APPLICATIONS OF MICROBIOLOGY

⁕ Soil Microbiology - Microbial Interactions, Biogeochemical roles of Microbes.

⁕ Aquatic Microbiology - Waste water treatment.

⁕ Agricultural Microbiology - Biofertilizers.

⁕ Environmental Microbiology - Bioremediation, Bioplastics, Biopolymers.

⁕ Industrial Microbiology - Microbial metabolites.

⁕ Medical Microbiology - Antibiotics and Vaccines

 Soil Microbiology
Microbial Interactions, Biogeochemical roles of Microbes.

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
Mechanisms of plant growth promotion by rhizobacteria
(PGPR)

Direct Indirect
(Production of plant growth (Production of metabolites like
promoting substances like Auxins, Antibiotic, Siderophores, HCN etc.)
Gibberellins, Cytokinins etc.)
❖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 Main types of soil microorganisms
attached to the soil Agrobacterium Alcaligenes
particles. 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

26
 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 (NO2-) 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) (S 0 → H2S)
Desulfuromonas, many
hyperthermophilic Archaea
Sulfur disproportionation(S2 O32- → 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

mining FERTILIZER
excretion GUANO
agriculture
weathering
uptake by uptake by
autotrophs autotrophs

MARINE DISSOLVED leaching, runoff DISSOLVED IN LAND

FOOD IN OCEAN SOIL WATER, FOOD
WEBS WATER LAKES, RIVERS WEBS

death, death,
decomposition decomposition

sedimentation settling out weathering

uplifting over
geologic time
MARINE SEDIMENTS ROCKS

Figure 4-33
Page 82

Slide 41
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.
 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
Aerobic Biological Treatment Technologies
1) Activated Sludge Process (CASP) System
2) Facultative lagoons-Aerated lagoons (oxidation pond)
3) Trickling filter
Activated Sludge Process (ASP) System

❖ 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 O2 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.
Parameters Aerobic Anaerobic
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
Nitrogen fixation In leguminous plants

❖Biological nitrogen fixation (BNF) is the

process whereby atmospheric nitrogen 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!

Nitrogenase Complex N2
Electrons from Nitrogenase
reduced source Reductase Nitrogenase
(Fe-S protein) (Mo-Fe protein)
NH3
ATP ADP
The• Dinitrogenase
Nitrogenase Reductase
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• Dinitrogenase
Nitrogenase (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
legume

Fixed Fixed carbon

nitrogen (malate,
(ammonia) sucrose)
 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
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
 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)

99
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.
• T he 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

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
106
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 steriliz
ed 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.
 Removal of microbial cells

Stages in downstream processing

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
INDUSTRIAL PRODUCTION

• The industrial production of metabolite was broadly classified in to

two processes namely,

• Upstream processing
• Downstream processing
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 sites and cleave the DNA by Restriction
hydrolyzing the phosphodiester bond. enzyme

• 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)
 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 Mg 2+.
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
Biotechnological application-Fermentation
Miscellaneous products from
fermentation:
Biopesticides
enzymes
amino acids
organic acids
solvents
natural flavor compounds
Biogas/biofeul
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.
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. Rennin in the top test tube… not there in
 This saves time, money, space the bottom one.
and animals.
 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 β-carotene.
 This offers some promise in helping to correct a
worldwide Vitamin A deficiency.
PRODUCTION OF ANTIBIOTICS
Some Antibiotics produced by
Microorganisms
Antibiotic Producing microorganism
Cephalosporin Cephalosporium acrimonium
Chloramphenicol Streptomyces venezuelae
Erythromycin Streptomyces erythreus
Griseofulvin Penicillium griseofulvin
Penicillin Penicillium chrysogenum
Streptomycin Streptomyces griseus
Tetracycline Streptomyces aureofaciens
Gentamicin Micromonospora purpurea
Fermentation Process

Penicillin
 Vaccine
A vaccine is a biological preparation that provides active acquired immunity to a
particular infectious disease.

A vaccine typically contains an agent that resembles either:

(a) Weakened (attenuated) or killed forms of the disease-causing microorganism and is often made
from
(b) The toxins produced by the microbe
(c) One of the microbe’s surface proteins.
(d) The nucleic acid portion of the microbe that codes for toxins or surface proteins

The agent stimulates the body's immune system to recognize it as a threat, destroy it, and develop the
ability to further recognize and destroy any encounter of the same microorganisms in future.
Vaccines can be
Prophylactic (to prevent the effects of a future infection). Most of the vaccines that we
know of, falls into this category
or
Therapeutic (to fight a disease that has already occurred, such as cancer).

The administration of vaccines is called vaccination. Vaccination is the most effective

method of preventing the spread of infectious diseases.

widespread immunity due to vaccination is largely responsible for the worldwide

eradication of smallpox and the restriction of diseases such as polio, measles,
and tetanus from much of the world.