Dendrogram based on 16S rDNA sequences of nitrite-oxidizing bacteria
(Koops and Pommerening-Röser 2001)
Biological methods
Microorganisms:

• Chlorination kills any remaining microorganisms.

Immobilization of enzyme or microbial
cells for waste water treatment
 Definition
• An immobilised biocatalyst is defined as a biocatalyst for which
movement in space is completely or severally restricted to form a
distinct phase within the bulk phase in which substrate, effector,
inhibitor molecules are dispersed and their exchange is possible.
Alternatively,
• The biocatalyst is imprisoned within its phase allowing its re-use or
continuous use but preventing it from contaminating the product;
other molecules, including the reactants, are able to move freely
between the two phases.
• This may be achieved by fixing the biocatalyst to, or within, some other
material.
• The term 'immobilisation' does not necessarily mean that the enzyme
cannot move freely within its particular phase, although this is often
the case.
• A wide variety of insoluble materials, also known as substrates (not to
be confused with the enzymes' reactants), may be used to immobilise
the enzymes by making them insoluble. These are usually inert
polymeric or inorganic matrices.
 Use of immobilized enzyme or microbial cells for
waste water treatment
• Suspended growth system and fixed film systems are the
two most common types of biological treatment used for
waste water.
• Both are based on microbial growth, metabolic activities of
micro-organisms, eventual death of organisms, sludge
production and bioconversion occurring during the whole
process.
• Aerobic and anaerobic modes of operation may differ in
the extent of sludge production but still both produce
sludge.
• Immobilisation of biocatalysts (cells or enzymes) add to the
advantage of biological treatment system.
Significance of enzymes as biocatalyst

• Enzyme require low energy inputs

• Enzymes have low heat of reaction
• Enzymes are heat labile, so it is easy to terminate the reaction
• enzymes require much less complex media
• Less wastes are produced
• Continuous operation is easier.
Significance of cells as biocatalyst

• Enzymes within the cells are better protected from

denaturation
• Degradation activity may be composed of a series of
reactions working together and it will be difficult to develop
different systems for different enzymes working together.
• Isolation, purification of enzymes and then using them often
incurs an additional expense.
• Chances of denaturation are more.
• Some enzymes occur as induced enzymes so exploitation of
such enzyme is easier if cells are used
Advantages of Immobilisation

• Reuse or continuous use of cells enhances overall

efficiency.
• Biocatalyst does not contaminate the product.
• Cells are more evenly distributed by immobilisation,
so diffusional restrictions are minimised.
• Immobilised cells are used with more ease to
exploit the kinetic features of continuously stirred
and packed bed reactors.
Methods of immobilisation
principal methods available for immobilisation:
• adsorption
• covalent binding
• entrapment
• membrane confinement
(a) enzyme non-covalently adsorbed to an insoluble particle;
(b) enzyme covalently attached to an insoluble particle;
(c) enzyme entrapped within an insoluble particle by a cross-linked polymer;
(d) enzyme confined within a semipermeable membrane.
Application of Immobilised cells and enzymes in
waste water treatment

• BOD/COD reduction
• Specific pollutant detoxification
• As biosensor
• For conversion of waste to get specific products.
 Examples
• Hypomicrobium spp are grown on sand bed with added methanol to cause nitrate
reduction.

• Micrococcus denitrificans cells encapsulated in liquid membranes for reduction

nitrate and nitrite.

• Use of immobilized amylase has been proposed for the treatment of waste water
from wheat starch industry and also to clarify colloidal starch clay suspensions of
white waters from paper mill industry.

• Nitrosomonas europaea cells immobilized in alginate have been used to oxidise

ammonia in waste water to nitrite and nitrate so as to reduce the BOD and algal
growth.

• Pseudomonas denitrificans immobilized in alginate have been used to reduce nitrite

and nitrate to gaseous nitrogen

• Methanogenic cells can be entrapped in agar, collagen or polyacrylamide membranes

to give methan production.

• Immobilized enzymes can be used to hydrolyze lactose in whey and cheese making
industry.
Purification of drinking water
 1. Natural
• Storage.
• Percolation
• Self purification of rivers
• Filtration through earth
2. Artificial
• Physical- boiling, distillation, aeration, sedimentation, U.V.
irradiation.
• Chemical –germicidal, precipitants, disinfectants.
• Filtrations– a) On a large scale (Slow and rapid sand filters).
b) On a small scale ( filters)
 Storage
• 2 weeks .
• 3 processes:
1. Sedimentation: 90% of suspended materials settle down in 24 hours
by gravity.
2. Oxidation of organic material by dissolved O2 & by aerobic bacteria.
3. Biological action: Reduction of Bacterial count to 90% in first 5-7
days.
Optimum period of storage of river water 10-14 days.
Percolation

• By passing through surface of soil downwards by sedimentation,

mechanical straining, biological and oxidation.
 Artificial methods:
1.UV irradiation:
germicidal properties with wavelength of 200-295nm on water of thickness
120mm.
Source- Quartz lamp, Hg vapour.
Adv.-short period, no change in taste and odour, no harmful effect on
overdose,
Dis adv.-costly
Ozonation

• Unstable gas. Strong oxidizing agent

• Strong virucidal effect, no residual effect.
Chemical disinfectants:
should satisfy following criteria:-
1.Destruction of pathogens without changing temp., pH, mineral
2.No products of reaction like toxic subs.,
color, taste.
3.Cheap,easily available, easy to apply
4.Leave residual concentration to deal with recontamination.
5.Small amount and rapid effect.
Disinfectants (Contd.)
Bleaching powder (CaOCl2):
pungent smell, unstable , 33 % of Cl2.
Dose: 2.5 gm for 1000L of well water.
To stabilize, addition of Ca(OH)2
High test hypochlorite or perchloron
• A calcium compd. With 60-70% of free chlorine
• More stable.
• Dose- 1 gm of HTH for 1 cu.m of water
Chlorine tab. (Nirmal): 500mg for 20 L H2O.
Iodine: ethanol
2 drops of 2% for 1 L for 10 min.

Dis. Adv- costly, alteration of taste, thyroid activity.

KMnO4: powerful oxidising agent
Dose: 5 ppm(1/5 gm/gallon to reduce 98%
bacteria).
Dis. Adv. –colour, smell, taste altered, no effect for all
bacteria
Slow sand filter ( biological) :
Filtration on large scale
• In Scotland, in 1804
• Elements:
1.Supernatant water – 1-1.5 m
constant water Waiting
period 3-12 hrs
2.Graded sand bed-
a) Filter sand:
thickness=1-1.2 m,
diameter of sand=0.2-0.3mm,
ft. rate=0.1-0.4cu.m/hr/sq.m of sand bed surface
large surface area (1cu m=15000sq.m)
Filtration mechanism
• Zoogleal layer- Schmutzdecke—2-3 cm
• Gelatinous consisting of algae, plankton, diatoms and
bacteria.
• Ripening of the filter.
3.Under drain system :
4. Filter-control system:
Cleaning of Filter:-
• Loss of head- Resistance of sand bed to filter.
• Scraping off the top portion of sand (1-2cm).
• Construction of new bed after 20-30 scraping.
 Rapid sand filter (mechanical)
• In USA, in1885.
• 2 types:
a) Gravity type (Paterson’s filter)
b) Pressure type( Candy’s filter)
• Components:
1. Coagulation: Raw water +coagulant (alum=5-40mg)
2. Rapid mixing in Mixing chamber.
3. Flocculation: gentle stirring 2-4 rpm for about 30mins
Formation of white flocculant ppt
4. Sedimentation : 2-6 hrs.
Contd.
5.Filtration :
a) Raw water
b) Sand bed : thickness 1m,
diameter of sand =0.4-0.7 mm
Surface area=80 - 90 sq m
Rate of filteration: 5-15 cu m/hr/sq m of sand bed
c) Gravel: 30-40 cm
d) Under-drain system
Filtration mechanism
• Formation of slimy layer of alum-floc
• Cleaning: by back washing or reversing the flow of
water for 15 mins
• Weekly washing
• Adv.
• SSF RSF
• Simple complicated
• Cheap expensive
• More space less space
• High quality less
• Reducing of E. coli= 99-99.9% 98%
• Removal of bacteria 99.9-99.99% 98-99%
Filtration on small scale

• Three types-
• Pasteur chamberland- porcelain candle
• Berkefeld- infusorial earth candle
• katadyn – coated with silver catalyst
• Disadv.
• No effect to virus
• Logged with impurities and bacteria
• Frequent cleaning by scrubbing with a hard brush and boiling
Water quality criteria
• By WHO 1993 and 1996
• Acceptability aspect
• Physical:
• turbidity< 5NTU,
• colour <15 TCU,
• taste / odour free
• cool temperature
Inorganic:
Cl= 250 mg/l NH3=1.5 mg/l
pH= 6.5-8.5 Hardness=moderate
Zn< 3mg/l H2S= 0.05-0.1mg/l
Na=200 mg/l TDS=1000 mg/l
 Microbiological aspects
• All water intended for drinking
 E.coli - must not be detected in 100ml
• Treated water
 E.coli - must not be detected in 100ml
 In case of large supply E.coli must not be
detected in 95% of sample taken through out
12 months period.
 Fecal Streptococci and Clostridium perfringens should be nil.
 Virus, protozoa, helminthes and free living org. should be nil.
Chemical aspects
• Inorganic substances:
• Antimony = 0.005 mg/l
• Arsenic= 0.01 mg/l
• Fluoride = 0.5-1.5 mg/l
• Lead = 0.1 mg/l
Organic substances :
PAHs (carcinogenic) should be free.
Pesticides should be under recommended guideline value
Chlorination
• Use of chlorine to purify water
• Adv.-
• powerful germicide
• Quick in action
• Oxidizing agent (Fe, Mn,H2S)
• Destroys some taste and odour producing agent
• Coagulating properties
• Disadv.-
•Alteration of taste
•No effect on spores, cyst.
Action of chlorine:
H2O+ Cl2  HCl + HOCl (hypochlorous)
HOCl  H + OCl (hypochlorite)
pH of water = 7
Principle of chlorination:
1. Water to be chlorinated should be free from
turbidity.
2. Chlorine demand (CD) should be estimated.
CD= amount of chlorine needed to destroy bacteria, to
oxidize organic matter and ammonical substances
present in the water.
It is the diff. between amt. of Cl added and amt. of
residual Cl remained at the end of the specific period of
contact(60 mins) at a given temp. and pH of water.
Break point:
The point at which chlorine demand of water is
met.In further adding chlorine beyond the break pt.,
free residual chlorine begins to appear in the water.
3. Contact period :
At least one hour is essential to kill bacteria and
virus.
4. The min. recommended conc. of residual Cl2 = 0.5 mg/l
5. The accurate dose = residual Cl2 + Cl2 demand.
 Potable (Drinkable) Water
1. Assay of water "purity" - the coliform test (1905)

• Coliform bacteria include rod-shaped , gram-negative, lactose-

fermenting species that do not form spores.

• Many enterics fit these criteria, including E. coli and Salmonella

sp.

• Coliforms are used to indicate if the water contains fecal

contamination.

• Although not necessarily pathogens, coliforms are useful as

indicators of true pathogens present in feces that we might not
have an assay for
• Indicator organisms are widely used in microbiology; criteria for a good
indicator organism are that it must:
• Be easy to detect and identify;
• Be present whenever pathogens are present;
• Respond to wastewater treatment similarly to pathogens.
EPA guidelines:

(i) Water samples must be assayed > 20 times/month.

(ii) The water is bad and the public must be alerted if > 4 colonies/100
mL are found in >5% of the samples.
Preparation of potable water

a. Settling - Water from rivers or underground wells is allowed to

settle, to separate and subsequently remove large insolubles (sand,
silt, etc.).
contd.

b. Coagulation –
• Aluminum and ferric ions are added and the pH is raised. This results in the
precipitation of aluminum hydroxide [Al(OH)3] and ferric hydroxide
[Fe(OH)3].
• As these substances precipitate from the solution, they carry along
microorganisms and adsorbed organics. After settling, the
supernatant is collected.
contd.

c. Filtration –
- Next, the water is filtered through sand.
-This further removes organisms and organics by adsorption.
-This is an especially important step for removing protozoa that have
a cyst phase, e.g. Giardia lamblia, because these cysts are often
resistant to chlorine.
contd.

d. Chlorination –
-Treatment with chlorine (Cl2) kills off any remaining microorganisms.
"Pure" drinking water prepared in this way is free of microorganisms.
Water purification:
 Waste Generation
• Current global MSW generation levels are approximately 1.3 billion tonnes
per year, and are expected to increase to approximately 2.2 billion tonnes
per year by 2025.
• The higher the income level and rate of urbanization, the greater the
amount of solid waste produced.
• Organization for economic cooperation and development (OECD)
countries produce almost half of the world’s waste, while Africa and South
Asia regions produce the least waste.
 Waste Generation by Region

