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Unit V:
Biogeochemical cycles (P, O, N- symbiotic and asymbiotic nitrogen fixation, sulphur and carbon
cycles). Symbiotic and asymbiotic associations, Biopesticides, Bioinsecticides.
EXPLAIN ABOUT BIOGEOCHEMICAL CYCLE: (Section C)
INTRODUCTION
Biogeochemical cycling of essential nutrient elements occurs both within
ecosystem and on a global basis. A chemical form of an element represents a reservoir. The
turnover of a reservoir depends on both the intensity of cycling and reservoir size.
Biogeochemical cycling describes the movement and conversion of materials by biochemical
activities throughout the atmosphere, hydrosphere and lithosphere. This cycling occurs on a
global scale, profoundly affecting the geology and present environment of our planet.
Biogeochemical cycles includes physical transformation such as dissolution,
precipitation, volatilization and fixation; chemical transformation such bio-synthesis, biodegradation and oxido- reductive biotransformation and various combinations of physical and
chemical changes. All living organisms participate in the biogeochemical cycling of materials
but microorganisms because of their ubiquity, metabolic capabilities and high enzymatic activity
rates, play a major role in biogeochemical cycling.
Most elements are subject to some degree of biogeochemical cycling. As may be
expected, elements that are essential components of living organism, the so-called biogenic
elements, are most regularly subject to biogeochemical cycling. The intensity (or) rate of
biogeochemical cycling for each element roughly correlates to the amount of the element in the
chemical composition of biomass. The major elemental components of living organisms (C, H,
O, P, N and S) are cycled most intensely.
Soil microorganisms serve as biogeochemical agents for the conversion of
complex organic compounds into simple inorganic compounds (or) into their constituent
elements. The overall process called Mineralization. This conversion of complex organic
compounds into inorganic compounds (or) elements provides for the continuity of elements as
nutrients for plants and animals including people.
CARBON CYCLE: (Section B)
INTRODUCTION: The maintenance of organic compounds on the soil depends on the
photosynthetic property of crop plant grown in the soil. The photosynthesis of carbon compounds
by green plants and bacteria helps in maintaining the balance. The organic substance synthesize
by them serve as source if energy and food material for other bidder and smaller forms of life.
CARBON CYCLE: The cycle of carbon in nature comprises the two main processes.
1. Conversion of oxidized form of carbon into reduced organic form by photosynthetic
organism: Carbon dioxide is reduced into organic compounds mainly by the process of
photosynthesis. Photosynthetic algae (or) the higher plants are the important agents of
fixing carbon-di-oxide. In the ocean the major plant forms that fix CO2 are the free
floating microscopic algae called phytoplankton. They are estimated to fix annually
1.2X1010 tons of carbon. Nearly 1.6 x1010 tons of carbon is said to be fixed annually by
photosynthetic terrestrial plant life. Besides autotrophic are heterotrophic bacteria are
also capable of synthesis organic matter from inorganic carbon, in addition to the
occurrence of photosynthesis among microorganism, the latter also represent the example
of CO2 fixation into organic compound which are as follows:
a. The CO2 represent the sole source of carbon for autotrophic bacteria. The latter
fix CO2 to carbohydrate by a reduction reaction.
1
CO2
+ 2H2

(CH2O)x + H2O
b. Heterotrophic bacteria fix CO2 commonly
CH3-CO-COOH +

HOOC-CH2-CO-COOH
2. Restoration of original oxidized
form (CO2) through mineralization of
organic form:
One can consider three
different modes through which the organic
matter is mineralized and the co2 is released
in the atmosphere.
(i) Process of respiration
(ii) Accidental (forest fire) and intentional
(fuel) burning
(iii) Decomposition of organic matter by
microorganism
The process of respiration in plants
and animals and the accidental (or) intentional
burning of plants and their parts result in the
breakdown of organic carbon compounds
releasing co2 in the atmosphere.
DECOMPOSITION OF ORGANIC MATTER BY MICROORGANISM:
The carbon cycle has 2 components that affect soil microbiology.
a. A slow cycle : in which carbon turnover is measured in 100s of 1000s of years and
involves rocj weathering and dissolution of carbonated on land and in oceans.
b. A fast cycle: in which carbon turnover is measured in years (or) decades and is
principally biological in nature. The fast cycle most directly affecting and most
affected by soil microorganism.
