• Microbial metabolites-microbial applications in

various fields-Biotech, pharma, agricultural and

environmental

• Control of microorganisms-Physical, chemical and

biological

• Host-microbe interactions-plant microbe and

animal microbe interactions
Applications of Microbes and
Microbial metabolites in
various fields
 Metabolites-Biotechnological
applications
• Metabolites are the intermediates products
of metabolism. A term metabolite is usually called
as small molecules.
• Metabolites have various functions: fuel, structure,
signaling, stimulatory and, catalytic activity of their own
(usually as a cofactor to an enzyme), defense, and
interactions with other organisms
(e.g. pigments, odorants, and pheromones).
• A primary metabolite is directly involved in normal
"growth", development, and reproduction. Primary
metabolite produced in large-scale by industrial
microbiology
• A secondary metabolite is not directly involved in those
processes, but usually has an important ecological
function.
 Microbial metabolites

Primary and Secondary Metabolites

• Primary metabolites are produced during
active cell growth, and secondary
metabolites are produced near the onset of
stationary phase.

• Primary metabolites – ethanol, citric acid,

glutamic acid, lysine, vitamins,
polysaccharides, enzymes etc.
• Secondary metabolites: all antibiotics
Microbial enzymes
 INTRODUCTION
• Enzyme is defined as a biomolecule that
catalyze (i.e., increase the rates of) chemical
reactions
• Enzymes are mainly synthesized within the
cells
• Most of the enzymes are protein in nature
• The name “Enzyme” literally means ‘in yeast’
En=in; zyme= yeast.
• Enzymes are also called as biological catalysts
 Enzymes
• Catalysts for biological reactions
• Most are proteins
• Lower the activation energy
• Increase the rate of reaction
• Activity lost if denatured
• May be simple proteins
• May contain cofactors such as metal ions or organic
(vitamins)

8
 Historical accounts

• Enzymes are catalysts which speed up the rates

of reactions without themselves undergoing
any permanent change.
• In a cell, there are a large number of enzymes
to catalyze all kinds of metabolic reactions.
• How many enzymes in a cells? It depends on
the cells, e.g. about 1700 enzymes are present
in E. coli.
• Enzyme is derived from the Greek meaning ‘in
yeast’ and was first used by Kühne in 1878.
• In 1897, Buchner demonstrated that filtrates of
yeast of enzymes could catalyze fermentation.
 Historical accounts
• The first enzyme to be crystallized was urease by
Sumner in 1926, an enzyme catalyzes the
hydrolysis of urea to yield carbon dioxide and
ammonia.
• The development of ultracentrifuge by Svedberg
in 1920’s provided very high centrifugal fields for
sedimentation of macromolecules.
• In 1960 the amino-acid sequence of ribonuclease
was deduced by Hirs.
• In 1965 the 3-dimensional structure of lysozyme
was deduced by the technique of X-ray
crystallography.
3-dimensional structure of lysozyme
 Historical accounts
• In 1958 Koshland proposed the ‘induced fit’
theory to account for the catalytic power and
specificity shown by enzymes.
• In 1963, Monod and his colleagues postulated
‘allosteric model’ of enzyme control mechanism.
• In last ten years, the application of recombinant
DNA techniques for the study of enzymes has
produced some remarkable new insights.
Structure of enzyme

zinc as
cofactor
bind in the
active site

Human carbonic anhydrase II

Structure of enzyme
 Chemical Nature of Enzymes
• enzymes consist of 200 or more amino acids, the
active site of an enzyme is made up of only 2 to 3
amino acids that are precisely positioned in 3D
space

• Protein secondary structure (alpha helices and

beta sheets) provides that stable scaffolding
upon which the critical active site amino acids
can be precisely positioned in 3D space.

• The 2-3 amino acids that come together in 3D space

to create an enzyme active site are very far apart in
the linear sequence of the amino acids that make up
the protein.
 Classification of Enzymes
• Oxidoreductoases
oxidases - oxidize ,reductases – reduce
• Transferases
transaminases – transfer amino groups
kinases – transfer phosphate groups
• Hydrolases
proteases - hydrolyze peptide bonds
lipases – hydrolyze lipid ester bonds
• Lyases
Cleaves various bonds other than hydrolysis and oxidation
• Isomerase
Catalyze isomerization chages within a single molecule
• Ligases
Join 2 molecules with covalent bonds
16
Isolation and purification of
enzymes
 Why isolate enzymes?
• It is important to study enzymes in a simple
system for understanding its structure,
kinetics, mechanisms, regulations, and role in
a complex system

• Also isolating pure enzyme is important to

use it for medical and industrial purposes
Soil acts as potential sources for enzyme
producing microbes
 Upstream processing: Soil samples were serial diluted with de-ionized
H2O in the rate of 10-1 to 10-10 (1g of soil dissolved in 9ml of
sterilized de-ionized H2O)

Soil (1g) Sterilized de-ionized water (9ml)

Shaking at RT for 30 min

1 ml soil suspension Serial dilution

9 ml H2O
 Isolation of chitinolytic bacterium

18.2g of Colloidal Chitin Agar Dissolved in 1000 mL of de- H2O

(CCA) medium

Sterilization at 120 lbs for 20 min by Autoclave

0.1% of Substrate
Isolation of chitinolytic bacterium

chitinolytic bacterium

Growth of chitinolytic bacterium on CCA medium

Fermentation Process
 Downstream processing
• Downstream processing refers to the
recovery and purification of biosynthetic
products from fermentation medium.
• It is an essential step in the manufacture of
products such as antibiotics, hormones,
antibodies, vaccines, and enzymes etc.
Stages in downstream processing Removal of microbial cells
Filtration, centrifugation,
sedimentation etc
Product isolation
Solvent extraction, ultra filtration,
precipitation

Product purification
Column chromatography,
TLC, Pre-HPLC

Product polishing
Lyophilization,
Crystallization
Commercialization
 Extracellular Lyses buffer Intracellular
enzyme enzyme
Break open cells by
grinding or ultra-sonics Total enzyme
isolation
Filter Cell biomass (useful waste
product)
Enzyme in
solution

Concentrate by evaporation at low Crude enzyme in solution eg

temperature protease in chemical industry

Powdered crude enzyme eg pectinase

Precipitate
Ammonium sulphate Identification
Sodium sulphate Pri. puri enzyme •SDS-PAGE
PEG Dialysis •Zymogram
acetone
Pure enzyme for medicine eg
Chromatography glucose oxidase
 Confirmation of enzymes
SDS-PAGE-Molecular weight

Zymogram
PRODUCTION OF ANTIBIOTICS
Some Antibiotics produced by
Microorganisms
Fermentation Process

Penicillin
 INDUSTRIAL PRODUCTION

• The industrial production of metabolite was

broadly classified in to two processes namely,

• Upstream processing
• Downstream processing
 Microbial Metabolites
• Metabolites are the intermediates products of metabolism. A
term metabolite is usually called as small molecules.
• Metabolites have various functions: fuel, structure, signaling,
stimulatory and, catalytic activity of their own (usually as a
cofactor to an enzyme), defense, and interactions with other
organisms (e.g. pigments, odorants, and pheromones).
• A primary metabolite is directly involved in normal "growth",
development, and reproduction. Primary metabolite
produced in large-scale by industrial microbiology
• A secondary metabolite is not directly involved in those
processes, but usually has an important ecological function.
 Applications

Microorganisms as a Bio-factory

❖Primary metabolites
❖Secondary metabolites
 Microbial metabolites
for
Biotechnology Research
 Restriction enzymes
• Recognizes specific base sequences in
double-helical DNA and cleave, at
specific places, both strands of a duplex
containing the recognized sequences.