Africa (AFR)
South Asia Region (SAR)
Middle East and North Africa (MENA)
Eastern and Central Asia (ECA)
Latin America and the Caribbean (LAC)
East Asia and the Pacific Region (EAP)
Organization for Economic Co-operation and
Development (OECD)
World Bank, 2012
Urban Waste Generation by Income Level and Year
Dangi et al., 2010
• Chronology of waste composition. Dangi et al., 2010
Waste types % waste composition by
weight
Organic wastes 71.0
Plastics 12.0
Paper and paper products 7.5
Metals 0.5
Glass 1.3
Rubber and leather 0.3
Textiles 0.9
Dirt and construction debris 5.0
Hazardous wastes 1.0
Other wastes 0.7
Total 100.0
Dangi et al., 2010
 What is landfill?
 55% of MSW and many other solid wastes are disposed in landfills
 Landfill disposal is part of the last option in the waste hierarchy.
 It is a method of treating and disposing of those wastes which, for
the time being, are not possible to reuse, recover or recycle.
 However, landfill disposal with energy recovery from gas partially
moves it up the hierarchy.
 Landfills have evolved from open dumps to highly engineered
facilities designed to contain waste and separate it from the
environment, capture contaminated water that contacts the waste
(leachate), and control gas migration.
The deposition of waste materials containing biodegradable matter
invariably leads to the production of gas and leachate, the composition
of which will vary according to the nature of the material being
degraded and the surrounding environmental conditions
 SANITARY LANDFILLING PRINCIPLES
• A sanitary landfill is an engineered facility that requires detailed
planning and specifications, careful construction, and efficient
operation.
• In a landfill, solid wastes are disposed of by spreading in thin
layers, compacting to smallest practical volume, and covering each
day, or periodically, with soil or suitable substitute material in a
way that minimizes environmental problems.
• Waste stabilization begins shortly after it is placed in the landfill,
but will not be completed until years after closure.
• Care must be exercised to ensure that an environmental problem
does not develop during operation or after closure.
• Careful design, construction, and operation can minimize the
potential for problems arising at a landfill.
• Dealing with the vast quantities of waste that our society
produces has become a major challenge.
• Improper past disposal practices have caused environmental
degradation, need for costly remedial actions.
• Old landfills were developed more to provide cheap disposal
alternatives than to protect the environment.
• Although other methods of managing waste, including
recycling, composting, and incineration, are being instituted
by communities around the country, access to a landfill
remains a necessity for everyone.
• Until our society can design and manufacture products that
are totally recyclable or reusable, landfills will be needed.
 Dumping/ landfill
• A major disadvantage of a landfill is the release of harmful
gases.
• One ton of biodegradable waste can produce about 400–500
cubic meters of landfill gas that contains methane and carbon
dioxide as the major constituents and traces of other gases.
• Methane is a greenhouse gas that is 25 times more hazardous
than carbon
dioxide.
Thus, this increases the implications of global warming and
climate change.
Health issues-
• prolonged exposure may suffer from cancer, respiratory disorders, and developmental defects
in children
• Even short-term exposures to ammonia and hydrogen sulfide releasing in the air can cause
eye, nose, and throat irritation as well as asthma.
• health complaints include vomiting, nausea, unconsciousness, sleeping difficulties, weight loss,
and chest pain.
• Methane build up is also a concern which exposes the lives of nearby residence due to the risk
of methane explosion
• Landfill sites often become vulnerable to collapses of abrupt landfill settlement due to rain,
spontaneous combustion of gases or materials, or excessive waste accumulation causing
damages that are deep and almost irreversible.
 Purpose of Sanitary landfill
• The main purpose of sanitary landfills is to ensure the safe decomposition of the waste
which otherwise may pollute all the periphery of the dumping site.
• The alternative layering of garbage and soil aids in hastening decomposition.
• Methane gas is produced from decomposition, it is collected in the landfill to generate
electricity instead of being released into the atmosphere.
• The sanitary landfill also uses a clay liner to isolate the trash from the environment.
Besides, it involves well-designed engineering methods to protect the Environment from
contamination by solid or liquid wastes.
• When the sanitary landfill is full, impervious clay is used to seal it, and if deemed safe,
that area can be used for other purposes.

Typical cross section of a modern landfill permitted to receive non hazardous solid waste
 Sanitary landfill

Various stages involved in sanitary landfilling-

1. Selection of site
2. Method of land filling
3. Control, collection and regulation of gases
and leachates generated
 1. Selection of site
i. Location – site should be away from human habitation

ii. Topography – low lying area is preferred.

- run off from the site should never get collected in natural courses of water.
iii. Area of the site should be such that it should permit easy dumping of solid waste over it for more
than a year.
iv. Subsoil: The subsoil should be impervious or less porous. Porous soil to be used as cover for the
landfills after compaction must be available at or near the site.
v. Groundwater table: Ground water should not be available at shallow depths.
vi. Climate: Hot and dry climate is quite preferred to wet climate. Rainfall of the area should be less.
vii. Environment: The area, though well connected to the roads for easy transportation, should be
separated from the land of human activities as agricultural lands by rows of trees or by a compound
wall constructed to enclose the area. Its neighborhood should be free from pollution and preferably of
lushly vegetable growth.
viii. Distance of haul: The site should not be quite far off as the length of transport and hence cost of
operation of the solid waste disposal is going to increase.
ix. Ultimate use of the site: When the site gets filled up and can no more take any load (after many
years), to what use the site is going to be put is to be decided now itself and strictly adhered to in
future.
 Methods of landfilling
The three common configurations of sanitary landfills
are:
• canyon or depression method
• trench method
• Cell method
• area method
 The canyon method/ depression method
• In canyon method natural canyons /depressions
or manmade are used
• This can include filling old quarry sites.
• Waste is spread over the area in thin layers and
well compacted. Another layer is added above.
At the end of day’s operation 150 mm thick earth
layer is laid and covered and compacted on all
sides.
• Thus a unit of solid waste is to be enclosed on all
sides by an earthen cover.
 The Canyon Method of Landfill
It is used of when a natural or man made depression is available at a convenient place.
 Trench method
– In the trench method the excavation into which the
landfill is placed is excavated specifically for the
purpose of landfilling.
– The soil is temporarily stockpiled.
– The waste is placed on the ground surface or, more
commonly, a landfill liner, spread in layers, and
driven over with compaction equipment.
– Successive layers are built up until a depth of 3 to 4 m is
achieved.
 The Trench Method of Landfill
• When no depression is available, trenches are dug and the earth excavated can be
used as covering material
 Cell method:
• Instead of dumping the solid waste over a vast
area, filling, compaction and covering it with a
layer of soil is done in a smaller area and the unit
so formed is called ‘a cell’.
• Cell is a definite unit confined on all the sides and
is heterogeneous in composition.
• It is a better method than any other method of
land filling because of independent cells of less
area and volume and ease of handling.
What is a cell?

• A completely covered compacted solid waste unit is called a

cell.
• A series of adjoining cells, all the same height, makes up a lift.
• The completed fill consists of several vertical lifts which may
extend 20 to 50m above the original ground surface.
• The final cover for the landfill will be about 1m of soil or a
combination of soil and synthetic materials.
• The function of the final cover is to limit the entry of water
into the landfill.
• This results in reduced leachate generation, that is, the
release of contaminated liquids from the base of the waste in
the landfill.
 The area method
• This is used when the terrain or soil conditions
are such that cells or trenches cannot be dug.
• Site preparation can include the use of liners
and leachate control systems.
• When neither a depression exist nor it is
possible to dig trenches, area method is used.
• The site should be enclosed on all sides with
tall embankments.
• Cell over cell is added until the compacted
waste reaches a height of 2 to 3m.
 The Area Method of Landfill
When neither a depression exist nor it is possible to dig trenches, area method is used.
Differences Between Sanitary Landfill and Open Dumping
 ANAEROBIC BIODEGRADATION OF SOLID WASTE

• Microbial reactions in the landfill control are landfill

gas and leachate composition, but the overall
breakdown of organic matter can be represented by
the equation:
• C6H1206 = CH4 + C02 + Biomass+ Heat
• In general, a landfill will go through three different
decomposition stages with different bacterial types
predominating in each stage.
Landfill stabilisation, gas and leachate
production
 Stage 1
• Solid waste initially decomposed aerobically, with carbon
dioxide, water, and nitrate the decomposition products.
• As oxygen is used up, facultative and anaerobic microorganisms
predominate.
• These bacteria produce volatile acids and carbon dioxide.
• These organic acids reduce the pH to 4 or 5, which in turn
solubilizes some inorganic materials in the landfill.
• During this time, conductivity will be very high.
• The low pH is toxic to methane producing bacteria, so little
methane will be produced during this period.
• The first anaerobic stage is characterized by low pH, high
volatile acid production, high chemical oxygen demand (COD),
high conductivity, and low methane production.
 Stage 2
• With time, methane producing bacteria become more predominant (second
stage anaerobic decomposition).
• These bacteria degrade the volatile acids to methane and carbon dioxide, (at
approximately a 50-50 ratio), resulting in a rise in pH to more neutral values
and a fall in the COD.
• With the higher pH, fewer inorganic salts are solubilized which, combined with
the decreasing volatile acid production, results in a fall in conductivity.
• The ratio of methane to carbon dioxide depends not only on the activity of
methane-forming microorganisms in relation to other microbial forms
(primarily acid formers), but also on the nature of the organic components of
the wastes.
• For example, anaerobic decomposition of cellulosic components results in
almost equal amounts of methane and carbon dioxide; proteins and fats
produce more methane than carbon dioxide.
• With the proportion of protein, fats, and cellulose found in municipal solid
waste, the ratio of methane to carbon dioxide in the biogas varies.
 Stage 3
• This is the maturation stage where processes
from Stage 2 continue but gradually slows
with decomposition.
 Factors affecting decomposition
• Major environmental factors which affect the rate and
extent of the biochemical decomposition in the landfill are
1.moisture,
2.temperature,
3.soil cover permeability to water,
4.rainfall,
5.the degree of resistance of the wastes to
bacterial attack, and
6.solid waste processing before landfilling.
 Leachates
• There occurs physical, chemical and biological changes
during decomposition of solid wastes.
• The liquid emanated out of the waste are called
leachates which percolate horizontally and vertically.
• The volume of the leachates increases because of
drainage or sub surface water mixing with it.
• The downward percolation of leachates are prevented
by liners in sanitary landfill.
• The liners can be plastic or concretes.
The quantity of leachate generated is primarily
dependent upon:
1. the amount of precipitation falling into the landfill
while it is open;
2. the amount of drainage through the landfill cover
after it is closed; and
3. the high moisture content of the wastes.
 Gases
• Gases mainly as methane, carbon dioxide, hydrogen peroxide,
ammonia and carbon monooxide are given out from sanitary
landfills due to anaerobic decomposition of organic wastes.

• The ultimate gas yield has an estimated gas composition

by volume of 51% methane and 49% carbon dioxide.

• Vents at regular intervals are provided to clear off gas

accumulations. Otherwise may ejected out through the line of
least resistance and at times may cause fire hazards and
explosions.
 Typical landfill gas composition
Typical Value (% Observed Maximum (%
Component
volume) volume)

methane 63.8 88.0

carbon dioxide 33.6 89.3

oxygen 0.16 20.9

nitrogen 2.4 87.0

hydrogen 0.05 21.1

carbon
0.001 0.09
monoxide
• Both methane and hydrogen are flammable in the presence of
oxygen and are therefore potentially explosive if ignition occurs
within a confined environment.
• Methane is flammable in air within the range 5-15% by volume while
hydrogen is flammable within the range 4.1-75%.
• However, except in the early stages of waste stabilization when the
concentration can reach levels of approximately 20% (v/v), hydrogen
is seldom present within landfill gas at levels within the explosive
range.
• Methane is non-toxic, but through displacement of oxygen within the
root-zone, may cause death of surface vegetation.
• Where landfill gas migration has occurred, the migration pathway can
often be followed by visual observation of the surface vegetation,
including trees, which show withering at leaf margins, defoliation,
and branch dieback.
• In extreme cases, surface heating of the soils can also be detected.
 Advantages

• With regard to public health and environmental

protection, a properly designed and operated landfill
offer advantages in the following areas:
(1) aesthetics, especially litter;
(2) decomposition, gases and leachate;
(3) birds;
(4) fires and odors
(5) pests;
(6) rats; and
(7) injury from scavenging.
 BIOREACTOR LANDFILL
• It is a process accelerating the stabilization of the landfill material and
increasing the evolution of more amount of gas by hastening the microbial
activity.
• This is achieved by injecting leachate, waste water sludge, wastewater or
even storm water into the layers of landfill to maintain a moisture content
of 35% to 65% and to enhance microbial activity.
• In aerobic process air is also made to circulate through different layers of
landfill by providing interconnected horizontal and vertical wells through
landfill layers.
• No such aeration is provided in anaerobic process and the methane
generated is carefully collected as otherwise it tends to catch fire
or
may add to the green house gases responsible for global warming.
 Advantage of Bioreactor Landfills:
• Decomposition is hastened i.e. takes only years for
stabilization while the period is decades in conventional
landfill.
• Leachate is effectively used and hence its quantity gets
reduced considerably, lowering the cost of treatment.
• Less space is required (i.e. 70 to 85% of that required for the
conventional type)
• Less toxic matter is left over after decomposition and
pathogens are eliminated.
• Significant quantities of methane is given out which is of
immense value as a fuel.
 Advantages of Sanitary Landfill
1. Effective waste disposal method:
– Sanitary landfills provide an efficient and organized way to dispose of municipal
solid waste.
2. Environmental protection:
– Sanitary landfills are designed with protective liners and systems to prevent
contamination of soil and groundwater, minimizing the impact on the environment.
3. Odor and pest control:
– Properly operated sanitary landfills have measures in place to control odors and
prevent the attraction of pests, reducing nuisance factors
4. Methane capture:
– Landfills can capture and utilize methane gas, a byproduct of decomposing waste,
as a renewable energy source, contributing to the reduction of greenhouse gas
emissions.
5. Land conservation:
– Sanitary landfills make efficient use of land by compacting and layering waste,
allowing for more waste to be stored in a smaller area compared to open dumping
methods.
6. Compliance with regulations:
– Well-managed sanitary landfills adhere to strict regulations and guidelines set by
environmental agencies, ensuring that waste disposal is carried out in a controlled
and responsible manner.
 Disadvantages of Sanitary Landfill

1. Land requirement: Sanitary landfills require a significant amount of land, which

can be a challenge in densely populated areas where suitable land is limited.
2. Potential groundwater contamination: Despite protective liners, there is a risk of
leachate, a liquid formed from decomposing waste, seeping into groundwater and
potentially contaminating water sources if not managed properly.
3. Air pollution: Landfills can emit various gases, including methane and volatile
organic compounds, which contribute to air pollution and can have negative
health effects.
4. Visual impact: The presence of a landfill in the vicinity can be aesthetically
unappealing and diminish the scenic value of the surrounding area.
5. Odor and noise concerns: Despite efforts to control odors, landfills can still
produce unpleasant smells, and the operation of heavy machinery and waste
disposal activities can generate noise that can disturb nearby residents.
6. Long-term monitoring and maintenance: Proper closure and post-closure care of
landfills require long-term monitoring and maintenance to ensure ongoing
environmental protection, which can be costly and time-consuming.
 Sisdol landfill
• Sisdol landfill is a location to dump solid waste from Kathmandu, Lalitpur, Bhaktapur, Kakani,
and Banepa.
• It is the biggest dumping site of Nepal.
• 100 tons of solid waste is dumped at the site daily.
• The landfill is located at Kakani Rural Municipality (previously Okharpauwa Village
Development Committee) of Nuwakot district.
• The landfill site has an area of 15 hectares.
• Two hectares of land is used for the actual filling while 12 ha are used as a buffer and the
remaining 1 hectare is used for other facilities.
• The landfill site has two valleys, with a capacity of 166,085 and 108,910 cubic meters
respectively.
• The waste from various cities is first collected in Teku transfer station.
• From the station, the waste is dispatched to Sisdol.
• Around 60 % of wastes in the Sisdol are organic but the waste remains mostly unsegregated.
• The recyclable waste is brought back and sold to recycling industries.
• The dump is filled in a semi-aerobic condition.
 Generation of waste in KMC