Most organic carbon in soil comes from soil. This carbon represents the residue of plants
on the soil surface and organic carbon coming from the decomposition of roots in soil. Plant
carbon can be roughly characterized as follows:
1. Carbohydrates: (30% to 75% of dry weight)
a. Cellulose
- 15% to 60%
b. Hemi cellulose
- 10% to 30%
c. Sugar and starch - 1% to 5%
2. Lignin: 10% to 30% of dry weight
3. Pectin: 1%
4. Waxes and pigments
5. Other 5% to 20%
a. Fats, Oils, Organic acids, Hydrocarbon
The values varies because as plants age. Cellulose, a hemi cellulose and lignin content
increases while the simple sugars, amino acids, proteins, fats and oils decreased.
Cellulose Decomposition: Cellulose is the most abundant organic material in plants. It is
readily attached by many species of fungi along with bacteria. The process of cellulose
decomposition to CO2 can be summarised in the form of following reaction.
Cellulose
Cellobiose
CELLULASE
Cellobiose
Glucose
2
 - GLUCOSIDASE
Glucose
end products
ENZYME SYSTEM OF
CO2
+
H2 O
+ and / or other
MANY MICROBES
The fungi which decompose cellulose in soil are
Aspergillus, Penicillum, Fuzarium,
Trichoderma, Verticillium etc.,
The bacteria which decompose cellulose are Bacillus, Achromobacter, Vibrio, Cellulomonas,
Clostridium, Streptomyces, Pseudomonas etc.
Hemicellulose Decomposition: Hemicellulose is a water soluble polysaccharide and consist of
hexose, pentose and uronic acid. Glucose, Galactose, Mannose, Xylose, arabinose, Gluconic
acid, Galactoronic acid are commonly found in the hemic-cellulose of plants.
The fungi which degrade hemi-cellulose are Aspergillus
Trichoderma,
Fuzarium, Penicillum, Chetomium etc.,
The bacteria which degrade hemi-cellulose are Bacillus,
Pseudomonas
Streptomyces, Cytophaga, Actinomycetes etc.,
Lignin Decomposition: Microbial decomposition of lignin as attracted many investigations.
Lignin is one of the most resident organic substances for the microorganism to degrade. Many
basidiomycetes have been found to be possess special capacity in thuis regard. Only rarely have
bacteria been found to reduce lignin. The fungi are Polyporus, Agaricus, Fomes, Armillaria etc.,
PHOSPHORUS CYCLE: (Section B)
Phosphorus 2nd only to nitrogen as an inorganic nutrient needed by plants and
microorganisms. It is an essential component of RNA, DNA, ATP and phospholipids.
Phosphorus is not abundant component of the environment and long term cultivation without
fertilization depletes soil phosphorus, whereas in fresh water, lakes and streams phosphorus is
probable most nutrient for the plant and microbial growth. Most phosphorus is in rocks and soil
and its gets into the sea, consequently, the largest reservoir of phosphorus in ocean is sediment.
Phosphorus is present in the terrestrial environment in several forms and in several major pools.
That can categorized as
1.
Absorbed phosphorus:
This form of phosphorus is the anion
3orthophosphates – Po4 , although at the pH of most soils sit is found as mono and di
basic phosphorus H2 Po4- and H Po4-. Orthophosphates precipitates with the Ca2+,
Mg2+, Fe2+ at neutral and alkaline pH. At high pH it ones more begins to become
available because it is associated with Na ions.
2.
Organic phosphorus: Much of the organic phosphorus in soil is unidentified
forms. The most common identified form is inositol phosphate. Phytin is inositol hexa
phosphate, one of the most common forms of plant producing organic phosphorus.
Most inositol phosphate is found in forest soils than in grass land soils. Since inositol
phosphate is not typically of microbial origin, this probably reflects the different storage
forms of organic phosphates that occur in vegetation.
3.