• Restriction enzymes recognize specific

bases pair sequences in DNA called
Restriction
restriction sites and cleave the DNA by
enzyme
hydrolyzing the phosphodiester bond.

• Cut occurs between the 3’

carbon of the first nucleotide
and the phosphate of the next
nucleotide.

• Restriction fragment ends have 5’

phosphates & 3’ hydroxyls.
• Most restriction enzymes occur naturally in microbes

• Protect bacteria against viruses by cutting up viral DNA.

• Bacteria protects their DNA by modifying possible restriction sites
(methylation).

• More than 400 restriction enzymes have been isolated.

• Names typically begin with 3 italicized letters.

– Enzyme Source

– EcoRI E. coli RY13

– HindIII Haemophilus influenzae Rd
– BamHI Bacillus amyloliquefaciens H

• Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.

• Short restriction site sequences occur more frequently in the genome than
longer restriction site sequences, e.g., (1/4)n.
 Application of Enzymes in molecular
biology research

• Polymerase Chain Reaction (PCR)

• Restriction digestion of DNA
• DNA ligase
• Enzyme Linked Immunosorbent Assay
(ELISA)
1. Lysase:
2. Ligase:
 PCR
The polymerase chain reaction (PCR) is a technique
widely used in molecular biology for
amplification/detection - a specific gene from known
samples of DNA

People's choice reaction

Invented by Kary Mullis 1983
Application

PCR can be used in many different laboratory such as –

Diagnosing disease and genetic disorders in clinical as
well as biotechnological laboratories ‘detection of
genes’.
 PCR Taq DNA Polymerase
Taq DNA Polymerase for Thermus aquaticus, which is a
microbe found in 176°F.
The enzyme is obtained from hyper thermophilic archae
Heat stable enzyme
amplifies the DNA from the primers by the polymerase
chain reaction, in the presence of Mg2+.
Helps to join the DNA base-pairs
ELISA
 ELISA
• The ELISA plate is coated with Antibody to
detect specific antigen

The enzyme horseradish peroxidase (HRP),

 BIOTECHNOLOGICAL USE OF
MICROBIAL ENZYMES

∙ Several thermostable enzymes, like the Taq

polymerase have been identified and widely
used in PCR and other reactions.

∙ Cellulase is obtained from E.coli and degrades

cellulose, a polysaccharide in plant cells.
∙ Protease act as a bio-detergents
Biosensors

a compact analytical device

incorporating a biological
sensing element with a
transducer
 Application of immobilized enzymes

Biosensors
An analytical device which can detect and quantify
specific analytes in complex samples

Biological
Sample Detection Transducer
Solution Element
Signal
Processor

Readout
Signal
Enzyme biosensors
Example of biosensors

Pregnancy test

Detects the human chorionic

gonadotropin (hCG) protein in urine.

Glucose monitoring device (for diabetes patients)

Monitors the glucose level in the blood.

 Bio-detergents:
Protease - based Enzymes
Miscellaneous products
Biopesticides
enzymes
amino acids
organic acids
solvents
natural flavor compounds
Biogas/biofeul
 Biotechnological
application-Fermentation
Microbial applications
in
agricultural
 Introduction
• Food biotechnology is the
application of technology to modify
genes of animals, plants, and
microorganisms to create new
species which have desired
production, marketing, or nutrition
related properties.
⚫ Called genetically engineered (GE) or genetically
modified (GM) foods, they are a source of an
unresolved controversy over the uncertainty of their
long-term effects on humans and food chains.
 Why genetically modify food?
1) Extended Shelf Life
• The first steps in
genetic modification
were for food
producers to ensure
larger profits by
keeping food fresher,
longer.
• This allowed for further
travel to and longer
availability at markets,
etc…
Extended Shelf Life Milk
 Example: Long Shelf Tomatoes
⚫ These genetically modified
tomatoes promise less waste and
higher profits.
⚫ Typically, tomatoes produce a
protein that softens them after
they have been picked.
⚫ Scientists can now introduce a
gene into a tomato plant that
blocks synthesis of the softening
protein.
⚫ Without this protein, the
genetically altered tomato softens
more slowly than a regular
tomato, enabling farmers to
harvest it at its most flavorful and
nutritious vine-ripe stage.
 2) Efficient Food Processing
• By genetically modifying
food producing
organisms, the wait
time and quantity of
certain food processing
necessities are
optimized.
• Again this is a money
saver. Although efficient, this type of food
processing is not an example of
biotechnology.
 Example: Rennin Production
⚫ The protein rennin is used to
coagulate milk in the
production of cheese.
⚫ Rennin has traditionally
been made in the stomachs
of calves which is a costly
process.
⚫ Now scientists can insert a
copy of the rennin gene into
bacteria and then use
bacterial cultures to mass
produce rennin.
⚫ This saves time, money, Rennin in the top test tube… not there in
space and animals. the bottom one.
 3) Better Nutrient Composition
⚫ Some plants, during
processing, lose some of
the vital nutrients they
once possessed.
⚫ Others are grown in
nutrient poor areas.
⚫ Both these problems can
be solved by introducing
genes into plants to
increase the amount or
potency of nutrients.
⚫ “Biofortification”
 Example: Golden Rice
⚫ Scientists have engineered "golden rice", which has received genes from a
daffodil and a bacterium that enable it to make beta-carotene.
⚫ This offers some promise in helping to correct a worldwide Vitamin A
deficiency.
❖Bio-control agents
❖Bio-fertilizers
Biofertilizers
 Concept of biofertilizer

Biofertilizers

The term biofertilizer refers to preparation containing live

microbes which helps in enhancing the soil fertility either by
fixing atmospheric nitrogen, solubilization of phosphorus or
decomposing organic wastes or by augmenting plant growth by
producing growth hormones with their biological activities.

Rhizobium Bacteria Bacteria in root surface Bacteria in root surface Legume inoculation
Concept of biofertilizer
BIOFERTILIZER ORGANISMS

RHIZOBIUM

AZOTOBACTER

PSB

BLUE GREEN
ALGAE

AZOSPIRILLUM

VA-MYCORRHIZA
 N2- Fixing Organisms
Free Living N2- Fixers
1. Obligate Aerobes
Azotobacter
Beijerenkia
Azotcoccus
2. Obligate aerobes that fix N2 at low O2
Azospirillum
Thiobacillus
Rhizobium
3. Facultative anaerobes- fix N2 under O2 free conditions
Klebsiella pneumoniae
Bacillus polymax
Escherichia intermedia
4. Obligate anaerobes
Clostridium
Desulfovibrio
5. Phototropic bacteria
Rhodospirillum
Chromatium
Chlorobium
6. BGA/ Cyanobacteria
a. Unicellular aerobic
Gloethece
Aphanothece
b. Filamentous heterocystous - aerobic/ anaerobic
Nostoc, Anabaena, Cylindrospermum
c. Filamentous non-heterocystous
Oscillatria, Lyngbya, Plectonema
 Symbiotic N2- Fixers
Rhizobium- Leguminosae (12,000 spp.)
Ulmaceae Parasponia

Non-Rhizobium N2 Fixers
Frankia (Actinorrhizae)
Alnus (Betulaceae) Elaegnus (Elaegnaceae)
Coenothus (Rhamnaceae) Dryas (Rosaceae)
Coriaria (Coriariaceae) Casuarina
(Casuarinaceae)
Lichens
Collema- Nostoc
Dendriscocaulon- Scytonema
Water fern
Azolla- Anabaena
Cycads
Cycas- Nostoc, Anabaena
Higher Plants
Haloragaceae- Gunnera- Nostoc

Associative Symbiosis and casual Assn.