Population approx. 8,00,000

Population growth rate 3.25%
Waste generation 0.25 kg/day
Others (VDC, Commercial, day pop. etc.)0.15kg/day
____________
per capita waste generation 0.40Kg/day
Total Generation 320 ton/day
Municipal Collection 300 ton/day
 Bioremediation
• Bioremediation is the use of living organisms (primarily microorganisms) to
destroy or immobilize waste materials and environmental pollutants.
• It is an emerging alternative technology for removing pollutants from the
environment restoring contaminated sites and preventing further pollution.
• The general approaches to bioremediation are to enhance natural
biodegradation by native organisms (intrinsic bioremediation), to carry out
environmental modification by applying nutrients or aeration (biostimulation)
or through addition of microorganisms (bioaugmentation).
• Biodegradability or recalcitrance depends on the nature of the chemical
molecule. Often a simple change in the substituents of a chemical molecule
may make difference between recalcitrance
• Unlike conventional technologies, bioremediation can be carried out on-site.
Bioremediation is limited in the number of toxic materials it can handle, but
where applicable, it is cost effective.
Contd.
• Many of novel compounds introduced into nature are called as
xenobiotics (xenos meaning foreign in Greek), and a large number
of them are not easily degraded by the indigenous microflora and
fauna.
• The list of xenobiotics is very long and some of them are directly
applied to nature in the form of pesticides or fertilizers, some
others are released as industrial waste products (effluents).
• the xenobiotics would also include a wide variety of dumpings such
as plastics, detergents and oil spills.
• The chemical pollutants such as toxic pesticides are of two types,
biodegradable and nonbiodegradable (recalcitrant).
• A biodegradable pesticide may be converted by microbial action
into a nontoxic coumpounds within a few months,
• A recacitrant chemical may remain in nature for several years in the
toxic form.
• Biodegradation, mineralization, bioremediation, biodeterioration,
biotransformation, bioaccumulation and biosorption are some
terms with minor subtle differences but often overlappingly used.
• Biodegradation is the general term used for all biologically
mediated break down of chemical compounds and complete
biodegradation leads to mineralization.
• Biotransformation is a step in the biochemical pathway which
leads to the conversion of a molecule (precursor) into a product.
A series of such steps are required for a biochemical pathway. In
environmental terms, it is important whether the product is less
harmful or not.
• Biodeterioration refers usually to the breakdown of economically
useful compounds but often the term has been used to refer to
the degradation of normally resistant substances such as metals,
plastics, drugs, cosmetics, painting, sculpture, wood products and
equipment.
• Bioremediation refers to the use of biological systems to degrade toxic
compounds in the environment.
• Bioaccumulation or biosorption is the accumulation of the toxic
compounds inside the cell without any degradation of the toxic
molecule.
• This method can be effective in aquatic environments where the
organisms can be removed after being loaded with the toxic
substance.
• Biomagnification-
• The concentration of the xenobiotic in the environment when
diluted may vary from ppm (parts per million) to ppb (parts per
billion) levels, and at still lower levels it may not have any effects.
However, the compound may become progressively more
concentrated in the body of certain animals as it moves up the food
chain. The process is called biomagnification.
• This was first discovered in California where a lake had been treated
with the pesticide DDD (related to DDT) to kill some insects.
• Later on the fish that ate the phytoplankton containing DDD as well
as the birds that ate the fish started dying. The body fat of the birds
contained 100,000 times higher concentration of DDD than the lake
water or the phytoplankton.
Bioremediation mechanism
• Microorganisms destroy organic contaminants in the course of
using the chemicals for their own growth and reproduction
• Organic chemicals provide:
• carbon, source of cell building material
• electrons, source of energy
• Cells catalyze oxidation of organic chemicals (electron donors),
causing transfer of electrons from organic chemicals to some
electron acceptor
Electron acceptors
• Electron acceptors:
• In aerobic oxidation, acceptor is oxygen
• In anaerobic, acceptor is (with decreasing efficiency):
• nitrate
• Sulfate
• manganese
• iron
• Microorganisms also need essential nutrients such as nitrogen
and phosphorus
Three requirements for biodegradation:

• capable organisms
• synthesis of requisite enzymes
• suitable environmental conditions
Genetic Potential
• It refers to the presence and expression of appropriate
degrading genes by the indigenous microbial
community.
• The biodegradation generally follows a period of
adaptation or acclimation, the length of which
depends on the contaminant structure
 Acclimation:
• Prior to the degradation of many organic compounds there is an
acclimation period, during which little measurable degradative
activity occurs.
• Understanding this phase may allow for minimizing the
acclimation period in many cases.
• The length of the acclimation can vary from minutes to months.
• The duration depends on the type and concentration of the
compound and environmental factors including temperature,
aeration, etc.
• Very long acclimation periods are noted for anaerobic degradation
of chlorinated solvents.
• Acclimation ends when detectable degradation begins.
• If a second addition of the contaminant is made after acclimation,
frequently additional acclimation is not observed, and frequently
degradation proceeds more rapidly than with the first addition.
• This phenomenon may be due to larger populations of the
degrading organisms being present.
General observations regarding acclimation:
1) Frequently acclimation to one compound results in acclimation to a
variety of similar compounds.
2) Temperature, pH, aeration status, and nutrient availability (high or
low) can all affect duration of acclimation.
3) Contaminant concentration often plays a major role in impacting
measurable acclimation duration. There may be concentrations below
which acclimation does not occur; however observations have been
made of acclimation occurring at contaminant concentrations so low
that organisms would not be able to use them as sole C sources.
4) Acclimation varies between sites and between microbial
communities.
5) Acclimation of microorganisms to the degradation of pesticides and
enhanced degradation upon repeated exposure (accelerated pesticide
degradation) contribute to the frequently observed ineffectiveness of
applied chemicals in agricultural settings. Data suggests that this
phenomenon is due to increased activity, not increased microbial
numbers.
What occurs during acclimation?
• Population size increases, often from very low levels,
at which analytical detection of contaminant
decreases is impossible.
• Populations can be inhibited by either high levels of a
toxic contaminant, or by another toxic co-
contaminant.
• Toxicants can act in several ways, including slowing
down (but not eliminating) the growth of the
contaminant degrading species in a straightforward
way.
• Toxicants can also suppress fast growing species,
letting resistant, slow growing populations determine
the measurable acclimation period.
• Genetic selection can occur within a population.
Bioavailability
• There are two steps in the biodegradative process:
• uptake of the substrate by the cells
• Metabolism or degradation of the substrate
• Assuming the presence of an appropriate metabolic
pathway, degradation of a compound can proceed
rapidly if the compound is available in a water soluble
form.
• Limited water solubility or strong adsorbent to soil
and sediment can limit the biodegradation due to low
bioavailability.
Inaccessibility of substrate
• Organics bound to clays are resistant to degradation
• Clays/humus may inactivate exoenzymes (cellulase)
or bind substrate
• Coatings or encrustments
• coatings with non-degradable organics (e.g.
Lignin) insoluble carbonates or inorganic oxides.
• Substrate movement into zones of limited biological
activity (ground water, anaerobic sediments)
 substrates that are not proximal
• A major question that must be addressed by microorganisms is
one of proximity to the target substrates or pollutants.
• If cells are encapsulated in a formulation, then the question
becomes one of how attached cells come out of the
encapsulation matrix or how substrates get to the attached cells.
• Organisms must be close to the substrate, sometimes in direct
contact, if they are to degrade it.
• Being "close" means within the diffusional distances of the target
compound, or of the extracellular enzymes to reach the
substrates, or of the products of extracellular digestion.
• There is a profound difference between well mixed aqueous
environments (such as the lakes, rivers, stream, oceans) and the
largely unmixed environments (soils, sediments) where there are
barriers to movement, motion and diffusion.
• In soil, a distance of several cm can mean the difference between
a compound being degraded or not degraded.
 How do microorganisms cope in such
instances ?
Physical means
• Microorganisms can be carried far distances through a soil
profile by water-mediated transport.
• They can also move to far away places by air currents to
inoculate new sites where there are favourable
environmental and nutritional conditions.
• Microorganisms may also be carried far distances by
vectors such as insects, animals or humans.
 Contd.
Biological means
• Some microorganisms can display chemotaxis towards
substrates, and this is very important in microenvironments.
• Some microorganisms are filamentous and can show
"directed" growth toward the substrates via hyphal
development.
• Many fungi, can "explore" the environment, locate
uninoculated wood, and then colonize it.
• Alternatively, cells can reproduce, producing spores which
might be carried by air currents to new locations near the
target pollutants.
 Large and inaccessible substrates
• The biological answer to the relatively small sizes of
microorganisms in relation to large substrates is to use
extracellular enzymes.
• The enzymes diffuse to the substrate, and carry out an initial
attack to convert the substrate into smaller, lower MW, more
soluble or diffusible compounds.
• Inefficiency of this process is generally related to the fact
that once released, extracellular enzymes are no longer
under the control of the organisms.
(a) enzymes may be subject to adsorption, degradation
and denaturation; and
(b) the products can be captured by competing
microorganisms.
 Solid, water-insoluble substrates or substrates which are
adsorbed to soil
• Many hydrocarbons are poorly water soluble, so this is a common
problem in the environment.
• Some of these compounds (such as polycyclic aromatic hydrocarbons)
may be present in high concentrations, but are not accessible or
bioavailable due to their physical or chemical properties.
• Often, the rate of substrate dissolution limits the rate of
biodegradation.
• Large amount of energy is needed to disperse the solids.
• Many microorganisms produce biosurfactants or bioemulsifiers which
disperse the substrates which can then be taken up inside the cells.
• Some researchers have speculated that biosurfactants can also help
with uptake of hydrophobic compounds into the cells.
• Another important function of biosurfactants is to desorb
hydrophobic compounds from soils, thereby making them more
accessible and bioavailable to the substrates.
 General strategies of pollutant metabolism
Degradative mechanisms may vary depending on the substrates and
microorganisms involved.
Initial processing through the peripheral metabolic pathways
• After being taken up, odd substrates and many pollutants are first
catabolized through peripheral metabolic pathway.
• These steps are often the most difficult and rate-limiting for
degradation of the particular compound.
• The enzymes involved are generally inducible, repressible, and their
genes may be plasmid-encoded.
• The purpose of these initial transformations is to convert the
compounds into forms that can feed into the central intermediary
metabolic pathways.
• Central metabolic pathways are normally constitutive.
• Both central and peripheral pathways can be subject to control at the
transcriptional level, i.e. genetic controls are operative.
• The controls may be inefficient for many xenobiotic compounds
 Structural constraints
• Biodegradation of many pollutants may be slow, difficult or
incomplete because of the substrates' chemical structure.
• Chemical structures that may affect biodegradability include:
1.Size. Large fused rings (>4 rings) are very recalcitrant.
For example, compare the ease of degradation of benzene,
naphthalene and phenanthrene or anthracene.
2.Shape. Position of each ring in the fused rings may affect
bioavailability and biodegradation.
A good comparison is anthracene vs phenanthrene.
3.Degree of branching. The greater the degree of branching, the
more difficult it is to break down the compound.
example is between a straight chain alkane vs a branched-chain
aliphatic compound.
4.Degree and type of substitution- e.g. chlorine, bromine,
amine or nitro- groups.
In general, the greater the degree of chlorination, the greater
the resistance to aerobic degradation, and the lesser
number of microorganisms capable of degrading the
compound.
This holds true for most types of substitution.
5.Position of substitution- In general, ortho and meta
substitutions in an aromatic ring render a compound more
difficult to degrade.
This is due to the specificity of dioxygenase enzymes
that initiate biodegradation of these compounds.
6.Toxicity- The toxicity of some compounds make it difficult
for microorganisms to degrade them.
7.Hydrophobicity- Hydrophobic compounds can be less
bioavailable to microorganisms. Many hydrophobic
compounds need to be rendered more hydrophillic before
being further degraded. This can be done by an oxygenase
attack or by biosurfactants.
8.Type of chemical bonds- Some C-Cl and C-N (esp. the
electron-withdrawing NO2) bonds may be difficult to break
up. Unusual combination of bonds can cause problems with
biodegradation, e.g.. TCE.
9.Charge- Highly charged compounds may not be easily taken
up by cells, thereby hampering their biodegradation. Some
of the detergents fall in this category.
 Co-metabolic requirements
• Some compounds cannot furnish any energy to the
microorganisms that initiate their biodegradation.
• Energy must be provided by another carbon source.
• This mode of metabolism is known as co-metabolism.
• Co-metabolism is believed to be due to the broad
substrate specificity of some catabolic enzymes.
• Its occurrence is believed to be rather frequent in nature.
• Co-metabolism plays an important role in initiating the
metabolism of many xenobiotic compounds.
 Utilization of mixed substrates
• In natural and engineered environments, mixed substrates are
the norm.
The important question for the bioremediator is whether
microbial cells will take up the more persistent pollutants first or
will the cells take up the easy-to-degrade substrates, and leave
the pollutants behind ?
• Generally, microorganisms will take up the preferred substrates
first, and not bother with the less preferred ones.
• With mixed substrates for which there are specific transporters,
the utilization can be sequential, diauxic, or simultaneous.
• Co-metabolism is a special case of mixed substrate utilization.
 Environmental Factors in Molecular Recalcitrance

A. Fate of xenobiotics
• transport
• advection (gaseous transport)
• dispersion (diffusion and mixing)
• absorption (e.g., water taken up by a sponge)
• adsorption (adhere to surface as a thin film, biofilm)
• volatilization
• sedimentation
• photochemical oxidation,
• biodegradation/biotransformation
 Environmental Extremes