Mineral phosphorus: These are over 200 mineral forms of phosphorus in
soil. Some of the most common mineral forms are the apatits which have the general
formula: M10 (Po4)6X2
Where M = Ca and Mg ; X = Flurides , cl, oH, Co3 2Example: Ca10 (Po4)6F2
3
PHOSPHORUS CYCLE: Microbial cycling of phosphorus involves transforming phosphorus
between inorganic and organic pools and insoluble and soluble forms. The amount of dissolved
phosphorus in soil at anytime varies between 0.1 to 1 kg / hec.
Solubilization: Bacteria that actively solublise phosphorus represent about 10% of the
soil microbial population. They are primarily Rhizosphere organisms such as Bacillus,
Micrococcus, Mycobacterium, Pseudomonas and some fungi Aspergillus, Penicillium etc.,
They are 3 basic mechanism of solubilizing mineral phosphorus and making it more available.
1.
Chaelation: Organic compounds made by micro organism such as oxalic acid,
citric acid, lactic acid Fumaric acid, Succinic acid, Glutamic acid can chaelate Ca2+,
Mg2+ and Fe2+ thus destbilizaing the phosphate mineral and making phosphorus soluble.
2.
Iron reduction : Ferrous ion is more soluble than ferric ion anaerobically by
example
H2S + Fe (Po4)2
FeS + 2H Po4 2Available phosphate is greater in air dried soils, compared tp water logger soil. The
reason the phosphorus become more available when the soil is water logged is because the Fe
becomes reduced and destabilizes the mineral phosphorus.
3.
Acidification: Acid production by microorganism dissolves minerals. Thus,
organic acids, nitric acids (produced by nitrifiers), sulphuric acid (produced by
Thiobacillus) and carbonic acid (H2CO3) all release phosphorus from mineral forms.
S0
H2SO4
H2SO4 + Ca3 (Po4 )2
H2 Po4 Immobilization: The concentration of phosphorus in soil solution is typically 0.1 to 1
ppm. Microbial concentration of phosphorus is 10 times higher than in plants. At low
phosphorus concentration microorganism accumulate phosphorus from inorganic (or) organic
sources at the expense of plants. Phosphorus gets into the cell after conversion to phosphate ester
(or) ATP and is stores as polyphosphates. Immobilization depends on the growth demands of the
microorganism and the proportion of P in organic compounds. P equal to 0.3% of the weight of
an organic compound is required for the microbial community to develop its full extent.
Mineralization: The microorganism breakdown the phosphorus containing compounds
with the liberation of mineral elements such s Ca, Fe and Na and this process is known as
mineralization. Organic phosphorus which makeup 30% to 50% of the total phosphorus in soil,
must be mineralized before it is available. Phospholipids and nucleic acid degrade rapidly but
inositol phosphate is slowly mineralized. Mineralization is favored by thermophilic temperature,
neutral to alkaline pH and organic matter that is rich in phosphorus.
SOIL MANAGEMENT
Phosphorus concentration and mineralization are affected by soil management.
Phosphorus is relatively immobile; it is readily precipitates. Consequently, it will stratify in
surface horizons where the soil is not cultivated. Inoculation of organic compost with phosphates
solubilizing Bacillus, Polymyxa plus nitrifying
Azotobacter chrococcum help in improving the quality of manure by reducing the C:N ration
from 15 to 12
and substantially improving the available phosphates.
4
SULPHUR CYCLE: (Section B)
INTRODUCTION:
The sulphur cycle was 1st discovered by Martineus Beijerink and Serge
Winograsky in the late 80s. Sulphur is present in traces in the air and it makes up about 0.1% of
the earth crust. In the industrial areas, the atmosphere contains high concentration of sulphur
because of the burning of the coal. The Sulphur in the air may reach the soil through rain water.
Most well and river water contain traces of sulphur. Sulphur and H2S are abundantly emitted
from volcano.
Plant utilize sulphur in the dissolved form Sulphate. It requirement varies
with plant species. Generally, cruciferous plants require upto 40kg of sulphur / hec, where as
cereals take less than 10kg / hec. Sulphur is present as sulphates, parts of aminoacids like
methionine, cysteine and in some substances like thiamine, biotin and Glutathione (non-protein
aminoacid).