Phyllophore- Azotobacter sp.
Roots of grasses- Azotobacter
Azospirillum
 Nitrogen fixation In leguminous plants

❖Biological nitrogen fixation (BNF) is the

process whereby atmospheric nitrogen
(N=N) is reduced to ammonia in the
presence of nitrogenase.

❖Nitrogenase is a biological catalyst found

naturally only in certain microorganisms
such as the
symbiotic Rhizobium and Frankia, or the
free-living Azospirillum and Azotobacter.

❖Symbiotic N fixation
❖Asymbiotic N fixation
 The Nitrogenase Complex
All nitrogen fixing species (symbionts & non- symbionts)
contain the nitrogenase complex
• Crucial components of the complex are two proteins:
– 1) nitrogenase reductase (Fe-4S protein); homodimer
– 2) nitrogenase (Fe-Mo protein); tetramer (A2B2)
• The nitrogenase complex is anaerobic!
 The Nitrogenase Reductase
• Dinitrogenase reductase (Mr 60,000) is a dimer of two
identical subunits.

• provides electrons with high reducing power

– Electron transfer from the reductase to the nitrogenase is
coupled with ATP hydrolysis

Ribbon diagram of Nitrogenase complex

gray and pink are the dinitrogenase
subunits
blue and green are the dinitrogenase
reductase subunits.
(bound ADP red, Fe atoms orange, S
atoms yellow)
 The Nitrogenase
• Dinitrogenase (Mr 240,000) is a tetramer with two copies
of two different subunits.
– has two binding sites for the reductase.
– uses e- to reduce N2 to NH3
– highly sensitive to oxygen

Ribbon diagram of Nitrogenase complex

gray and pink are the dinitrogenase
subunits
blue and green are the dinitrogenase
reductase subunits.
(bound ADP red, Fe atoms orange, S
atoms yellow)
 Rhizobium-legume symbioses
Host plant Bacterial symbiont

Alfalfa Rhizobium meliloti

Clover Rhizobium trifolii
Soybean Bradyrhizobium japonicum
Beans Rhizobium phaseoli
Pea Rhizobium leguminosarum
Sesbania Azorhizobium caulinodans
Both plant and bacterial factors determine specificity
legu
me

Fixed Fixed carbon

nitrogen (malate,
(ammonia) sucrose)

rhizo
 Obvious signs of nodulation by common rhizobial species

Pea Plant

MEDICAGO
(alfalfa) LOTUS
(birdsfoot
trefoil)
Pink color is leghaemoglobin a
protein that carries oxygen to
the bacteroids R. leguminosarum
nodules
 Rhizobium Attachment and infection

Nod factor
(specificity)
Invasion through infection tube
Flavonoids
(specificity)

Bacteroid Nitrogen
differentiation fixation

Formation of
nodule primordia
From Hirsch, 1992.
New Phyto. 122, 211-237
Microbes in pharmaceutical
applications
 Pharmaceuticals application

Pharmaceutical substances : Medicinal therapy

▪ Traditional pharmaceutical sectors:
- Chemical-based drugs : chemical synthesis
- Extraction or isolation from biological sources

Biopharmaceuticals : A class of therapeutic agents produced by modern

biotechnological techniques, like recombinant DNA, protein engineering, and
hybridoma technologies etc. (used in the 1980s)
- Nucleic acids used for gene therapy and antisense technology
- Proteins for in vivo diagnostics

A Protein or nucleic acid-based pharmaceutical substances used for therapeutic or

diagnostic purpose, which is produced by modern biotechnological techniques

Form the backbone of medicinal agents in the modern biotech era

• Pharmaceutical products
– Antibiotics
– Vitamins
– Vaccines

88
Some Antibiotics produced by
Microorganisms
Application of Pharma-microbiology
 Organic Synthesis

• Organic synthesis is a special branch of chemical

synthesis and is concerned with the construction
of organic compounds via organic reactions.
• Organic molecules can often contain a higher level
of complexity compared to purely inorganic
compounds, so the synthesis of organic
compounds has developed into one of the most
important branches of organic chemistry.
 Green chemistry
• Green chemistry, also called sustainable
chemistry, is a philosophy of chemical
research and engineering that encourages the
design of products and processes that
minimize the use and generation of
hazardous substances
By usage of microbial agents especially,
immobilized enzymes
Microbes in environmental
applications
 Biogeochemical Cycle
• N cycle
• C cycle
• Sulfur cycle
• Phosphorous cycle
 The NITROGEN CYCLE
a variety of microorganisms participate in this
process
more oxidized more reduced

Reduction by most
plants & some Synthesis:
anaerobic bacteria (microorganisms,
plants & animals

Amino acids
Nitrate N2 Ammonia & reduced
NO3- NH4+
Denitrification Nitrogen fixation nitrogen
(some bacteria) compounds

Degradation:
Animals & microbes
Nitrification
(e.g. Nitrobacter) Nitrification
Nitrite (e.g. Nitrosomonas)
NO2-
 Key terms of The Nitrogen Cycle
• Nitrogen Fixation: Conversion of N2 to ammonia (NH3)
– By any bacteria in soil/water having the nitrogenase complex,
e.g. Rhizobium in root nodules of legumes.
-
• Nitrification: Conversion
-
of ammonia to nitrite (NO 2
) and
then nitrate (NO3 ).
– Both reactions carried out by bacteria
• Assimilation: Conversion of NH3, NO2-,, NO3- (inorganic) into
organic compounds (proteins, DNA, & other forms)
– All living cells (plants, animals, & bacteria).
• Ammonification: Conversion of the amine groups of
organic compounds into simpler compounds (often,
ammonia NH3).
– Mostly via decay processes carried out by decomposer bacteria
• Denitrification: Conversion of NH3, NO2-,, NO3- to N2
– Mostly by anaerobic bacteria in waterlogged soil, bottom
sediments of lakes, swamps, bogs and oceans.
Carbon cycle
carbon can be interconverted between methane,
complex organic matter, and carbon dioxide; carbon
fixation can occur by the activities of cyanobacteria, the
green algae, photosynthetic bacteria, and aerobic
chemolithoautotrophs
Chemoheterotrophs consume the organic compounds,
animals eat photoautotrophs, especially green plants,
and may in turn be eaten by other animals.
When the organisms die, the organic compounds of
their bodies are deposited in the soil and are
decomposed by microorganisms, principally by
bacteria and fungi. During this decomposition, carbon
dioxide is returned to the atmosphere.
Sulfur cycle:
sulfur can be interconverted between
elemental sulfur, sulfide, and sulfate forms by
the actions of various microorganisms