• Oxygen
• Organic matter content
• Nitrogen
• temperatures
• lack of water
• pH
• high salt concentrations
Oxygen
• Oxygen is very important in determining the extent and rate of
contaminant biodegradation.
• Aerobic biodegradation is much faster than the anaerobic
biodegradation.
eg. Petroleum based hydrocarbons entering the aerobic zones of
freshwater lakes and rivers are generally susceptible to
microbial degradation but oil accumulated in anaerobic
sediments can be highly persistent.
• Likewise, biodegradation of such compounds require much
longer adaptation periods, than the aerobic environment.
 Organic matter content
• The factor that has great influence on microbial activity is the organic
matter, the primary source of carbon for heterotrophic micro-
organisms in most environments.
• In an ecosystem the deeper region such as the vadose zone and the
ground water region have low amounts of organic matter as a result
decrease in microbial number and are often dormant.
• Plus need extended acclimation times for significant biodegrading
population build up.
 Nitrogen
• Microbial utilization of organic matter (contaminants) particularly
hydrocarbons composed primarily of carbon and hydrogen, creates a
demand for essential nutrients such as nitrogen and phosphorus.
• Thus, biodegradation can often be improved simply by the addition of
nitrogen fertilizers.
• In general, microbes have an average C:N ratio within their biomass
of about 5:1 to 10:1 depending on the type of microorganism.
Therefore, a ratio of approx. 100:10:1(C:N:P) is often used in such
sites.
• The carbon that is metabolized is released as carbon dioxide so are
lost from the ecosystem while nitrogen and phosphorus metabolized
is incorporated into microbial biomass and is conserved in the system.
 Temperatures
• Hydrocarbon degradation has been reported to occur at a range
of temperatures between close to freezing and more than 30º C.
• Bacteria can adapt to temperature extremes in order to maintain
metabolic activity.
pH
• In soils, the rate of hydrocarbon degradation is often higher in
alkaline conditions than in acidic conditions.
• In acidic soils, fungi are more competitive than bacteria, which
prefer a neutral environment.
• Thus, at lower pH fungi become more important in
hydrocarbon degradation.
• The pH of the sediment ranged from 6.5 to 8.0, but when
incubated at different pH levels, lower rates of hydrocarbon
degradation were evident at pH 5 and 6.5 than at pH 8.
 Salinity
• Salinity also affect the hydrocarbon degradation .
 Water activity
• Optimal conditions for activity of aerobic soil micro-organisms
occur between 38% and 81% of the soil pore space because in
this range of water contents, water and oxygen availability are
maximized.
• At higher water contents, the slow rate of oxygen diffusion
through water limits oxygen replenishment, thereby limiting
aerobic activity.
Contaminant properties
Generic Mechanisms
1. Oxygenases
• catalyze the incorporation of oxygen from O2 into organic compounds.
• These enzymes contain a variety of cofactors such as hemes, flavins,
copper, and manganese that "activate O2 to the singlet state
- Monooxygenases (incorporate only one <O> as OH and the other goes
off as H2O, also called hydroxylases)
R-H + NADPH2 + O2 -------> ROH + NADP + H2O
- Dioxygenases (incorporate both <O's>)
R + O2 ------- RO2
2. Dehalogenation
- reductive dechlorination(anaerobic)
R-Cl -------- R-H
-hydrolytic dehalogenation
R-Cl --------- R-OH
-oxygenolytic dehalogenation (O2 followed by spontaneous halide loss)
Example of Dehalogenation
3. Reductive Ring Cleavage-
• anaerobic-ring reduction followed by ring cleavage, presence of O2
speeds up process
Illustration of ring cleavage
Screening of xenobiotics for biodegradability

Requirements for biodegradation studies

1) capable organisms-this is best achieved by continuous
inoculation with organisms from diverse sources.
2) proper conditions for enzyme induction-carbon limited
conditions with a continuous low supply
3) auxiliary carbon source (structurally similar biogenic
compound) -since breakdown products are difficult to predict it
is best to use a diverse mix of compounds.
4) proper concentrations
5) Awareness of physical characteristics of compound (volatility,
adsorptivity, solubility)
 Bacteria in Bioremediation
• Several bacteria have been found to be good degraders of toxic
pesticides such as halocarbons.
• Some sulfate reducing bacteria transform tetrachloroethane to cis-
1,2-dichloroethene by anaerobic dehalogenation of halocarbons.
• Methanogenic bacterial consortium has been shown to degrade
perchloroethene.
• Mono and dichlorobenzenes are degraded aerobically by various
Pseudomonas and Alcaligenes strains.
• Pentachlorobenzenes (PCBs) are degraded by strains of
Acinetobacter and Alcaligenes the same way as Phanerochaete
chrysosporium, the fungus.
• Several soil-inhabiting bacteria have been reported to degrade
chlorophenols under both aerobic and anaerobic conditions.
• Pentachlorophenol is degraded by a monoxygenase enzyme which
removes chlorine from the molecule making it nontoxic, and this
enzyme is found in some soil bacteria.
• Some aromatic compounds such as benzene and toluene can
be degraded by bacteria, especially species of Pseudomonas.
• Biodegradation of oil spills is a major problem because it
usually occurs in marine water surface and seeding with
bacteria becomes difficult.
• Besides, there is no single baterium that can degrade all the
components of the oils which are petroleum products.
• The genetically engineered strain of Pseudomonas putida has
been reported as “superbug” by Anand Chakrabarty, an
Indian born scientist working in USA which can degrade
petroleum.
• Other bacteria used in the treatment of oil spills are strains of
Alcaligenes eutropus, Rhodococcus sp., Bacillus sp. and
several unidentified bacteria.
• Bacteria such as Pseudomonas and Bacillus have been shown
to degrade the azo- or reactive dyes from textile industry
effluents.
• The process is often referred to as biobleaching.
• The bacteria are often used in consortia for biobleaching.
Fungi in Bioremediation
• Fungi are good in the accumulation of heavy metals such as
cadmium, copper, mercury, lead and zinc.
• Systems using Rhizopus arrhizus have been developed for
treating uranium and thorium.
• The ability of fungi to transform a wide variety of hazardous
chemicals has aroused interest in using them in
bioremediation.
• The white rot fungi are unique among eukaryotes for having
evolved nonspecific methods for the degradation of lignin;
curiously they do not use lignin as a carbon source for their
growth.
• Lignin degradation is, therefore, essentially a secondary
metabolic process, not required for the main growth process.
• Scientists compared the abilities of three lignin-degrading fungi,
Phanerochaete chrysosporium, P. sordida and Tramates hirsute
to degrade PCP (Pentachlorophenyl) in soil.
• Inoculation of soil with 10% (wt/wt) Phanerochaete sordida
resulted in the greatest decrease of PCP.
• P. sordida was also most useful in the degradation of PAHs
(Polycyclic aromatic hydrocarbons) from soil.
• Davis et al. (1993) showed that P. sordida was capable of
degrading efficiently the three ring PAHs, but less efficiently the
four-ring PAHs.
• Phanerochaete chrysosporium has been shown to degrade a number
of toxic xenobiotics such as aromatic hydrocarbons ( Benzo alpha
pyrene, Phenanthrene, Pyrene) chlorinated organics (Alkyl halide
insecticides, Chloroanilines, DD, Pentachlorophenols, Trichlorophenol,
Polychlorinated biphenyls, Trichlorophenoxyacetic acid), nitrogen
aromatics ( 2,4-Dinitrotoluene, 2,4,6-Trinitrotoluene-TNT) and several
miscellaneous compounds such as sulfonated azo dyes.
• Among the fungal systems, Phanerochaete chrysosporium is emerging
as the model system for bioremediation.
• Other fungi which can be used in bioremediation are the members of
Zygomycetes .
• Aquatic fungi and anaerobic fungi are the other candidates for
bioremediation.
• Among other fungi used in bioremediation, the yeasts,
e.g., Candida tropicalis, Saccharomyces cerevisiae, S. carlbergensis and
Candida utilis are important in clearing industrial effluents of
unwanted chemicals.
• Agaricus bisporus and Lentinus oloides versicolor is important in
cleaning up pulp and paper mill are used in composting, the most
useful waste disposal practice
 Bioremediation technologies
• The objective of bioremediation is to exploit naturally occurring
biodegradative processes to clean up contaminated sites.
• There are several types of bioremediation.
• In situ bioremediation ( in place treatment of contaminated site)
• Ex situ bioremediation (treatment of contaminated soil and water that is
removed from the site)
• Actual application of bioremediation is still limited in practice but is
rapidly gaining in popularity.
• Most of the developed bioremediation technologies are based on
following practices:
1.Addition of oxygen
2.Addition of other nutrients
3.Stimulation of anaerobic degradation using alternative
electron acceptor
4.Addition of surfactants
5.Addition of micro-organisms
 Natural bioremediation
• Intrinsic bioremediation
• Indigenous level of contaminant biodegradation that occurs without
any stimulation or treatment.
 Land based treatment

• Composting
• Excavated soil can be treated in piles
- addition of moisture and nutrient
- regular mixing for aeration
• Land farming
• Excavated soil can be treated in
constructed treatment cells.
PROCESS PERFORMANCE
• Windrow composting has been demonstrated as an
effective technology for treatment of explosives-
contaminated soil.
• During a field demonstration conducted by USAEC, TNT
(Trinitro toluene) reductions were as high as 99.7% at 30%
soil in 40 days of operation, with the majority of removal
occurring in the first 20 days of operation.
• Maximum removal efficiencies for RDX ( hexahydro 135
trinitro 1,3,5 triazine) and HMX( octahydro 1,3,5,7
tetranitro 1,3,5,7 tetraazocine) were 99.8% and 96.8%
respectively.
Biopiles
• ex-situ aeration of soil
• Biopile treatment is a full-scale technology in which excavated
soils are mixed with soil amendments, placed on a treatment
area, and bioremediated using forced aeration. The
contaminants are reduced to carbon dioxide and water.
• The basic biopile system includes a treatment bed, an aeration
system, an irrigation/nutrient system and a leachate collection
system.
• Moisture, heat, nutrients, oxygen, and pH are controlled to
enhance biodegradation. The irrigation/nutrient system is
buried under the soil to pass air and nutrients either by vacuum
or positive pressure.
• Soil piles can be up to 20 feet high and may be covered with
plastic to control runoff, evaporation and volatilization, and to
promote solar heating. If volatile organic compounds (VOCs) in
the soil volatilize into the air stream, the air leaving the soil may
be treated to remove or destroy the VOCs before they are
discharged into the atmosphere.
• Treatment time is typically 3 to 6 months.
Bioventing
• in-situ aeration of soil
• Bioventing is applicable to any chemical that can be
aerobically biodegraded
• bioventing stimulates the in-situ biodegradation by providing
oxygen to microorganisms in the soil.
• The system supplies oxygen by injecting air directly into the
residual contamination.
• In contrast to soil vapor vacuum extraction (SVE), bioventing
uses low airflow rates to provide only enough oxygen to
sustain microbial activity
• Optimal flow rates maximize biodegradation as vapors move
slowly through biologically active soil while minimizing
volatilization of contaminants.
• A basic bioventing system includes a well and a blower, which
pumps air through the well and into the soil.
 Limitations
Factors that may limit the applicability and effectiveness of the process
involve:
• Low permeability soils (reduce bioventing performance)
• Air near the structure of concern has to be extracted in order to
avoid vapor build up in basements within the radius of influence
of air injection wells
• Monitoring of off gases at the surface may be required
• Aerobic biodegradation of many chlorinated compounds may not
be effective
• Low soil moisture content, which may be caused by bioventing,
limits biodegradation.
Pump and treat type process
• Used for inaccessible soil and ground water.
• Soil leachate or groundwater are pumped to the surface, treated under
controlled conditions in the bioreactor and cleaned water is returned to the
ground.
Bioreactors

• Bioreactors represent highly controlled methods of treating

contaminated soils and groundwater.
• Temperature, pH, nutrient levels and agitation can be controlled in
constructed batch or continuously fed reactors, microbial activity
and thus contaminant degradation can be optimized.
• This can again be:
• Solid phase process (land treatment)
• Slurry process
 Phytoremediation
 Phytoremediation is the use of plants and trees to clean up
contaminated soil and water.
 Currently, the majority of research is concentrated on determining
the best plant for the job, quantifying the mechanisms by which the
plants convert pollutants, and determining which contaminants are
amenable to phytoremediation.
 This technology is useful for soil and water remediaton.
 PROCESSES OF PHYTOREMEDIATION
Phytoremediation is based on certain natural processes carried out by
plants including:
• Uptake of metals and certain organic compounds (i.e., moderately
water soluble, such as BTEX) from soil and water
• Accumulation or processing of these chemicals via lignification,
volatilization, metabolization, mineralization
• Use of enzymes to breakdown complex organic molecules into
simpler molecules (ultimately CO2 and water);
• Increasing the carbon and oxygen content of soil around roots
(and so promoting microbial/fungal activity) through release of
chemicals (exudates) and decay of root tissue;
• Capture of groundwater (even contaminated groundwater) and
utilization for plant processes
 Types of Phytoremediation
Depending on the underlying processes, applicability, and type of
contaminant, phytoremediation can be broadly categorized as:
 Phytodegradation: use of plants to uptake, store and degrade
contaminants within its tissue
 Phytostimulation or rhizodegradation: use of rhizospheric
associations between plants and symbiotic soil microbes to
degrade contaminants.
 Phytovolatilisation: use of a plant's ability to uptake
contaminants from the growth matrix and subsequently
transform and volatilize contaminants into the atmosphere
 Phytoextraction: use plants to absorb, translocate and store
toxic contaminants from a soil matrix into their root and shoot
tissue
 Rhizofiltration: use of roots to uptake also store contaminants
from an aqueous growth matrix
 Phytostabilisation: plant-mediated immobilization or binding of
contaminants into the soil matrix, thereby reducing their
bioavailability
 APPLICABILITY
CONTAMINANTS
Contaminants that have been remediated in laboratory and/or field
studies using phytoremediation or plant-assisted bioremediation
include:
• Heavy metals (Cd, Cr(VI), Pb, Co, Cu, Pb, Ni, Se, Zn)
• Radionuclides (Cs, Sr, Ur)
• Chlorinated solvents (TCE, PCE)
• Petroleum hydrocarbons (BTEX)
• Polychlorinated biphenyls (PCBs)
• Polynuclear aromatic hydrocarbons (PAHs)
• Chlorinated pesticides
• Organophosphate insecticides (e.g., parathion)
• Explosives (TNT, DNT, TNB, RDX, HMX)
• Nutrients (nitrate, ammonium, phosphate)
• Surfactants.
 IMPLEMENTATION