Sulphur
Volcanoes
H2S
Bacterial reaction
Plant protein
Microbial
degradation
SO2
Animal protein
Organics of protein (waste)
SULPHUR TRANSFORMATION:
1. Inorganic transformation
Anaerobic reductive environment
5
Sulphates
Absorption by plant.
Building up of protein
SO42-
S2Anaerobic oxidizing environment
2. Photosynthetic transformation
Anaerobic, light environment
H2S + CO2
3. Organic transformation
S0
+
(CH2O) n
Aerobic and anaerobic environment
SO42R – OS + R-SH
4. Mineralization
R – OS
H2 S + R
R - SH
SO42- + R
The bacteria capable of oxidizing inorganic sulphur compound could either be aerobic
(or) anaerobic. Their morphology varies from filamentous forms(Beggiatoa), (Thiothrix) to nonfilamentous form (Thiobacilllus). Some fungi and Actinomycetes have also been reported to be
sulphur oxidize (Aspergillus, Penicillium etc.,) Sulphur reducing bacteri (Desulfovibrio
desulfuricans) which reduce inorganic Sulphate into
H 2S, have a role in completing
the sulphur cycle in nature.
Entry of Sulphur into the soil: Sulphur enter into the soil as organic compounds, in
plant and animal residues as elemental sulphur, as hydrogen sulphide through rain water (or) as
sulphur, Sulphate etc., in the fertilizers. The complex sulphur containing organic compounds
reaching the soil are attacked by several soil dwellers. The proteins are decomposed to amino
acids and amino acids breakdown to liberate H2S which is oxidized to Sulphate under aerobic
condition. Under anaerobic condition some compounds like mercaptons may be evolved. Under
aerobic condition the mercaptons are also oxidize to Sulphate.
Oxidation: H2S, when washed into soil, is utilized by the characteristic autotraphs known as
Sulfobacteria of the genus Thiobacillus and other forms as well T. thioparus ; T. thioxidans and
T. denitrificans are strict chemosynthetic autotrophs, utilizing the energy liberated in the
oxidation of sulphur.
S2 + O2 + 2H2O
2H2SO4
2H2S + O2
S2 + 2H2O
Thus, sulphur and H2S are oxidized to yield sulphuric acid which react with calcium
phosphate and other substances.
Reduction: Sometime Sulphate reduce to sulphite by the Sulphate reducing bacteria like
Microspica desulphuricans and Sporovibrio desulphuricans.
The reaction process is particularly favored by the alkaline and anaerobic conditions of
soil, as the organism use Sulphate as the source of O2. In this process, H2S is released which
gives the characteristic oduor. Besides the strict autotrophs of the genus Thiobacillus, there are
several other members of facultative autotroph capable of reducing H2S and drive energy from it.
Ex: Acromobacteriaceae, Chlorobacteriaceae, Thiobacteriaceae. These
organisms utilize H2S as a hydrogen donor for the reduction of CO2. They are found in sulphur
springs, sewage, stagnet water etc., They may also present in water pipes and cause serious
offensive odor and obstruction to the flow. Sulphate reaction in sewage sysyem leads to the
formation of sulphides. That are corrosive, toxin and smelly. One solution to add NO3 2- to the
system (nitrate) because nitrate is more readily available electron acceptor than SO42- .
The Sulphate and sulphuric acid when dissolved in water are more available for plant
growth. The plant utilize the Sulphate to form various amino acids, growth factor etc., They are
either taken by the animal (or) returned to the soil as organic waste. When the various complex
organic sulfate compounds reach the soil they are attacked by soil micro organism and the cycle
continues.
6
Acid Rain:
Once the atmosphere, H2S and CH3SH (methyl sulfide) are oxidized to SO2 and
precipitated to inorganic sulphur. Perhaps, half of the sulphur in the atmosphere in industrial and
this has lead to some serious environmental condition. Burning of coal produces H2SO2 which
eventually oxidized to H2SO3 (sulfurous acid). As a result of sulfur in atmosphere and acid amog
the building decay will occur. The effect of this is acid rain. Acid rain is important where soils
are not buffered but just 1.5 ppm SO2 lower the pH to 4. Lichens are extremely sensitive to SO2
and consequently make good bio-indicator of SO2 pollution.