Dissimilatory sulfate reduction produces sulfide

which accumulates in the environment

Assimilatory sulfate reduction results in the

reduction of sulfate for use in amino acid
biosynthesis
Key processes and prokaryotes in the sulfur cycle
Processes Organisms
Sulfide/sulfur oxidation(H2S→S0 → SO42-)
Aerobic Sulfur chemolithotrophs
(Thiobacillus, Beggiatoa, many others)
Anaerobic Purple and green phototrophic
bacteria, some chemolithotrophs
Sulfate reduction(anaerobic)(SO42- → H2S)
Desulfovibrio, Desulfobacter
Sulfur reduction(anaerobic) (S0 → H2S)
Desulfuromonas, many
hyperthermophilic Archaea
Sulfur disproportionation(S2O32- → H2S + SO42-)
Desulfovibrio and others
Organic sulfur compound oxidation or reduction(CH3SH→CO2+ H2S)
(DMSO→DMS)
Desulfurylation(organic-S → H2S)
Many organisms can do this
Sulfur cycle
PHOSPHOROUS CYCLE
 HUMAN IMPACTS TO PHOSPHOROUS
CYCLE
1. Humans mine LARGE quantities of phosphate rock to use in
commercial fertilizers and detergents. Phosphorous is NOT
found as a gas, only as a solid in the earth’s crust. It takes
millions to hundreds of millions of years to replenish.
2. Phosphorous is held in the tissue of the trees and vegetation,
not in the soil and as we deforest the land, we remove the
ability for phosphorous to replenish globally in ecosystems.
3. Cultural eutrophication – add excess phosphate to aquatic
ecosystems in runoff of animal wastes from livestock feedlots,
runoff of commercial phosphate fertilizers fro cropland, and
discharge of municipal sewage.
 IMPORTANCE OF PHOSPHOROUS
CYCLE

• 1.Phosphorous is an essential nutrient of both plants

and animals.
• 2. It is part of DNA molecules which carry genetic
information.
• 3. It is part of ATP and ADP) that store chemical energy
for use by organisms in cellular respiration.
• 4. Forms phospholipids in cell membranes of plants
and animal cells.
• 5. Forms bones, teeth, and shells of animals as calcium
phosphate compounds.
Environmental Pollution
• Environment: Soil, Air, water and atmosphere
• Air, soil and water pollution

• Industries-sources of pollution:
Chemical manufacturing
Leather industries
• Toxic substances
• Oil contamination
• Heavy metal pollution
• Plastic wastes-Bioplastic production
• Biological wastewater treatment-Domestic
wastewater and industrial effluents
 Biological treatment of waste
water
❖ Methods
▪ Aerobic : removal of organic pollutants in
wastewater by bacteria that require oxygen to
work
End products: Water and carbon dioxide and
biomass
▪ Anaerobic :bacteria digests biosolids in the
absence of oxygen
End products: methane and carbon dioxide
gas and biomass
Principle of Aerobic
process

Principle of
Anaerobic process
 Aerobic Biological Treatment
❖ Steps
▪ Primary
▪ secondary
▪ Tertiary treatments
Differenc
es in aero
bic and a
naerobic
treatmen
t
Parameters
Aerobic
Anaerobic
Aerobic Biological Treatment Technologies
1) Activated Sludge Process (CASP) System
2) Cyclic Activated Sludge System (CASS)
3) Integrated Fixed Film Activated Sludge (IFAS) System
4) Membrane Bioreactor (MBR)
Activated Sludge Process (ASP) System
❖ most common and oldest biotreatment process

❖ used to treat municipal and industrial wastewater

❖ wastewater after primary treatment i.e. suspended impurities

removal is treated in an activated sludge process based
biological treatment system comprising aeration tank followed
by secondary clarifier.
Activated sludge process
❖ The aeration tank is provided with fine bubble diffused
aeration pipework at the bottom to transfer required oxygen
to the biomass and also ensure completely mixed reactor.
❖ The aerated mixed liquor from the aeration tank overflows
to the secondary clarifier unit to separate out the biomass
and allow clarified, treated water to the downstream
filtration system for finer removal of suspended solids.
❖ The separated
biomass is
returned
to the aeration
Tank by means of
return
activated sludge
(RAS) pump.
Activated sludge plant Settling tanks
Characteristics of aerobic activated sludge:
Overall, activated sludge must contain a microorganisms
capable of producing all enzyme systems required for the
biodegradation of both soluble and insoluble pollutants.
Activated sludge contains prokaryotes (bacteria) and Eukaryotes
(Protozoa and fungi)
The primary consumers of organic wastes are the heterotrophic
(An organism that cannot synthesize its own food and is dependent
on complex organic substances for nutrition) bacteria
The majority of the bacterial genera in activated sludge are
Gram-negative
Pseudomonas, Arthrobacter, Comamonas, Lophomonas,
Zoogloea, Sphaerotilus, Azotobacter, Chromobacterium,
Achromobacter,Flavobacterm, Bacillus, and Nocardia
Certain genera Sphaerotilus or Nocardia-causes poor settling
Many types of protozoa : 50,000 cells/ml
Activated sludge flocs

Note filamentous bacteria

Note Vorticella
and other
protozoa
 Facultative lagoons-Aerated
lagoons (oxidation pond)
Facultative lagoons or stabilization ponds use only natural phenomena and
almost no mechanical action. Oxygenation for bacterial oxidation of
organics comes from photosynthesis by algae and a bit from wind.
CO2 released by bacteria is used by the algae. Excess biomass and other
settleables are treated by anaerobic bacteria at the bottom.

Drawback: Eutrophication-algae is utilizing the micronutrients (eg.PO4) in the

Water and grow as dense mat on the surface of the water, thus creates O 2 scarcity –
water living organisms wont survive.
Large-scale wastewater treatment
 Anaerobic waste-water treatment
Anaerobic treatment is often performed for solids and
high-strength industrial waste-waters.
both facultative and obligate anaerobic microorganisms, in
the absence of oxygen are being involved in the anaerobic
waste water treatment.
They biodegrade the organic pollutant to generate
methane, carbon dioxide and biomass.
The microbiology of these anaerobic digestion processes
is complex.
Their efficient and stable operation requires a microbial
population containing at least 3 different interacting
microbial groups.
1) Fermentative/hydrolytic bacteria (group 1)
2) Acetogenic bacteria (group 2)
3) Methanogenic bacteria (group 3)
There are 3 stages: 1)Hydrolysis
2) Acetogenesis
3) Methanogenesis
 Insoluble substrate

Cell
Exoenzyme Endoenzyme membrane

Exocellular slime Cell wall

produced in the cell

and released through
the cell membrane and Soluble wastes enter
cell wall to hydrolyze the bacterial cell and
insoluble substrate that are degraded by
is adsorbed to the endoenzymes.
exocellular slime.
 Characteristics of Anaerobic activated sludge

The types of bacteria within an anaerobic sludge (digester):