• Phytoremediation techniques are still being tested, in stages ranging

from lab- to field-scale.
research includes:
• Lab studies to investigate the processes behind phytoremediation;
• Screening studies to find suitable plants for further investigation;
• Bench- and pilot-scale testing of promising plant species;
• Limited and full-scale field trials.
 WATER REMEDIATION METHODS
Rhizofiltration
• Surface water- rhizofiltration may be conducted in situ, with
plants being grown directly in the contaminated water body.
• If groundwater is located within the rhizosphere (root zone),
rhizofiltration of groundwater can also be in situ.
• Alternately, rhizofiltration may involve the pumping of
contaminated groundwater into troughs filled with the large
root systems of appropriate plant species.
• The large surface areas provided by these root systems allow for
efficient absorption of metals from the contaminated
groundwater into root tissues.
• In addition to removal through absorption, metals are also removed
from groundwater through precipitation caused by exudates (liquids
released from plant tissues).
• These precipitates are filtered from the groundwater after it passes
through the plant troughs and before treated water is removed from
the process loop.
• Roots are harvested, and depending on the species of plant used,
shoots may be transplanted to grow new roots.
• Plants can be replaced in the system to ensure constant operation
results.
• Rhizofiltration using sunflowers has been used in the remediation of
radionuclides from surface water near Chernobyl (strontium and
cesium) and in water using a rhizofiltration system.
 Phytotransformation
• Surface water remediation via phytotransformation can be
accomplished in situ in ponds or wetlands.
• In addition, groundwater can be remediated using
phytotransformation in situ if the water table is within the
zone tapped by deep-rooted plants such as poplars or ex situ
by pumping water to troughs or constructed wetlands
containing appropriate plants.
• In the phytotransformation process, plants take up organic
contaminants and degrade them to less toxic or non-toxic
compounds.
• This technique is being tested on explosives-contaminated
groundwater.
 SOIL REMEDIATION METHODS
Phytoextraction
• This process involves the removal of metals, radionuclides, and
certain organic compounds (i.e., petroleum hydrocarbons) by direct
uptake into plant tissue.
• Amendments (i.e., fertilizer, water, etc.) may be required, as
determined from preliminary field testing, to ensure successful plant
growth.
• Lengths of time before harvesting the plants are also determined
from preliminary testing, and after this period of time, plant tissue is
removed and, if necessary, a new crop of plants are planted.
• Although testing has focused on single plants, several species may be
used at a site, either at the same time or subsequently, to remove
more than one contaminant.
Characteristics of plants able to perform phytoextraction include:

• Ability to accumulate and tolerate high concentrations of

metals in harvestable tissue;
• Rapid growth rate;
• High biomass production (This results in more metal
removed per planting).
 Phytostabilization

• Phytostabilization, is the use of certain plant species to absorb

and precipitate contaminants, generally metals, reducing their
bioavailability, and so reducing the potential for human
exposure to these contaminants.
• This technique can be used to re-establish a vegetative cover
at sites where natural vegetation is lacking due to high metals
concentrations in surface soils.
• Metal-tolerant species can be used to restore vegetation to
the sites, thereby decreasing the potential migration of
contamination through wind erosion and transport of exposed
surface soils and leaching of soil contamination to
groundwater (erosion and leaching are common in
unvegetated areas).
 Characteristics of plants appropriate for phytostabilization at a
particular site include:

• Tolerance to high levels of the contaminant(s) of concern

• High production of root biomass able to immobilize these
contaminants through uptake, precipitation, or reduction;
• Retention of applicable contaminants in roots, as opposed to
transfer to shoots, to avoid special handling and disposal of
shoots.
• tests involve the vegetation of a mine tailings site in Kansas
containing, elevated levels of cadmium, lead, and zinc, to reduce
wind and water erosion.
 The Use of Phytoremediation to Treat Organic
Contaminants
• Organic contaminants (specifically hydro-carbons that contain
carbon and hydrogen atoms) are common environmental
pollutants. There are several ways that plants can be used for
the phytoremediation of these contaminants:
phytodegradation, rhizo-degradation, and phytovolatilization.
• Phytodegradation
Plants contain enzymes (complex chemical proteins) that
catalyze and accelerate chemical reactions. Some enzymes
break down and convert ammunition wastes, others degrade
chlorinated solvents such as trichloroethylene (TCE), and
others degrade herbicides.
Destruction of Organic Contaminants by Phyotodegradation
 Rhizodegradation
• Rhizodegradation, also called phyto-stimulation or plant-assisted
bioremediation/degradation, is the breakdown of contaminants in the
rhizosphere (soil surrounding the roots of plants) through microbial
activity that is enhanced by the presence of plant roots and is a much
slower process than phytodegradation.
• Micro-organisms (yeast, fungi, or bacteria) consume and digest
organic substances for nutrition and energy.
• Certain micro-organisms can digest organic substances such as fuels or
solvents that are hazardous to humans and break them down into
harmless products in a process called biodegradation.
• Natural substances released by the plant roots – sugars, alcohols, and
acids – contain organic carbon that provides food for soil
microorganisms and the additional nutrients enhance their activity.
• Biodegradation is also aided by the way plants loosen the soil and
transport water to the area.
 Phytovolatilisation
• Phytovolatilization is the uptake and transpiration of a
contaminant by a plant, with release of the contaminant or a
modified form of the contaminant from the plant to the
atmosphere.
• Phytovolatilization occurs as growing trees and other plants take
up water and the organic contaminants.
• Some of these contaminants can pass through the plants to the
leaves and evaporate, or volatilize, into the atmosphere.
• Poplar trees at one particular study site have been shown to
volatilize 90% of the TCE they take up.
 Potential Applications of Phytoremediation
Application Contaminants Typical Plants
Phytotransformation · Herbicides (atrazine, alachlor) Phreatophyte trees
(poplar, willow,
Aromatics (BTEX) cottonwood, aspen);
Chlorinated aliphatics (TCE) Grasses (rye, Bermuda,
sorghum, fescue);
Nutrients (NO3-, NH4+, PO43-)
Ammunition wastes (TNT, RDX) Legumes (clover,alfalfa, cowpeas)

Rhizosphere · Organic contaminants (pesticides, Phenolics releasers

(mulberry, apple, orange);
Bioremediation aromatics, and polynuclear
aromatic hydrocarbons [PAHs]) Grasses with fibrous roots (rye,
• fescue, Bermuda) for contaminants
0–3 ft deep;
Phreatophyte trees for 0–10 ft;
Aquatic plants for sediments
 TECHNOLOGY LIMITATIONS
TECHNOLOGY LIMITATIONS
• Possible disadvantages associated with all
phytoremediation/plant-assisted remediation techniques
include:
• Long length of time required for remediation (usually more than one
growing season);
• Treatment is generally limited to soils at less than 3 feet from the
surface and groundwaterwithin 10 feet of the surface;
• Climatic or hydrologic conditions (e.g., flooding, drought) may restrict
the rate of growth of types of plants that can be utilized;
• Ground surface at the site may have to be modified to prevent
flooding or erosion;
• Contaminants may still enter the food chain through animals/insects
that eat plant material containing contaminants;
• Soil amendments may be required, including chelating agents to
facilitate plant uptake by breaking bonds binding contaminants to soil
particles.
 Future/Ongoing Improvements to
Phytoremediation to Enhance Acceptance

• Use of chelators to enhance metal solubility

• Combination with other in situ technologies
(e.g., microbial bioremediation, electrokinetics)
• Selection of improved plant varieties
• Genetic engineering of improved varieties
• Validate rhizosphere effects
• Determine acceptable end points
• Identify critical parameters, species, climate, soils.
Phytoremediation of Metals
Reed Bed Technology
Introduction

Reed bed technology is

versatile, has a wide range of
applications and can be
combined with other
technologies and processes to
extend the range of effluent
problems that can be
effectively treated.
The market for reed bed
technology is set to expand
worldwide.
Origins

• Reed bed technology was developed in Germany in the 1960s by Dr. Käthe
Seidel.
• There has been a lot of international research on this ecotechnological methods
since this time and uses different methods with either horizontal or vertical flow
vector or combinations of both or with conventional treatment processes.
 Reed Bed Wastewater Treatment Systems
• The principle of this technology is to activate microbial processes that
stimulate the natural breakdown of polluting compounds in a specific
waste water situation.
• This is possible due to the special characteristics of wetland plants,
such as reeds, which transfer substantial amounts of atmospheric
oxygen through to their root systems encouraging an extraordinary
quantity and species diversity of micro-organisms to flourish around
their roots.
• Reed bed treatment systems essentially comprise self-contained,
artificially engineered wetland ecosystems.
• They utilise particular combinations of plants, soils, bacteria,
substrates and hydraulic flow systems to optimise the physical,
chemical and microbiological processes naturally present within the
root zone of the plants.
• Organic pollutants are broken down as a food source by the
microorganisms
• whilst other contaminants, such as metals or PCB‘s are fixed in
humic acid and cation exchange bonds in the soil or mineral
substrates in which these plants are rooted.
• The complexity of microbial life forms and the reactions within the
root zone of the reed bed result in a powerful water cleaning
capability which is often much less constrained than in many
chemical or physical treatment systems.
• Reed bed treatment systems have been successfully used for
treating a wide range of wastewater concentrations, from the very
dilute up to COD concentrations of 20,000 mg/l and nitrogen
concentrations of 4,000 mg/l. They can also handle a wide range of
effluent throughputs ranging from small and intermittent flows of
about 1 m3/d to several 10,000 m3/d.
 Factors influencing the purification process of
reed bed treatment systems:
The main factors that typify the treatment characteristics are:
• hydraulic loading rate (m³/[ha x d]) and hydraulic detention time(d)
• temperature
• influent pollutant concentration
• oxygen supply
• development stage of the reed ecosystem
 ADVANTAGES AND CONSTRAINTS OF REED
BED TREATMENT TECHNOLOGIES

CONSTRAINTS to the Technology

• Reed bed treatment systems have a number of constraints which need to be taken into
account when advising clients of their suitability for treating a particular waste problem.
Such constraints are:
LARGE LAND AREA REQUIREMENT
• The requirement for a much larger land area compared to other treatment technologies.
RUNNING - IN - PERIOD
• Many reed bed systems require a longer running-in-period than other treatment technologies
before they can achieve their hill operational performance.
• This is especially so in systems using soil based substrates, as their full hydraulic throughput is
directly proportional to the growth and climax development of the planted root zone.
• Also, particularly harsh concentrations or constituents in the effluent can retard the growth
and climax development of the plants and their host micro-organisms.
• These factors, singly or combined, mean that in some cases a 1 to 3 year period to
complete commissioning is necessary.
 NOT A STAND ALONE SOLUTION TO EVERY TYPE OF
WASTE PROBLEM

• Reed bed technology alone is not capable of providing the

optimum treatment solution to every type of waste
problem.
• There are effluents and sludges with concentrations and/ or
types of constituents which are too problematic or
unsuitable for primary or secondary treatment in a reed bed
system.
• However, reed bed systems can be synergistically combined
with other technologies, especially other biotechnical
systems, (such as a second step to anaerobic digestion for
effluents with very high COD levels), so increasing the
effective range of application for both technologies.
 ADVANTAGES of the Technology

ROBUST AND EFFECTIVE

• lt is easy for those unfamiliar with this technology to underestimate
the robustness and effectiveness of the natural
processes operating within such engineered ecosystems and their
powerful cleaning and recycling capability.
• The composition of micro-organisms in a reed bed system is very
versatile with more than 5,000 different types of bacteria compared
with 200 - 300 in other biological treatment plants.
CONSISTENT DISCHARGE QUALITY
• Such engineered ecosystems have a superior capacity to tolerate wide
variations in effluent characteristics, and to withstand shock loadings,
without damage or disruption to treatment and so maintaining a
consistent discharge quality.
NO BY-PRODUCTS
• Reed bed systems produce no noise or smells and no sludges
or other by-products with associated additional costs of
disposal.
VERSATILITY
• A particular strength of the technology is that, although it is
not a general panacea for every waste treatment situation, it
can be used in many different ways for different purposes.
• lt can be used as primary or secondary treatment for a wide
variety of effluents and sludges.
• Reed bed systems can be used to treat effluents which are
surprisingly concentrated as well as effluents with difficult
pollutants too dilute to treat by conventional means but
which remain too dangerous to release into the
environment.
• Reed bed systems can be used to recycle water.
• They can also play a cost effective role.
LONG LASTING WITH LOW OPERATIONAL & MAINTENANCE
COSTS
• With little or no electrical or mechanical parts, reed bed
systems are long lasting, wear free and naturally regenerative.
• Being self regulating ecosystems, they are simple to operate
without chemical additives or complex electronic controls and
require minimal staffing levels due to their very low
maintenance requirements.
• Consequently operational and energy costs of reed bed
systems are very low and system lifetimes are very long.
LOWER CAPITAL COSTS THAN ALTERNATIVE CONVENTIONAL
SYSTEMS
• Territorial authorities and companies choose reed bed
treatment systems on economic grounds.
ENVIRONMENTALLY SAFE & BENEFICIAL

• Reed bed systems in their construction and operation are

environmentally benign, as in essence they are specially engineered
ecosystems, using substantially natural components to harness
nature‘s own powerful processes.
• The tall attractive reeds in such systems not only blend pleasantly with
the local landscape, but also offer considerable amenity and wildlife
conservation potential.
• As a spin-off benefit, reed bed systems can recreate ,,natural“ wetland
habitats which are otherwise fast disappearing around the world, and
for this reason alone their use is likely to be enthusiastically supported
by many influential people and organisations.
• Horizontal Reedbed technology
Ecofriendly technology
 Bioenergy and Biofuel
• It is a renewable energy source based on the carbon cycle,
unlike other natural resources such as petroleum, coal, and
nuclear fuels.
• Agricultural products specifically grown for biofuel production
include corn, and soybeans, primarily in the United States;
rapeseed, wheat and sugar beet primarily in Europe; sugar
cane in Brazil; palm oil and miscanthus in South-East Asia;
sorghum and cassava in China; and jatropha in India.
• Hemp has also been proven to work as a biofuel.
• Biodegradable outputs from industry, agriculture, forestry and
households can be used for biofuel production, either using
anaerobic digestion to produce biogas, or using second
generation biofuel processes; examples include straw, timber,
manure, rice husks, sewage, and food waste.
• The use of biomass fuels can therefore contribute to waste
management as well as fuel security and help to prevent
climate change, though alone they are not a comprehensive
solution to these problems.
 Bioenergy
The methods for converting biomass into heat (and electricity) and
into other fuels (ie, into biofuels) fall into the following main
classifications:
A. Thermochemical Conversion
There are three major thermochemical conversion methods:
1. DIRECT COMBUSTION.
2. PYROLYSIS:
3. GASIFICATION:
B. Biochemical Conversion
Two major biochemical conversion methods
1. ALCOHOLIC FERMENTATION.
2. ANAEROBIC DIGESTION.
C. Extraction
1. TRANSESTERIFICATION OF OIL
Bioenergy Technologies
 Thermochemical Conversion
1. DIRECT COMBUSTION.
• The biomass, such as waste wood, bark, and agricultural residues, is
directly burned in a stove or furnace for the production of heat, steam, or
electricity.
• The biomass requires little or no pre-processing prior to combustion,
though drying down to about the fiber-saturation point (about 30% moisture
content in wood) is required to burn biomass in small stoves and furnaces.
• Generally, the smaller the burner, the greater the pollution per unit of
biomass burned.
• Large biomass-fired systems mainly suffer from particulate emissions –
that is, tiny fuel and char particles blown out of the furnace.
• In some cases, NOx emissions can be a problem.
• Small systems suffer from hydrocarbon emissions, including gases and
fuel aerosol (ie, the white smoke of fireplace or wood stove), carbon
monoxide, and soot (tiny, mainly carbon particles).
2. PYROLYSIS:

• The term pyrolysis means decomposition.