- saccharolytic bacteria
- proteolytic bacteria fermentative bacteria
- lipolytic bacteria
- methane-forming bacteria
3 important bacterial groups in anaerobic digesters with respect to
the substrates utilized by each group.
These groups include
- the acetate-forming (acetogenic) bacteria (AFB),
- the sulfate-reducing bacteria (SRB),
- the methane-forming bacteria (MFB):
3 groups of methane-forming bacteria
1) the hydrogenotrophic methanogens,
2) the acetotrophic methanogens, and
3) the methylotrophic methanogens.
Microbial treatment of oil pollution
 Microbial treatment of crude oil- Marine oil degrader Alcanivorax
borkumensis

❖Alcanivorax borkumensis is a key marine

oil-degrading bacterium that can
dramatically increase in numbers after an oil
spill and become the most abundant microbe
in oil-polluted waters
❖nitrogen and phosphorous salts
❖secretes emulsifiers – break up oil droplets

❖This “hydrocarbonoclastic” bacterium

degrades an exceptionally broad range
of alkane hydrocarbons but few other
substrates (linear alkanes,
cyclo-alkanes, and isoprenoids)
 Heavy metal pollution and
their removal methods
❖ trace metals in the environment may
accumulate unnoticed to toxic levels.
❖ Number of heavy metals : 65 – defined with
respect to a number of criteria :
their cationic-hydroxide formation
specific gravity greater than 5 g/ml,
complex formation,
more recently, association with eutrophication
and environmental toxicity.
Sources of heavy metal pollution
Sources of heavy metal pollution
contd…
 Microbes for metal
remediation-mechanism
❖ Metal ions – bind with cell surface
❖ Mechanisms :
electrostatic interactions,
Van der Walls forces
Covalent bonding
redox interactions
Extracellular precipitation
combination of these processes
❖ The negatively charged groups : carboxyl, hydoxyl and
phosphoryl of the bacterial cell wall adsorb metal cations,
which are then retained by mineral nucleation
❖ Extent of sorption varies with the metal as well as with the
microorganisms
Eg: Bacillus sp. : take up 15% Cu and 14% Zn
Microbes for metal remediation-mechanism contd…

❖ Thiobacillus ferroxidans and Leptospirillum ferroxidans –

oxidise iron and sulfur

❖ P.stutzeri AG 259 is capable of producing silver-based single

crystals which can reduce the toxicity of metals

❖ Microbial metabolite : surfactants

Eg: Rhamnolipids - P.aeruginosa show specificity for certain
metals such as Cd and Pb
Plastic wastes-Major pollution
 Problems with Conventional Plastic

• Pros
– Cheap and Easy to Manufacture
– Good Commercial Properties
• Cons
– Complex entanglements of polymer chains (usually
Polyethylene Terephthalate PET or Polybutylene
Terephthalate PBT) make it hard to decompose
– Relies heavily on petrochemicals
– Needs processing
– Recycling requires energy and money
– Releases toxic chemicals
– Fragmentation or Cyclization occurs
– 200 million tons produced each year and most of it is not recycled
 Why Use Bioplastics?
– Bioplastics
• Applications
– Bottling, resins, packaging, etc
• Main constituents
– Polylactic acids from starch (Corn, Potatoes, etc)
– Oils, sugars, fibers, etc
• Pros
– Reduces or eliminates GHG in production
– Requires less or no petrochemicals
– Plants decreases CO2 in the atmosphere
– Biodegradable - byproducts water, CO2, and organic materials
– Can be utilized as fuel
– Slow Release of CO2 allows for plants to absorb CO2 than
release it in the atmosphere
• Potential Cons
– Uses Genetically Modified processes
– Cost up to three times more than regular Plastic
– Use of fertilizers and pesticides for crops
Control of Microorganisms-Physical,
chemical and biological methods
 Introduction
• The control of microbial growth is necessary in many
practical situations, and significant advances in
agriculture, medicine, and food science have been
made through study of this area of microbiology.
• "Control of microbial growth", as used here, means to
inhibit or prevent growth of microorganisms. The
control of MICROBES in two basic ways: (1) by killing
microorganisms or (2) by preventing the growth of
microorganisms.
• Control of growth usually involves the use of physical
chemical, and biological agents which either kill or
prevent the growth of microorganisms.
 Definitions
• Sterilization: A process that kills all living cells, including
viruses and spores, from a substance or object using
autoclave
• Disinfection: A treatment that reduces the total number of
microbes on an object or surface, but does not necessarily
remove or kill all of the microbes
• Sanitation: Reduction of the microbial population to levels
considered safe by public health standards
• Antiseptic: A mild disinfectant agent suitable for use on
skin surfaces
• -cidal: A suffix meaning that “the agent kills.” For example,
a bacteriocidal agent kills bacteria
• -static: A suffix that means “the agent inhibits growth.” For
example, a fungistatic agent inhibits the growth of fungi,
but doesn’t necessarily kill it.
 Control of Microorganisms
• Physical Methods
• Chemical Agents
• Biological Agents
• Mode of action
• Conditions Influencing Antimicrobial
Activity
 Physical Methods
• Moist Heat
• Dry Heat
• Low Temperatures
• Filtration
• Radiation
 Physical Methods: Moist Heat
• Mechanism of killing is a combination of
protein/nucleic acid denaturation and
membrane disruption
• Effectiveness Heavily dependent on type of cells
present as well as environmental conditions
(type of medium or substrate)
• Bacterial spores much more difficult to kill than
vegetative cells
 Physical Methods: Moist Heat
• Measurements of killing by moist heat
– Thermal death point (TDP): Lowest temperature at
which a microbial suspension is killed in 10 minutes;
misleading because it implies immediate lethality
despite substrate conditions
– Thermal death time (TDT): Shortest time needed to
kill all organisms in a suspension at a specified
temperature under specific conditions; misleading
because it does not account for the logarithmic
nature of the death curve (theoretically not possible
to get down to zero)
 Physical Methods: Moist Heat
• Measurements of killing by moist heat (cont.)
– Decimal reduction time (D value): The time required to
reduce a population of microbes by 90% (a 10-fold, or
one decimal, reduction) at a specified temperature and
specified conditions
– z value: The change in temperature, in ºC, necessary to
cause a tenfold change in the D value of an organism
under specified conditions
– F value: The time in minutes at a specific temperature
(usually 121.1°C or 250 °F) needed to kill a population of
cells or spores
 Physical Methods: Moist Heat
• Methods of Moist Heat
– Boiling at 100°C
• Effective against most vegetative cells; ineffective against
spores; unsuitable for heat sensitive chemicals & many foods
– Autoclaving/pressure canning
• Temperatures above 100°C achieved by steam pressure
• Most procedures use 121.1°C, achieved at approx. 15 psi
pressure, with 15 - 30 min autoclave time to ensure
sterilization
• Sterilization in autoclave in biomedical or clinical laboratory
must by periodically validated by testing with spores of
Clostridium or Bacillus stearothermophilus
 Physical Methods: Moist Heat
⚫ Methods of Moist Heat
⚫ Pasteurization is a process of heating a food, which is
usually a liquid, to a specific temperature for a
predefined length of time and then immediately cooling
it after it is removed from the heat. This process slows
spoilage caused by microbial growth in the food.
Pasteurization
Used to reduce microbial numbers in milk and other beverages while
retaining flavor and food quality of the beverage
Retards spoilage but does not sterilize
Traditional treatment of milk, 63°C for 30 min
Flash pasteurization (high-temperature short term pasteurization);
quick heating to about 72°C for 15 sec, then rapid cooling
Physical Methods: Moist Heat
 Physical Methods: Dry Heat
• Incineration
– Burner flames
– Electric loop incinerators
– Air incinerators used with fermenters; generally
operated at 500°C
• Oven sterilization
– Used for dry glassware & heat-resistant metal
equipment
– Typically 2 hr at 160°C is required to kill bacterial spores
by dry heat: this does not include the time for the glass
to reach the required temp (penetration time) nor does
it include the cooling time
 Physical Methods:
Low Temperatures