• In the absence (or significant deficiency) of air, we say
thermal pyrolysis or simply pyrolysis.
• If significant air is present (as in combustion), we say
oxidative pyrolysis – that is, pyrolysis in the presence of air

• The original biomass is processed to new fuels (biofuels)

by heating.
• If wood is slowly heated and the temperature of the wood remains
fairly low, the carbon in the wood forms char, and the other
elements in the wood are released (mainly as CO2 and water
vapor).
• The wood contains cellulose and lignin.
• About 2/3rd of the wood is cellulose, about 1/3rd is lignin.
• The lignin may be thought of as the “glue” that holds the cellulose
fibers in place.
• The lignin is mainly composed of C and H. The cellulose contains
C, H, and O.
• Upon slow heating at relatively low temperature, the C from the
cellulose and lignin forms chains and layers of carbon – that is, it
forms char.
• The H and O mainly leave as H2O and CO2.
• The new biofuel is charcoal.
• If the biomass is more rapidly heated and its temperature
increased, the char forming tendency is reduced, and the
biomass decomposes into various hydrocarbons.

• These hydrocarbons are part of the volatile matter released

from the biomass.

• Several of these hydrocarbons, if cooled, will condense into tars

and oils.

• That is, the product, pyrolysis oil, a biofuel.

• Air in the pyrolysis reaction chamber must be limited so that

the volatiles do not significantly oxidize, and do not ignite.

• Thus, the fuel to burn in an energy system is an oil – pyrolysis

oil.
3. GASIFICATION:
• If we increase the rate of heating of the biomass,
and increase the temperature of the particles,
the volatile matter released from the biomass
becomes significantly composed of gases.
• Additionally, if we add about ½ the amount of air
or O2 required for stoichiometric combustion of
the original biomass, the char particles
remaining from the pyrolysis stage (ie, the
decomposition stage) become gasified.
• The goal with most gasifiers is to create a
synthetic gas mainly composed of CO, H2, CO2,
H2O, (and sometimes) CH4.
 B. Biochemical Conversion
1. .
ALCOHOLIC FERMENTATION
• Yeast is used to catalyze this reaction.
• The biofuel product is ethanol.
• In a typical process, there is considerable water present,
leading to a product of about 10% ethanol in water.
• Distillation increases the ethanol content to about 95% purity.
• This is followed by purification steps leading to anhydrous
ethanol (ie, ethanol without water).
• Ethanol is an attractive biofuel.
• It is a good engine fuel if blended with 5-15% gasoline (to
overcome the cold starting problem of pure ethanol).
• Since it is a liquid, it can be easily transported.
 Bioethanol
• Ethanol is the most common biofuel worldwide.
• This alcohol fuel is produced by fermentation of sugars
derived from wheat, corn, sugar beet and sugar cane.
• The production methods used are enzymatic digestion
(to release sugars from stored starches e.g. from wheat
and corn), fermentation of the sugars, distillation and
drying.
• Ethanol can be used in petrol engines as a replacement
for gasoline; it can be mixed with gasoline to any
percentage.
• All petrol engines can run on blends of up to 15%
bioethanol with petroleum/gasoline.
• For higher percentage blends, engine modifications are
needed.
• Many car manufacturers are now producing flex-fuel
vehicles, which can run on any combination of
bioethanol and petrol, up to 100% bioethanol.
 Butanol
• Butanol is often claimed to provide a direct replacement for
gasoline, because it can be used directly in a gasoline engine (in a
similar way to biodiesel in diesel engines).
• butanol has sufficiently similar characteristics with gasoline such
that it should work without problem in any gasoline engine, no
widespread experience exists.
• Butanol is formed by ABE fermentation (acetone, butanol, ethanol)
and experimental modifications of the process show potentially
high net energy gains with butanol as the only liquid product.
• Butanol will produce more energy and allegedly can be burned
"straight" in existing gasoline engines (without modification to the
engine or car), and is less corrosive and less water soluble than
ethanol, and could be distributed via existing infrastructures.
 Biomethanol

• Methanol is currently produced from natural gas, a fossil

fuel.
• It can also be produced from biomass (biomethanol).
• The methanol economy is an interesting alternative to the
hydrogen economy.
• Biomethanol can be blended with petrol up to 10-20%
without any infrastructure changes.
• Brazil has been a leading producer and
user of ethanol.
• The ethanol is used as a transportation
fuel.
• However, when the price per barrel of oil is
low, it is difficult for the ethanol to
economically compete.
• Ethanol used in the USA for blending with
gasoline is produced from corn.
2. ANAEROBIC DIGESTION.
• certain types of bacteria in the absence of air
promote the reaction of biomass with water to
form methane and carbon dioxide.
Composition
• 60%-70% of Methane (CH4),
• 38%-28% of Carbon dioxide (CO2)
• 2% of Hydrogen (H2)
• Hydrogen Sulfide (H2S)
• Anaerobic fermentation activities, consists
of three different bacterial communities:
fermentative, acetogenic, and methanogenic bacteria
 Biogas
• Biogas typically refers to a gas produced by the biological
breakdown of organic matter in the absence of oxygen.
• Biogas is comprised primarily of methane and carbon
dioxide.
• Biogas originates from biogenic material and is a type of
biofuel.
• Biogas is a product of the anaerobic digestion or
fermentation of biodegradable materials such as manure or
sewage, municipal waste, and energy crops.
• The methane in biogas gives it the ability to be used as a
fuel.
• The combustion of which releases energy.
• In developing countries biogas can be used as a low-cost
fuel for cooking.
• It can also be utilised in modern waste management facilities
where it can be used in gas engines to generate electricity.
• Biogas is a renewable fuel and electricity produced from it
can be used to attract renwable energy subsidies in some
parts of the world.
 Production
• Biogas can be produced utilising anaerobic digesters.
• These plants can be fed with energy crops such as maize silage or
biodegradable wastes including sewage sludge and food waste.
• Landfill gas is produced by organic waste decomposing under
anaerobic conditions in a landfill.
• The composition of biogas varies depending upon the origin of the
anaerobic digestion process.
• Landfill gas typically has methane concentrations around 50%.
• Advanced waste treatment technologies can produce biogas with 55-
75%CH4
• The methane in biogas forms explosive mixtures in air.
• The lower explosive limit is 5% methane and the upper explosive limit
is 15% methane.
• The methane contained within biogas is 20 times more potent as a
greenhouse gas than carbon dioxide.
• Therefore landfill gas which escapes into the atmosphere significantly
contributes to the effects of global warming.
• In addition to this volatile organic compounds (VOCs) contained
within landfill gas contribute to the formation of photochemical smog.
 Applications
• Biogas can be utilised for electricity production, space heating, water heating
and process heating.
• If compressed, it can replace compressed natural gas for use in vehicles,
where it can fuel an internal combustion engine or fuel cells.
• biogas produced from the anaerobic digestion of manure in small-scale
digestion facilites is called Gober gas.
• The digester is an airtight circular pit made of concrete with a pipe
connection.
• The manure is directed to the pit, usually directly from the cattle shed. The
pit is then filled with a required quantity of wastewater.
• The gas pipe is connected to the kitchen fire place through control valves.
• The combustion of the biogas produced in this manner flammable has very
little odour or smoke.
• Owing to its simplicity in implementation and use of cheap raw materials in
the villages it is one of the most environmentally sound energy source for the
rural needs.
• Methane within biogas can be concentrated to the same standards as
natural gas, when it is called biomethane.
• Compressed biogas is becoming widely used in Sweden, Switzerland and
Germany. A biogas-powered train has been in service in Sweden since
2005.
• A wide variety of different agricultural crops can be turned into
fuel for motor vehicles.
• In different parts of the world, products as diverse as sugar
cane, wheat and rapeseed are used to make substitutes for
petrol or diesel.
• These fuels are usually mixed with conventional fuels to make
a blend, but some vehicles can now use pure, or almost
pure,vegetable oils and alcohols.
• Governments are pushing bio-fuels for two main reasons.

First, they see these fuels as helpful in increasing the

security of energy supplies.
Second, governments believe that bio-fuels help reduce
greenhouse gas emissions.
For these two reasons President Bush promised significant new
funds for new ethanol technologies in his (January 2007)
State of the Union address.
bio fuels are good for soaking up unwanted agricultural
surpluses.
 Biodiesel
• Biodiesel is the most common biofuel in
Europe.

• It is produced from oils or fats using

transesterification and is a liquid similar in
composition to mineral diesel.

• Its chemical name is fatty acid methyl (or ethyl)

ester (FAME).
• Oils are mixed with sodium hydroxide and methanol
(or ethanol) and the chemical reaction produces
biodiesel (FAME) and glycerol.
• 1 part glycerol is produced for every 10 parts
biodiesel.
• Thus biodiesel can be thought of as a modified
vegetable oil
• Biodiesel can be used in any diesel engine when
mixed with mineral diesel.
• The majority of vehicle manufacturers limit their
recommendations to 15% biodiesel blended with
mineral diesel.
• In many European countries, a 5% biodiesel blend
is widely used and is available at thousands of gas
stations.
• Compared to petroleum based diesel, vehicle use of
pure biodiesel (B100) reduces particulate matter
(soot) emissions by about 48%, hydrocarbon
emissions by about 67%, but nitrogen oxide
emissions are about 10% higher.
• In the USA, more than 80% of commercial trucks
and city buses run on diesel.
• Therefore "the nascent U.S. market for biodiesel is
growing at a staggering rate—from 25 million
gallons per year in 2004 to 78 million gallons by the
beginning of 2005.
 Biodiesel vs Bioethanol
Biodiesel is usually made by processing rapeseed oil or soya bean oil.
In the US, most biodiesel is made from soya, but in the UK rapeseed provides the
feedstock
Biodiesel is a replacement for conventional diesel.
By contrast, bio-ethanol is a replacement for conventional petrol.
It is usually made from sugar cane, wheat or corn (maize).
In the case of corn, the predominant feedstock in the US, the kernels are ground up,
starches are turned to sugars, and these are then fermented
turn into ethanol.
The world's most advanced bio-ethananol market – Brazil – uses sugar cane.
Thus biodiesel can be thought of as a modified vegetable oil and bio-ethanol an alcohol
made from sugars.
In both cases, the manufacturing process is using an animal or human foodstuff as the
ingredient for a fuel.
This is important for two reasons: first, food is valuable and, second, the land to
productively grow food is in short supply.
Biodiesel has about 90% the energy value of fossil diesel but ethanol is slightly less than
70% as calorific.
While modern diesel cars can run on quite high concentrations of biofuel, the typical car
on the road will only run on percentages of ethanol lower than about 10-15%.
Potential and possible uses
of biofertilizers
 Why biofertilizers ?
 To meet the increasing menace of human population and a
concurrent agricultural productivity.
 Increases in crop production have been made possible
through the use of commercial man made fertilizers.
 The use of N and P fertilization in addition to the
introduction of highly productive and intensive agricultural
systems has allowed these developments to occur at
relatively low cost.
 However, such high tech technologies have also created
environmental problems such as deterioration of soil
quality, surface water and ground water as well as air
pollution, reduced diversity and suppressed ecosystem
function.
 Clearly, there is an urgent need for sustainable agricultural
practices on a global level and the application of
biofertilizers in place of chemical ones has been
recommended.
Biofertilizers
 Biofertilizers means the input of plant
nutrients of biological origin for the
growth of plants.
 It is also known as microbial inoculants
or microbial preparations.
 They do not directly increase the soil
fertility but instead exercise their
biological effects on improving the
nutritional conditions.
N2 fixing bacteria as microbial biofertilizers
 N2 is the most limiting nutrient to plant growth as the
atmospheric nitrogen is not available for plant
uptake.
 Some bacteria are capable of fixing atmospheric
nitrogen and form various associations with plants.
 Many free living N2 fixing bacteria occur in soil
 Some have adapted to form symbioses others have
intimate endophytic associations with plants
 Others live in close association in the plant root
zone without forming intimate endophytic symbiosis.
 The amt.of N2 fixed by these systems is
considerable although variation resulting from
environmental conditions or different plant microbe
combinations is vast.
 Symbiotic N2 fixing bacteria
 The best known and most exploited symbiotic N fixing
bacteria are those belonging to the family Rhizobiaceae
(Rhizobia) and include the genera: Rhizobium,
Bradyrhizobium, Sinorhizobium, Azorhizobium,
Mesorhizobium and Allorhizobium.
 These bacteria infect legumes and have a global
distribution.
 Frankia is the genus of N2 fixing actinomycetes that are
capable of infecting and nodulating a group of eight families
of mainly woody plants used in land reclamation, for timber
and fuel wood production.
 Cyanoacteria are ecologically important. Aquatic
cyanobacterium, Trichodesmium contributes approx. 36% of
global N2 fixation.
 It has been essential in the cultivation of rice.
 Azolla- Anabaena
N2 fixing associated bacteria
 In addition to symbiotic bacteria infecting plant roots
numerous less intimately associated N2 fixing
bacteria are considered for crop yield improvement.
Eg.
 Acetobacter diazotrophicus and Herbaspirillum spp.
Associated with sugarcane, sorghum,and maize.
 Alcaligenes, Azospirillum, Bacillus, Enterobacter,
Klebsiella, Pseudomonas and Rhizobium associated
with rice and maize.
 Azospirillum can increase the growth of various crops
like, sunflower, carrot, oak, sugerbeet, tomato,
pepper and cotton in addition to wheat and rice.
Free living N2 fixing bacteria
 Many free living bacteria also fix
atmospheric N2.
Eg.
 Azotobacter, Beijerinckia and
Clostridium.
Plant growth promoting Rhizobacteria
 PGPR can stimulate plant growth.
 These bacteria vary in their mechanism of
plant growth promotion but generally
influence growth via P solubilization, nutrient
uptake enhancement or plant growth
hormone production.
Eg. Rhizobacterium belonging to genus
Achromobacter could enhance root hair no.
and length in oilseed rape: increases NO3
and K uptake.
Phosphorous solubilization
 Phosphorus is second only to nitrogen in mineral
nutrients most commonly limiting the growth of crops.
 Phosphorus is an essential element for plant
development and growth making up about 0.2 % of plant
dry weight.
 Plants acquire P from soil solution as phosphate anions.
However, phosphate anions are extremely reactive
and may be immobilized through precipitation with
cations such as Ca2+, Mg2+, Fe3+and Al3+, depending
on the particular properties of a soil.
 Several scientists have reported the ability of
different bacterial species to solubilize insoluble
inorganic phosphate compounds, such as tricalcium
phosphate, dicalcium phosphate, hydroxyapatite, and
rock phosphate.
Mechanisms of phosphate solubilization
 The principal mechanism for mineral phosphate solubilization is
the production of organic acids, and acid phosphatases.
 It is generally accepted that the major mechanism of mineral
phosphate solubilization is the action of organic acids
synthesized by soil microorganisms.
 Production of organic acids results in acidification of the
microbial cell and its surroundings.
 Gluconic acid seems to be the most frequent agent of
mineral phosphate solubilization.
 Also, 2-ketogluconic acid is another organic acid identified
in strains with phosphate solubilizing ability .
 Chelating substances and inorganic acids such as
sulphideric, nitric, and carbonic acid are considered as other
mechanisms for phosphate solubilization.
 However the effectiveness and their contribution to P release in
soils seems to be less than organic acid production.
phosphate solubilizers
 Mostly they are associated with the plant
rhizosphere, so they are called as rhizobacteria.
 This group of bacteria has been termed plant
growth promoting rhizobacteria, and among them
are strains from genera such as Alcaligenes,
Acinetobacter, Arthrobacter, Azospirillum,
Bacillus, Burkholderia, Enterobacter, Erwinia,
Flavobacterium, Paenibacillus, Pseudomonas,
Rhizobium, and Serratia.
 They are used as biofertilizers or control
agents for agriculture improvement, and there
are numerous researchers for the area with the
agricultural environment conservation.
 Strains of Bacillus were found to
produce mixtures of lactic, isovaleric,
isobutyric and acetic acids. Other organic
acids, such as glycolic, oxalic, malonic,
and succinic acid, have also been
identified among phosphate solubilizers.
 Strains from the genera Pseudomonas,
Bacillus and Rhizobium are among the
most powerful phosphate solubilizers.
Plant hormone production
 In addition to improving plant nutrition by
fixing atmospheric N or solubilizing plant
unavailable pools of P, bacteria can
influence plant growth by Production of
plant hormones.
 Eg. Bacillus pumilu, B. licheniformis in the
rhizosphere of alder (Alnus glutinosa) can
produce giberellins.
 Paenibacillus polymyxa is able to produce
cytokinins
Fungi and their potential as Biofertilizers