• Refrigerator:
– around 4°C
– inhibits growth of mesophiles or thermophiles;
psychrophiles will grow
• Freezer:
– “ordinary” freezer around -10 to -20°C
– “ultracold” laboratory freezer typically -80°C
– Generally inhibits all growth; many bacteria and other
microbes may survive freezing temperatures
 Physical Methods: Filtration

• Used for physically removing microbes and dust particles

from solutions and gasses; often used to sterilize
heat-sensitive solutions or to provide a sterilized air flow
• Depth filters: eg. Diatomaceous earth, unglazed
porcelean
• Membrane filters: eg. Nitrocellulose, nylon,
polyvinylidene difluoride
• HEPA filters: High efficiency particulate air filters used in
laminar flow biological safety cabinets
 Physical Methods: Radiation

• Ultraviolet Radiation
– DNA absorbs ultraviolet radiation at 260 nm
wavelength
– This causes damage to DNA in the form of
thymine dimer mutations
– Useful for continuous disinfection of work
surfaces, e.g. in biological safety cabinets
 Physical Methods: Radiation
• Ionizing Radiation
– Gamma radiation produced by Cobalt-60 source
– Powerful sterilizing agent; penetrates and damages both
DNA and protein; effective against both vegetative cells and
spores
– Often used for sterilizing disposable plastic labware, e.g.
petri dishes; as well as antibiotics, hormones, sutures, and
other heat-sensitive materials
– Also can be used for sterilization of food; has been approved
but has not been widely adopted by the food industry
 Chemical Agents
• Phenolics
• Alcohols
• Halogens
• Heavy metals
• Quaternary Ammonium Compounds
• Aldehydes
• Peptides
Phenolics
• phenol and phenolics (phenol derivatives) such as cresols, xylenols,
and orthophenylphenol - disinfectants in laboratories and hospitals.
• Phenolics act by denaturing proteins and disrupting cell membranes.
• advantages as disinfectants:
• phenolics are tuberculocidal, effective in the presence of organic
material, and remain active on surfaces long after application.
• have a disagreeable odor and can cause skin irritation.
Alcohols
• most widely used disinfectants and antiseptics.
• They are bactericidal and fungicidal but not sporicidal; some
lipid-containing viruses are also destroyed.
• The two most popular alcohol germicides are ethanol and
isopropanol, usually used in about 70 to 80% concentration.
• denaturing proteins and possibly by dissolving membrane lipids.
• A 10 to 15 minute soaking is sufficient to disinfect thermometers and
small instruments.
Halogens
• fluorine, chlorine, bromine, iodine, and astatine
• The halogens iodine and chlorine are important antimicrobial agents.
• used as a skin antiseptic and kills by oxidizing cell constituents and iodinating
cell proteins.
• At higher concentrations- even kill some spores.
• Iodine - tincture of iodine, 2% or more iodine in a water-ethanol solution of
potassium iodide.
• Iodophor-release iodine slowly to minimize skin burns and irritation.
• used in hospitals for preoperative skin degerming and disinfecting.
Heavy Metals
• mercury, silver, arsenic, zinc, and copper - used as germicides.
• 1% solution of silver nitrate is often added to the eyes of infants to prevent
ophthalmic gonorrhea (in many hospitals, erythromycin is used instead of
silver nitrate because it is effective against Chlamydia as well as Neisseria).
• Silver sulfadiazine is used on burns.
• Copper sulfate is an effective algicide in lakes and swimming pools.
• Heavy metals combine with proteins, often with their
sulfhydryl groups, and inactivate them. They may also
precipitate cell proteins.
Quaternary Ammonium Compounds
• Anionic, non-ionic and cationic
• cationic detergents are effective disinfectants.
• The most popular of these disinfectants are quaternary ammonium
compounds characterized by a positively charged quaternary
nitrogen and a long hydrophobic aliphatic chain
• They disrupt microbial membranes and may also denature
proteins.
• Eg.: benzalkonium chloride and cetylpyridinium chloride
Aldehydes
• aldehydes, formaldehyde and glutaraldehyde - are highly reactive
molecules
• that combine with nucleic acids and proteins and inactivate them,
probably by crosslinking and alkylating molecules
• They are sporicidal and can be used as chemical sterilants.
• Glutaraldehyde is less irritating than formaldehyde - used to
disinfect hospital and laboratory equipment.
Antibiotics
 Antibiotics
• Antibiotics are generally considered to be organic
compounds of low molecular weight produced by
microbes.
• At low concentration, antibiotics are deleterious to the
growth or other metabolic activities of other
microorganisms.
Most of the Antibiotics produced by
Microorganisms
 Penicillin
• All penicillin like antibiotics inhibit synthesis of peptidoglycan,
an essential part of the cell wall.
• They do not interfere with the synthesis of other intracellular
components.
• These antibiotics do not affect human cells because human
cells do not have cell walls.
• Penicillins are active against Gram positive bacteria
• Some members (e.g. amoxicillin) are also effective against
Gram negative bacteria
PRODUCTION OF PENICILLIN
• Penicillin was the first important commercial product produced
by an aerobic, submerged fermentation
• First antibiotic to have been manufacture in bulk.
• Used as input material for some semi synthetic antibiotics.
• It is fermented in a batch culture
⚫ Thanks to work by Alexander Fleming (1881-1955), Howard
Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin
was first produced on a large scale for human use in 1943. At
this time, the development of a pill that could reliably kill

⚫ bacteria was a remarkable development and many lives were

saved during World War II because this medication was
available.

A. Fleming E. Chain H. Florey

 A tale by A. Fleming
• In 1928, Sir Alexander Fleming, a Scottish
biologist, observed that Penicillium notatum,
•a common mold, had destroyed
staphylococcus bacteria in culture.

He published a report on penicillin and its

potential uses in the British Journal of
Experimental Pathology.
Penicillin
Mechanism of Antibiotics
 Summary of antimicrobial agents affecting cell wall
synthesis
Agents affecting the
cell wall β-lactamase
inhibitors
Clavulanic acid
β-lactam antibiotics Other antibiotics Sulbactam
Bacitracin Tazobactam
Vancomycin
Daptomycin

Penicillins Cephalosporins Carbapenems Monobactams

Amoxicillin Ertapenem
Imipenem/cilastatin* Aztreonam
Ampicillin Meropenem
Dicloxacillin
Indanyl carbenicillin
Methicillin 1st generation 2nd generation 3rd generation 4th generation
Nafcillin Cefadroxil
Oxacillin Cefaclor Cefdinir Cefepime
Cefazolin Cefprozil Cefixime
Penicillin G Cephalexin Cefuroxime
Penicillin V Cefotaxime
Cefoxitin Ceftazidime
Piperacillin Ceftibuten
Ticarcillin Ceftizoxime
Ceftriaxone

(according to Lippincott´s Pharmacology, 2009)

 Cephalosporin
• The cephalosporins are a class of β-lactam
antibiotics originally derived from the fungus
Acremonium, which was previously known as
"Cephalosporium".
• Cephamycins constitute a subgroup of
β-lactam antibiotics called cephems.
• A lactam is a cyclic amide. The term is a
lactone + amide.

β-lactam 172
 Mode of action - Cephalosporins
• Cephalosporins are bactericidal and have the same mode of action
as other β-lactam antibiotics (such as penicillins), but are less
susceptible to β-lactamases.
• Cephalosporins disrupt the synthesis of the peptidoglycan layer of
bacterial cell walls.
• The final transpeptidation step in the synthesis of the peptidoglycan
is facilitated by transpeptidases known as penicillin-binding proteins
(PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides
(peptidoglycan precursors) to crosslink the peptidoglycan.
• Active against both gram positive and negative bacteria

173
 The Beta-Lactam Antibiotics

• Cell wall active agents

– Prevent the final step in the synthesis of the
bacterial cell wall

• Range from very narrow spectrum to very

broad spectrum
 β-Lactams

β-lactam ring
 How do they work?
1. The β-lactam binds to Penicillin Binding
Protein (PBP)
2. PBP is unable to crosslink peptidoglycan
chains
3. The bacteria is unable to synthesize a stable
cell wall
4. The bacteria is lysed (cidal effects).
bacteriacidal effect against G+&G-
 Peptidoglycan Synthesis

“Penicillin binding
protein”
 Mode of action of rifampin
• Rifampin has a greater affinity to react with
phosphorus containing biomolecules in the
cell. Thus sulphur containing proteins in the
cell membrane, inside the cells and
phosphorus containing elements like DNA are
likely to be the preferential sites for action of
antibiotics.
 P. aeruginosa treated with Rifampin
• propidium iodide ------ viable cells counting
• ethidium bromide ----- dead cells counting

Control (without Anti) 5 µg/mL, 30 min 25 µg/mL, 30 min

Analysis of bacterial DNA damage
caused by
Conditions Influencing Antimicrobial
Activity

• Several critical factors play key roles in determining

the effectiveness of an antimicrobial agent,
including:
– Population size
– Types of organisms
– Concentration of the antimicrobial agent
– Duration of exposure
– Temperature
– pH
– Organic matter
Host-Microbe interaction:
Plant microbe and animal
microbe interaction
 Microbial interaction
1. Neutralism
2. Commensalism
3. synergism
4. mutualism
5. competition
6. antagonism
7. parasitism
8. predation
❖ Neutralism
there is no any physiological effect between
the populations.

❖ Commensalism
Commensalism is a unidirectional relationship
betwen populations in which one population
benefits and the other one is unaffected.
❖ Synergism
Synergism indicates that both populations
benefit from the relationship but the
association is not obligatory. Both
populations are capable of surviving
independently.

.
❖ Mutualism Symbiosis
Mutualism Symbiosis is an obligatory inter-
relationship between two populations that
benefits both of them.
Lichens is composed of a fungus and an
alga.
❖ Competition
Competition occurs when two
populations are striving for the same
resource of nutrients or the habitat.
❖ Antagonism
Antagonism occurs when one population
produces a substrate inhibitory to
another population.
❖ Parasitism
the parasite population is benefited and the
host population is harmed.
❖ Predation

Predation is a widespread phenomenon

where the predator engulfs or attacks the
prey. The prey can be larger or smaller
than the prey, and this normal results in
the death of the prey.
Plant-microbe interactions
 Plant-microbe interactions
Plants are heavily colonized by microorganisms

❖ The presence of microorganisms increases the rate of organic

matter released from the roots (exudation)

❖ Microorganisms influence plant growth through the release of

compounds such as auxins, giberellins and cytokinins
(PGPR)
❖A soil aggregate composed
of mineral and organic
components, showing that
localization of soil microbes.
❖Very few microorganisms
are found free in the soil
solution; most of them
occur as microcolonies
attached to the soil Main types of soil microorganisms
particles. Agrobacterium Alcaligenes
Arthrobacter Bacillus
Caulobacter Cellulomonas
Clostridium Corynebacterium
Flavobacterium Micrococcus
Mycobacterium Pseudomonas
Staphylcoccus
 Proportion of different soil microorganisms in soil

Microbos Number /g Biomass(g/m3)

Bacteria 108 160
Fungi 105 200
Actinomycets 105 - 106 160
Algae 104 - 105 32
Protozoa 104 38
❖The number of bacteria in the rhizosphere (the narrow region of
soil that is directly influenced by root secretions and associated
soil microorganisms) and rhizoplane (the external surface of
roots together with closely adhering soil particles and debris) is
higher than in the soil devoid of plants; this happens because
soils devoid of plants are poor in many attractive substances
secreted from the roots.
❖As soon as a seed starts to germinate, a relatively large amount
of carbon and nitrogen compounds i.e., sugars, organic acid,
aminoacids, and vitamins are excreted into the surrounding
environment.
❖This attracts a large population of microorganisms inducing
vigorous competition between the different species

❖Beneficial microorganisms are known to be biocontrol agents

and/or growth promoters.
Rhizosphere Effect
( R/S ratio )

The rhizosphere is the soil region in

close contact with plant roots.

Within the rhizosphere, the plant roots

exert a direct influence on the soil
bacteria. This influence is known as
the rhizosphere effect.

In the rhizosphere, microbial populations reach much

higher densities in the rhizosphere than in the free soil.
 Microbial populations in the rhizosphere may
benefit the plant by:
(1) removing hydrogen sulfide, which is toxic to
the plant roots
(2) increasing solubilization of mineral nutrients
needed by the plant for growth
(3) synthesizing vitamins, amino acids, auxins,
gibberellins that stimulate plant growth
(4) antagonizing potential plant pathogens
through competition and the production of
antibiotics
 Mycorrhiza
❖Mycorrhiza literally means "root fungus" and refers to the
symbiotic association that exists between plant roots and
fungi.

❖Probably the roots of the majority of terrestrial plants are

mycorrhizal.

❖There are two classes of mycorrhizae:

Ectomycorrhizae, in which fungal cells form an extensive
sheath around the outside of the root with only little
penetration into the root tissue itself

Endomycorrhizae (vesicular-arbuscular (VA)), in which the

fungal mycelium is embedded within the root tissue.
 Mycorrihizas
a. Ectomycorrhiza b.Endomycorrhiza
Ericacious Orichidacious
Mycorrihiza Mycorrhiza

VA Mycorrhiza
 Functions of mycorrhiza

Increase in P and Protection of plant

nutrient uptake against soil
stresses

VA Mycorrhiza

Production of plant Increase solubility

growth hormones of soil minerals
 Ectomycorrhiza
• Almost all trees form
ectomycorrhizas

• Fungus does not enter plant cell

• Fungus forms a net around the

root(hairs) to extent their access to
soil nutrients

• Fungus colonizes the outercell layers

and forms a Hartig Net (formation of
a fungal mantle on top of the root)
 Endophytic fungi
Endophytic fungi live in the intercellular spaces inside
plants
-Some fungi protect their hosts from herbivores by
producing toxins
- rye grass is more resistant to aphid feeding in the
presence of endophytes