 Mycorrhizal fungi- the various potential benefits of

mycorrhizal symbiosis on plant performance and crop
yield suggest their substantial application in agriculture, in
land reclamation and restoration thereby use as
biofertilizer.
Future Prospectives
 More in-depth knowledge about interaction among
plants and rhizosphere organisms is must.
 Unless the positive effects can be repeatably
shown in practical applications, the commercial
viability of inoculation programs will be uncertain.
 The inocula made commercially available should
conform to minimal quality standards.
 Simple and clear governmental and international
regulations are essential to guarantee efficiency of
the inocula.
 Local or regional strains should preferably be
selected and used for the target crops.
Biological Control Of Pathogens

 Plant diseases are caused mainly by fungi,

bacteria, viruses and nematodes.
 Biocontrol of plant disease involves the
use of an organism or organisms to
reduce disease.
 Biocontrol includes management of
resident populations of organisms (the
'black box' approach) and introductions of
specific organisms (the 'silver bullet'
approach) to reduce disease.
 Biological control
 The terms “biological control” and its abbreviated synonym “biocontrol” have
been used in different fields of biology, most notably entomology and plant
pathology.
 In entomology, it has been used to describe the use of live predatory insects,
entomopathogenic nematodes, or microbial pathogens to suppress
populations of different pest insects.
 In plant pathology, the term applies to the use of microbial antagonists to
suppress diseases as well as the use of host-specific pathogens to control
weed populations.
 In both fields, the organism that suppresses the pest or pathogen is referred
to as the biological control agent (BCA).
 More broadly, the term biological control also has been applied to the use of
the natural products extracted or fermented from various sources.
 These formulations may be very simple mixtures of natural ingredients with
specific activities or complex mixtures with multiple effects on the host as
well as the target pest or pathogen.
 members of the U.S. National Research
Council took into account modern
biotechnological developments and referred
to biological control as “the use of natural or
modified organisms, genes, or gene products,
to reduce the effects of undesirable
organisms and to favor desirable organisms
such as crops, beneficial insects, and
microorganisms”,
 More narrowly, biological control refers to the
purposeful utilization of introduced or resident
living organisms, other than disease resistant
host plants, to suppress the activities and
populations of one or more plant pathogens.
Important aspects for effective and economic
control
 Cause of the disease
 Mode of perrennation and dissemination of the
infection
 Host parasite relationship and means of
secondary spread
 Effect of environment on pathogenesis and their
spread
Mechanisms of biological control

 There are three main mechanisms by

which one microorganism may limit the
growth of another microorganism:
1. antibiosis,
2. hyperparasitism, and
3. competition for resources.
Antibiosis
 Antibiosis is defined as inhibition of the growth
of one microorganism by another as a result of
diffusion of an antibiotic.
 Antibiotic production is very common among
soil-dwelling bacteria and fungi, and in fact many
of our most widely used medical antibiotics (e.g.,
streptomycin) are made by soil microorganisms.
 Antibiotic production appears to be important to
the survival of microorganisms through
elimination of microbial competition for food
sources, which are usually very limited in soil.
 Some of antibiotics produced by BCAs

Antibiotic Source Target pathogen Disease

2, 4-diacetyl- Pseudomonas Pythium spp. Damping off
phloroglucinol fluorescens F113
Agrocin 84 Agrobacterium Agrobacterium Crown gall
radiobacter tumefaciens
Lytic enzymes and other byproducts
 Diverse microorganisms secrete and excrete other metabolites
that can interfere with pathogen growth and/or activities.
 Many microorganisms produce and release lytic enzymes that
can hydrolyze a wide variety of polymeric compounds, including
chitin, proteins, cellulose, hemicellulose, and DNA.
 Expression and secretion of these enzymes by different
microbes can sometimes result in the suppression of plant
pathogen activities directly.
 For example, control of Sclerotium rolfsii by Serratia
marcescens appeared to be mediated by chitinase expression
And, a b-1,3-glucanase contributes significantly to biocontrol
activities of Lysobacter enzymogenes strain C3 (Palumbo et al.
2005).
 destructive hyperparasitism
 This is parasitism of a pathogenic fungus by another fungus. It
involves direct contact between the fungi resulting in death of
the plant pathogen, and nutrient absorption by the parasite.
 Mycoparasites produce cell wall-degrading enzymes which
allow them to bore holes into other fungi and extract nutrients
for their own growth.
 But many so-called mycoparasites also produce antibiotics
which may first weaken the fungi they parasitize.
 The fungus Trichoderma harzianum, available commercially as
the seed treatment product Bio-Trek (Wilbur-Ellis, CA) is a
mycoparasite of several damping-off pathogens including
Pythium, Rhizoctonia, and Fusarium.
 competition for resources
 Microorganisms compete with each other for carbon, nitrogen,
oxygen, iron and other micronutrients.

 Nutrient competition is likely to be the most common way by

which one organism limits the growth of another, but
demonstrating that this is actually responsible for biological control
is quite challenging.
 One of the most recent EPA registrations for a microbial
pesticide is the product VICTUS (Sylvan Spawn, PA) which
contains the bacterium Pseudomonas fluorescens.
 This organism, when applied to commercially grown
mushrooms, helps prevent bacterial blotch caused by a closely
related but pathogenic species, Pseudomonas tolaasii.
 P. fluorescens is a better competitor for nutrients than P.
tolaasii, and when applied to mushroom caps, excludes the
pathogen by utilizing all the available food. There is no
evidence of antibiosis.
 The product Kodiak (Gustafson, TX), which contains the
bacterium Bacillus subtilis, is believed to control fungal
diseases through competition for nutrients, in this case
exudates from seeds and roots.
 in most terrestrial habitats, microbial competition for the soluble
form of iron, Fe3+, is keen.
 Some fungi and bacteria produce very large molecules called
siderophores which are efficient at chelating Fe3+.
 Individual strains can have their own particular siderophores
and receptors which can bind Fe3+ in such a way that the iron
becomes inaccessible to other microorganisms, including
pathogens.
 In some cases, siderophore production and competitive
success in acquiring Fe3+ is the mechanism by which
biocontrol agents control plant diseases.
 Siderophores produced by certain strains of Pseudomonas
have been implicated in disease suppression of several fungal
diseases, but none of these biocontrol organisms have yet
been developed commercially.
Baculoviruses as biocontrol agent
 Baculoviruses are pathogens that attack insects
and other arthropods.
 they are rod shaped measuring 40-110nmx200-
400nm, and are composed primarily of double-
stranded DNA that codes for genes needed for
virus establishment and reproduction.
 an infective baculovirus particle (virion) is
protected by protein coat called a polyhedron
(plural polyhedra).
 Most insect baculoviruses must be eaten by the
host to produce an infection, which is typically
fatal to the insect.
Biopulping and biobleaching
 Biopulping
• Definition and Overview:
• Biopulping is a type of industrial biotechnology.
• The main processes for paper production that are
currently in use are mechanical and chemical.
• Biopulping provides a solution to the problems of
chemical and mechanical paper production.
• It uses fungi present in natural wood decay to alter the
lignin in the cell walls of the wood, which therefore
"softens" the wood chips.
• Overall, biopulping uses the knowledge gained from a
natural process to produce high quality paper while
reducing the energy needed and the pollutants that
escape out into the air during the process.
• Biopulping reduces electrical energy needed by an
average of 25%- 30%
 History and Development:

• Finland started using biopulping in the late 1980's.

• The US started biopulping in April of 1987.
• Biopulping was started in the US by the USDA Forest Service
Products Laboratory in Madison, Wisconsin with collaboration by
the Universities of Wisconsin and Minnesota.
• BioPulping International, Inc. was founded in 1996.
 Fungi in Biopulping
• Ceriporiopsis subvermispora is a white-rot basidiomycete that
has ligninolytic activity.
• Lignin, along with cellulose, is a major polymeric component of
wood that is rather refractory to biodegradation.
• It is degraded efficiently by only a small group of fungi. There
are three main ligninolytic enzymes: manganese peroxidase,
lignin peroxidase and lacasse.
• Ceriporiopsis subvermispora produces isoenzymes of
manganese peroxidase and lacasse, but no isoenzymes of
lignin peroxidase have been found. However, its ligninolytic
activity is as high as in organisms possessing lignin peroxidase.
• Many groups around the world have studied C. subvermispora
and other white-rot fungi for uses in biological ligninolysis in the
pulp and paper industry.
• Pretreatment of hard wood chips with Phanerochaete
chrysosporium shows an improvement in kraft pulp yield
 Industrial-Scale Process
• The fungal treatment process fits well.
• Wood is debarked, chipped, and screened
according to normal mill operation.
• Then chips are briefly steamed to reduce
natural chip microorganisms,
• cooled with forced air, and inoculated with
the biopulping fungus.
• The inoculated chips are piled and
ventilated with filtered and humidified air for
1 to 4 weeks prior to processing.
 Advantages of Biopulping

• Reduced electrical energy consumption (at least 30%)

• Potential 30% increase in mill throughput for mechanical
pulping
• Improved paper strength properties
• Reduced environmental impact
 Biobleaching
• The biobleaching of kraft with laccase/mediator continues to
receive strong interest
• new environmentally benign elemental chlorine free (ECF) and
totally chlorine free (TCF) bleaching technologies are necessary
for minimizing the hemi-cellulose content in dissolving pulp,
adjusting the brightness at a high level and improving
simultaneously, the quality of the effluents in terms of toxicity
and adsorbable organic halogen (AOX).
• Biological methods of pulp prebleaching using xylanases
provide the possibility of selectively removing upto 20% of xylan
from pulp and saving up to 25% of chlorine containing bleaching
chemicals.
• Alternatively, pulp can be bleached with white-rot fungi and their
lignolytic enzymes, enabling chemical savings to be achieved
and a chlorine free bleaching process to be established.
 Advantages of Biobleaching
• Reduced consumption of bleaching
chemical;
• reduced adsorbable organic halogen;
• improved pulp and paper quality;
• improved brightness;
• reduced effluent toxicity and pollution load.
 Biodegradable plastics

Biodegradable plastics are `environment-

friendly'; they have an expanding range of
potential applications, and are driven by the
growing use of plastics in packaging.

Biodegradable plastics made with plant-based

materials have been available for many years.
Their high cost, however, has meant they
have never replaced traditional non-
degradable plastics in the mass market.
 Naturally occurring polymers

polysaccharides e.g., starch from potatoes and corn, their derivatives,

cellulose from marine crustaceans;

proteins such as gelatin (collagen), casein (from milk), keratin (from silk and
wool) and zein (from corn);
polyesters such as poly hydroxy alkanoates formed by bacteria as food
storage;

lignin;

shellac and natural rubber polylactic acid, jute, flux, silk, cotton can fall into the
category of natural polymers where the monomer is produced by fermentation.
The rate of degradation of each of these depends very much on their structural
complexity, as well as the environmental conditions.
 How are Biopolymers and Bioplastics
Made?

1. Using Fermentation to Produce Plastics

• Bacterial Polyester Fermentation
• Eg. Ralstonia eutropha
• Lactic Acid Fermentation
• After the lactic acid is produced, it is converted
to polylactic acid using traditional
polymerization processes.
2. Growing Plastics in Plants
Plants are becoming factories for the production
of plastics.
Eg. Arabidopsis thaliana plant through genetic
engineering.
• The researchers have transferred the gene that
codes for this enzyme into the plant, as a result
the plant produces plastic through its cellular
processes. The plant is harvested and the
plastic is extracted from it using a solvent. The
liquid resulting from this process is distilled to
separate the solvent from the plastic.
 Compostable plastic: A plastic that
undergoes biological degradation during
the composting process (up to 2-3
months in a windrow) to yield carbon
dioxide, water, inorganic compounds
and biomass at a rate consistent with
other known compostable materials and
leaves no visually distinguishable or
toxic residues.

Biodegradable plastic: A degradable

plastic in which the degradation must
result from the action of naturally
occurring microorganisms over a
period of time (up to 2-3 years in a
landfill).
 Environmental benefits of biodegradable
plastics
• biodegradable plastics increases the soil organic
content as well as water and nutrient retention,
while reducing chemical inputs and suppressing
plant disease.
• increase the rate of organic waste degradation in
landfills while enhancing methane harvesting
potential and decreasing landfill space usage.
• Biodegradable landfill covers may also
considerably extend landfill life.
• The energy required to synthesize and
manufacture biodegradable plastics is generally
much lower for most biodegradable plastics than
for non-biodegradable plastics.
 Challenges ahead
Acceptance of biodegradable polymers is likely to
depend on:
(1) Customer response to costs that today is
generally 2 to 4 times higher than for conventional
polymers;
(2) Possible legislation (particularly concerning
water-soluble polymers);
(3) The achievement of total biodegradability; and
(4) The development of an infrastructure to collect,
accepts, and process biodegradable polymers as a
generally available option for waste disposal.
Reed Bed Technology
 Introduction
Reed bed technology is versatile, has
a wide range of applications and can
be combined with other technologies
and processes to extend the range of
effluent problems that can be
effectively treated.
The market for reed bed technology is
set to expand worldwide.
Origins

• Reed bed technology was developed in

Germany in the 1960s by Dr. Käthe Seidel.
• There has been a lot of international research
on this ecotechnological methods since this
time and uses different methods with either
horizontal or vertical flow vector or
combinations of both or with conventional
treatment processes.
 Reed Bed Wastewater Treatment Systems
• The principle of this technology is to activate microbial processes that stimulate the
natural breakdown of polluting compounds in a specific waste water situation.
• This is possible due to the special characteristics of wetland plants, such as reeds,
which transfer substantial amounts of atmospheric oxygen through to their root
systems encouraging an extraordinary quantity and species diversity of micro-
organisms to flourish around their roots.
• Reed bed treatment systems essentially comprise self-contained, artificially
engineered wetland ecosystems.
• They utilise particular combinations of plants, soils, bacteria, substrates and
hydraulic flow systems to optimise the physical, chemical and microbiological
processes naturally present within the root zone of the plants.
• Organic pollutants are broken down as a food source by the microorganisms
whilst other contaminants, such as metals or PCB‘s are fixed in humic acid and
cation exchange bonds in the soil or mineral substrates in which these plants are
rooted.
• The complexity of microbial life forms and the reactions within the root zone of the
reed bed result in a powerful water cleaning capability which is often much less
constrained than in many chemical or physical treatment systems.
• Reed bed treatment systems have been successfully used for treating a wide range
of wastewater concentrations, from the very dilute up to COD concentrations of
20,000 mg/l and nitrogen concentrations of 4,000 mg/l. They can also handle a wide
range of effluent throughputs ranging from small and intermittent flows of about 1
m3/d to several 10,000 m3/d.
Factors influencing the purification process
of reed bed treatment systems:

The main factors that typify the treatment

characteristics are:
• hydraulic loading rate (m³/[ha x d]) and
hydraulic detention time(d)
• temperature
• influent pollutant concentration
• oxygen supply
• development stage of the reed ecosystem
 ADVANTAGES AND CONSTRAINTS OF REED
BED TREATMENT TECHNOLOGIES
CONSTRAINTS to the Technology
• Reed bed treatment systems have a number of constraints which need to be taken
into account when advising clients of their suitability for treating a particular waste
problem.
Such constraints are:
LARGE LAND AREA REQUIREMENT
• The requirement for a much larger land area compared to other treatment
technologies.
RUNNING - IN - PERIOD
• Many reed bed systems require a longer running-in-period than other treatment
technologies before they can achieve their hill operational performance.
• This is especially so in systems using soil based substrates, as their full hydraulic
throughput is directly proportional to the growth and climax development of the
planted root zone.
• Also, particularly harsh concentrations or constituents in the effluent can retard the
growth and climax development of the plants and their host micro-organisms.
• These factors, singly or combined, mean that in some cases a 1 to 3 year period to
complete commissioning is necessary.
 NOT A STAND ALONE SOLUTION TO EVERY
TYPE OF WASTE PROBLEM

• Reed bed technology alone is not capable of providing

the optimum treatment solution to every type of waste
problem.
• There are effluents and sludges with concentrations
and/ or types of constituents which are too problematic
or unsuitable for primary or secondary treatment in a
reed bed system.
• However, reed bed systems can be synergistically
combined with other technologies, especially other
biotechnical systems, (such as a second step to
anaerobic digestion for effluents with very high COD
levels), so increasing the effective range of application
for both technologies.
 ADVANTAGES of the Technology

ROBUST AND EFFECTIVE

• lt is easy for those unfamiliar with this technology to
underestimate the robustness and effectiveness of the natural
processes operating within such engineered ecosystems and
their powerful cleaning and recycling capability.
• The composition of micro-organisms in a reed bed system is
very versatile with more than 5,000 different types of bacteria
compared with 200 - 300 in other biological treatment plants.
CONSISTENT DISCHARGE QUALITY
• Such engineered ecosystems have a superior capacity to
tolerate wide variations in effluent characteristics, and to
withstand shock loadings, without damage or disruption to
treatment and so maintaining a consistent discharge quality.
NO BY-PRODUCTS
• Reed bed systems produce no noise or smells and no
sludges or other by-products with associated
additional costs of disposal.
VERSATILITY
• A particular strength of the technology is that, although
it is not a general panacea for every waste treatment
situation, it can be used in many different ways for
different purposes.
• lt can be used as primary or secondary treatment for a
wide variety of effluents and sludges.
• Reed bed systems can be used to treat effluents
which are surprisingly concentrated as well as
effluents with difficult pollutants too dilute to treat by
conventional means but which remain too dangerous
to release into the environment.
• Reed bed systems can be used to recycle water.
• They can also play a cost effective role.
LONG LASTING WITH LOW OPERATIONAL &
MAINTENANCE COSTS
• With little or no electrical or mechanical parts, reed bed
systems are long lasting, wear free and naturally
regenerative.
• Being self regulating ecosystems, they are simple to
operate without chemical additives or complex electronic
controls and require minimal staffing levels due to their
very low maintenance requirements.
• Consequently operational and energy costs of reed bed
systems are very low and system lifetimes are very long.
LOWER CAPITAL COSTS THAN ALTERNATIVE
CONVENTIONAL SYSTEMS
• Territorial authorities and companies choose reed bed
treatment systems on economic grounds.
ENVIRONMENTALLY SAFE & BENEFICIAL

• Reed bed systems in their construction and operation are

environmentally benign, as in essence they are specially
engineered ecosystems, using substantially natural
components to harness nature‘s own powerful processes.
• The tall attractive reeds in such systems not only blend
pleasantly with the local landscape, but also offer considerable
amenity and wildlife conservation potential.
• As a spin-off benefit, reed bed systems can recreate ,,natural“
wetland habitats which are otherwise fast disappearing around
the world, and for this reason alone their use is likely to be
enthusiastically supported by many influential people and
organisations.
• Horizontal Reedbed technology
 Heavy Metal
Background
• Metals in the environment predate the
biosphere.
• Volcanic activity brings metals to the surface in
high enough concentration to be toxic to
microorganisms.
• Microbes have evolved various systems to
detoxify, resist, or avoid toxic metals.
• Metals may serve as electron donors or
acceptors.
classification
 Heavy Metal Removal
• Heavy metal contamination of water and sediments
has been an on-going problem since the
industrialization.
• Heavy metals are known to cause a number of
human health disorders and result in
environmental degradation.
• Since heavy metals can not be decomposed they
must be sequestered from the environment.
• Current heavy metal clean-up technologies are
largely non-renewable, expensive, or not feasible
for many aqueous environments.
• There is a need for renewable, cost effective, and
environmentally compatible technologies for the
recovery of heavy metals from contaminated Area.
 Sources of heavy metal pollution
• Most of the naturally occurring high
concentration of heavy metals occur in
immobilized form in sediments and ores and
is biologically unavailable.
• However ore mining, smelting and other
modern industries have caused increased
deposition of heavy metals in the terrestrial
and aquatic environment.
 Major sources of heavy metal pollution are:

• Combustion of fossil fuels,

• Mineral mining and processing
• Nuclear and other industrial effluents/sludge
• Brewery and distillery wastes
• Biocides
• Preservatives
Heavy metal pollutants of industrial
activities
• Copper
• zinc
• Cadmium
• Iron
• Mercury
• Lead
• Nickel
• Silver
• Gold
Radioactive elements like:
• Uranium, thorium
Sources and effects of some metal contaminants

Metal source effect

Cadmium electroplating, battery kidney disease, joint pain
Lead leaded petrol, batteries impaired nervous system
Mercury fungicide impaired nervous system
» Paralysis, death
Arsenic smelting, refining, pesticides poisoning
 Heavy Metal Removal Technologies
• Solvent extraction
• Chemical addition, precipitation
• sulfide released from sulfate reducing
bacteria.
• settling tank- primary sedimentation is the
usual sewage treatment process which
reduces 40-60% of total metal present.
• Ion exchange
• Oxidation,
• precipitation
Biological removal of metal removal fall into the
following categories:
1. Processes using metabolically active cells.
- Intracellular accumulation
no commercial application
- Extra cellular accumulation
eg. Gallionella, Leptothrix, organisms
in activated sludge.
- Precipitaion
eg. Desulfovibrio, Desulfotomaculum,
sulfate reducers, Hydrogen using strains
2. Processes by cells or biomass which is
metabolically not active, eg. Biosorption
3. Processes using isolated biological components
 Biological systems for heavy metal removal
• Microbes- algae, bacteria and fungi as well as higher plants have
capabilities to uptake these pollutants. After uptake, these either
accumulate or are assimilated by them.
• Accumulated heavy metals are recovered for recycling or disposal.
• Algae and cyanobacteria are used in removal of Cu, Cd, Pb, Ni,
Zn, Hg, Fe from waste water.
• Zooglea ramigera adsorbs copper and cadmium up to the levels of
300 and 100 mg of metal per g dry wt. respectively.
• Pseudomonas putida, Arthrobacter viscous and Citrobacter sp.
remove several toxic heavy metals from industrial effluents.
• Radioactive metals as uranium and thorium are removed by
Rhizopus arrhizus and Penicillium chrysogenum can
accumulate radium.
• Fungi like Trichoderma, Aspergillus, Aureobasidium, Ophiotoma
and Rhodotorula are shown to have biosorption ability of heavy
metals and these seem to play important role in detoxification of
industrial effluents.
• Green plants are also able to remove heavy metals. Eg. Brassica
juncea for chromium
 Biosorption
Metabolism independent binding or adsorption
of heavy metals to living or dead cells,
extracellular polysaccharides, capsules and slime
layers all referred to as “Biosorption”.

Walls and envelopes of bacteria, yeasts, algae,

fungi are very efficient in biosorption due to the
charged groups present in them.
• Exopolysaccaride of Zooglea ramigera
contains glucose, galactose, pyruvate and
adsorbs uranium, copper, cadmium. For
regeneration acidification is done by HCl.
• Chitin and chitosan from cell wall of fungi and
exoskeleton of arthropods have affinity for
uranium, thorium, radium and several heavy
metals.
• Alkaligenes eutrophus, Bacillus coagulans trap
cadmium in their envelopes.
 Factors affecting biosorption
• pH
• Ion competition
• Composition of microbial cell wall,
extracellular material
• Chemical modification of the metal
 Advantages of biosorption
• High efficiency at low metal concentration while
evaporation , reverse osmosis are feasible if metal
concentrations are high
• Successful operation over a wide range of pH and
temperature.
• Calcium and magnesium ions compete for binding sites
in ion exchange resin column which does not happen for
biosorption.
• Microbial biomass required for biosorption may be
available as a fermentation waste product or specially
grown using cheap processes.
• Cost effective compared with other physico-chemical
methods.
• Recovery of accumulated metal and regeneration of
biosorbent is relatively easy.
• Scaling up is also possible
 Use of bacteria in biosorption
• Pseudomonas fluorescence immobilized on polyvinyl chloride
granules and packed into columns can be used to removezinc and
lead.
–This system was used as the basis for water treatment plant for
Hungarian chemical factory producing effluent containing zinc,
chromium, barium, aluminium, nickel, lead and nitrate.
• Pseudomonas aeruginosa immobilized on an oxygen plasma treated
polypropylene is also used. It can be used to recover uranium.
• Citrobacter sps is used to precipitate cadmium, lead, copper as
insoluble metal phosphate.
• Sphaerotilus natans accumulates metals in mucilagenous layer
outside sheath. It accumulates iron, copper, magnesium, cobalt,
cadmium when present as sulfates.
• Metals are also bound to zoogloeal growth in aerobic biological
treatment units.
• Uranium removal by Streptomyces virido-chromogens immobilized in
polyacrylamide gels in column.
 Use of fungi in Biosorption
• Rhizopus arrhizus for cadmium, uranium
and thorium.
• Aspergillus niger for silver.
• Aspergillus niger, Penicillium
chrysogenum, Claviceps paspali for zinc
removal.
• Trichoderma viride immobilized in china
clay in columns can be used to remove
copper.
 Use of algae for biosorption
• A number of investigations have shown
that algal biomass is capable of binging
considerable amount of heavy metals such
as zinc, copper, cadmium, lead, gold,
uranium etc.
• Cells of Chorella vulgaris immobilized in
polyacrylamide gel packed into columns
have been used for uranium removal.
• Cells of Chorella vulgaris binds gold,
silver, mercury, zinc, copper removal.
Bioremediation of toxic metals

• Traditional remediation techniques in removing toxic metal

contaminants using physical and chemical methods are expensive and
may cause other forms of damage to the environment,
• bioremediation can serve as an inexpensive, effective and
environmental friendly remediation method.
Different phytoremediation techniques
Some plant species for phytoextraction of toxic metals
Some plant species for phytofiltration of toxic metals
Some plant species for phytovolatilization of toxic metals
Some plant species for phytostabilization of toxic metals
Costs of phytoremediation and some traditional remediation techniques
Advantages and Limitations of Phytoremediation
Genetic engineering approach
major mechanisms

• biosorption,
• bioaccumulation,
• biotransformation,
• bioleaching,
• biomineralization and
• microbially-enhanced chemisorption of metals
Major mechanisms of bioremediation
Some microorganisms identified for biosorption of toxic metals
Some microorganisms identified for bioaccumulation of toxic metals
Some microorganisms identified for biotransformation of toxic metals
a. direct enzymatic reduction in biotransformation
b. Different approaches in indirect reduction in biotransformation
Some microorganisms identified for bioleaching of toxic metals
Some microorganisms identified for biomineralization of toxic metals
Costs of microremediation, phytoremediation and different traditional remediation methods
Some earthworm species identified for removing toxic metals