208
 Tolerance to salinity :
❖Soil salinity in arid regions is frequently an important limiting factor
for cultivating agricultural crops.
❖endophytic bacteria can mitigate the effects of salt stress in
different plant species.
❖High K+/Na+ ratios were found in salt-stressed maize in which
selectivity for Na+, K+ and Ca2+ was altered upon inoculation with
Azospirillum
Micropropagation:
❖Micropropagation is an efficient method of propagating large
numbers of genetically uniform plants
❖In vitro bacterization of potato plantlets has been shown to
enhance their transplant stress tolerance thereby eliminating
the need of an expensive greenhouse hardening step, which
even now is commonly used by pre-elite seed potato producers.
❖Plants bacterized in vitro with Pseudomonas fluorescens
strains CHA0 and IP10 were found to have a significantly
higher fresh shoot weight compared to non-bacterized plants 209 in
Bioremediation and phytoremediation
❖ Phytoextraction, actuated by hyperaccumulating or
non-hyperaccumulating species, could be improved by using a
plant-microbe system, thus contributing to novel promising methods
for the cleaning-up of soils contaminated by heavy metals.
❖ Rhizobacteria of the genus Azospirillum have been extensively used
for crop hytostimulation as above stated
❖ The implementation of lead phytoextraction in contaminated industrial
soils by applying A. brasilense Sp245 to plants of indigenous species
belonging to Mediterranean forestry was investigated.
❖ The possible phytoextraction ability was evaluated in Myrtus
communis L. and Laurus nobilis L., previously selected among other
plant species that were found able to grow in the contaminated areas,
on the basis of the Pb content
• The growing speed and the vegetative habitus.
• By trials carried out in greenhouse, it was shown that
• A. brasilense Sp245 can enhance the plant growth in Pb
contaminated soil and affect the plant total lead content. 210
Animal-microbe interactions

211
 Microbe-Human Interactions
❖ The human body exists in a state of dynamic
equilibrium

❖ Many interactions between human body and

microorganisms involve the development of
biofilms

❖ Colonization of the body involves a constant

“give and take”

212
 Contact, Colonization, Infection,
Disease
❖ Microbes that engage in mutual or commensal
associations – normal (resident) flora, indigenous
flora, microbiota

❖ Infection – a condition in which pathogenic

microbes penetrate host defenses, enter tissues,
and multiply

❖ Pathogen – infectious agent

❖ Infectious disease – an infection that causes

damage or disruption to tissues and organs

213
214
 Resident Flora
❖ Most areas of the body in contact with the
outside environment harbor resident microbes
❖ Internal organs, tissues, and fluids are
microbe-free
❖ Transients – microbes that occupy the body for
only short periods
❖ Residents – microbes that become established

215
216
217
 Resident Flora
• Bacterial flora benefit host by preventing
overgrowth of harmful microbes – microbial
antagonism
• Endogenous infections – occur when normal
flora is introduced to a site that was
previously sterile

218
 Initial Colonization of the Newborn
• Uterus and contents are normally sterile
and remain so until just before birth
• Breaking of fetal membrane exposes the
infant; all subsequent handling and feeding
continue to introduce what will be normal
flora

219
220
Indigenous Flora of Specific Regions

221
 Importance of The Normal
Flora (Advantages)
1. They constitute a protective host defense mechanism by
occupying ecological niches.
The normal flora prevent colonization by pathogens by
competing for attachment sites or for essential nutrients.
This is thought to be their most important beneficial effect,

2. They produce vitamin B and vitamin K in

intestine. which can be absorbed as nutrients
by the host. For example, enteric bacteria
secrete Vitamin K and Vitamin B12, and lactic
acid bacteria produce certain B-vitamins.
 Importance of The Normal Flora
(Advantages)
3. The oral flora contribute to immunity
by inducing low levels of circulating
and secretory antibodies that may
cross react with pathogens.
4. The oral bacteria flora exert microbial
antagonism against nonindigenous species by
production of inhibitory fatty acids,
peroxides, bacteriocins, etc. which inhibit or
kill other bacteria.
5. The normal flora of
intestine - helps in
digestion of food
 Importance of The Normal Flora
(Disadvantages)
1. They can cause disease in the following:
a) When individuals become
immunocompromised or debilitated.
b) When they change their usual anatomic
location.
2. The oral flora of humans may harm their host
since some of these bacteria are pathogens or
opportunistic pathogens
 Normal flora - Risks

• Dental plaque • Inflammatory bowel

• Dental caries: disease
destruction of enamel, • Obesity
dentin or cementum of
teeth
• Periodontal disease
 Opportunistic flora
• Some normal flora become opportunistic
pathogens
• (Staphylococcus aureus, Streptococcus mutans,
Enterococcus faecalis, Streptococcus
pneumoniae, Pseudomonas aeruginosa, etc.)
• Breach of skin/mucosal barrier: trauma,
surgery, burns
• Bacterium at one site may be commensal,
but might be pathogenic at another site
• Growth of commensals may put patient
at risk
– Broad-spectrum antibiotic therapy
decreases total number of bacterial in gut
• During repopulation, faster-growing
aerobic Enterobacteriaceae over
slower-replicating anaerobes increases
probability of gram-negative bacteremia
 Probiotics/Prebiotics

• Probiotic
– Oral administration of living organisms to promote health
– Mechanism speculative: competition with other bacteria;
stimulation of nonspecific immunity
– Species specific: adherence and growth (tropism)

• Prebiotic
– Non-digestible food that stimulates growth or activity of GI
microbiota, especially bifidobacteria and lactobacillus bacteria
(both of which are noninflammatory)
– Typically a carbohydrate: soluble fiber
 Rumen microbes

❖ A ruminant is any hooved animal that digests its

food in two steps-
a) By eating the raw material and regurgitating a
semi digested form known as cud
b) then eating the cud, a process called ruminating
❖ Ruminants share another common feature that
they all have an even number of toes.
❖ Examples are: cattle, goat, sheep, camel, giraffe,
buffalo and dear etc.
 Fermentation in Ruminants
• Rumen is a fermentation chamber filled with
microorganisms.
• Anaerobic process-thus host can absorb energetic
by-products from bacteria fermentation.
• Utilizes enzymes produced by rumen microorganisms
to digest the ingested material .
• Benefits two distinguished groups: host (ruminant)
and the microorganisms.
 Rumen Microbes
• Protozoa
– Large (20-200 microns) unicellular organisms
– Ingest feed particles
– Engulf feed particles and digest carbohydrates, proteins
and fats Entodinium (Rumen Protozoa)

– Numbers affected by diet

•Fungi
•Numbers usually low
•Digest recalcitrant fiber
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Bacterial Populations
Cellulolytic bacteria (fiber digesters)
– digest cellulose
– require pH 6-7
– utilize N in form of NH3
– require S for synthesis of sulfur-containing amino acids (cysteine and
methionine)
– produce acetate, propionate, little butyrate, CO2
– predominate from roughage diets
• Amylolytic bacteria (starch, sugar digesters)
– digest starch
– require pH 5-6
– utilize N as NH3 or peptides
– produce propionate, butyrate and lactate
- predominate from grain diets
• Methane-producing bacteria
– produce methane (CH4)
– utilized by microbes for energy
– represent loss of energy to animal
– released by eructation