NITROGEN CYCLE: (Section B)
Introduction: For agricultural N2, P and K constitute major nutrients from soil. Among them
nitrogen is more susceptible to microbial transformation. The transformation of nitrogen involves
inorganic, organic and volatile compounds and the direction of transformation depends in
microorganism. Small portion of nitrogen in atmosphere is converted to organic compounds by
certain free living (or) symbiotic microorganism. Nitrogen in proteins and nucleic acids, in plant
tissues is consumed by animals where it is converted to simple and complex compound.
When animals and plants, decay microbiologically, the organic nitrogen is
released as ammonium which is utilized by vegetation (or) is oxidized to nitrate. Nitrate may
also be used by plants (or) may be reduced to ammonium (or) gaseous nitrogen which is escapes
to atmosphere.
Like this terrestrial nitrogen cycle, a cycle operates in aquatic environment also.
Atmospheric nitrogen
Nitrogen fixation
Denitrification
Symbiotic
Non-symbiotic
(Rhizobium)
(Anabaena,
Azotobacter)
Plant organic Nitrogen
Animal Organic N2
Dead organic matter in
soil
In cells of micro
organism
Nitrate
Nitrification
Nitrobacter
Denitrification
Nitrite
Nitrification
Ammonia
Nitrosomonas 7
Ammonification
Amino acids
Proteolysis (or) Decomposition of proteins by microorganism: Proteins consist of amino acid
units linked together by the peptide linkage. They are invariably of L-optical form and their
molecular weight may be as high as 1, 00,000. The structure of amino acid molecule varies from
very simple form to highly complicated molecules. They may combine to form small units
known as peptide. Many peptide units join to form protein. These are large and vary in different
organic tissues.
The proteins are broken down by the microorganism with the help of proteolytic
enzymes. The broken down of protein accomplished into 2 stages:
Proteins
PROTEINASE
Polypeptide
PEPTIDASE
Amino acid
The production of proteinases by microorganism depends on several factors. It
seems possible that when protein is added to the soil, the fungi first attack the molecule and
reduce it to polypeptide and then bacteria become active and competitive with fungi in further
breaking down of polypeptides to amino acids. This breakdown is accomplished by a number of
ways by microorganism but in most cases the product is ammonia. In this respect, bacteria are
relatively more active than other organism.
Spore forming bacteria, Pseudomonas,
Actinomycetes, Fungi seems to readily attack amino acids in soil.
Ex: Bacteria: Pseudomonas, Bacillus, Clostridium, Serretia and Micrococcus
Ex: Fungi: Alternaria, Aspergillus, Mucor, Penicillium, Rhizopus
However, in acid soil, fungi are more important agents of ammonification
than bacteria.
Amino acids are broken down by oxidative deamination by aerobic microorganism.
COOH
|
CH2
COOH
|
|
CH2
+
O2
CH2
+ NH3
+ CO2
||
|
CH – NH2
CH2
|
|
COOH
COOH
Glutamic acid
Succinic acid
Succinic acid may be made available for plant growth and CO2 released to
the soil atmosphere and finally to the atmosphere above soil. Bacteria breakdown complex and
in this process ammonia is released.
Amino acids may be broken down by hydrolytic deamination.
R.CH.NH2.COOH + H2O
RCHOH.COOH
+
NH3
R.CH.NH2.COOH
+H2O
RCH2OH +
CO2 +
NH3
R.CH.NH2.COOH
+H2O
RCHO
+
HCOOH
+
NH3
Thus depending on the enzyme system involved in the hydrolytic process,
we may obtain organic acids, alcohols, aldehyde co2 etc., besides NH3.
The anaerobic bacteria may also produce ammonia - desaturative deamination
CH3.CH2.CHNH2.COOH
CH3.CH=CH.COOH
8
+ NH3
Several fungi are capable of producing alcohol and ammonia form amino
acids. Thus, the amino acids are broken down to gives rise to NH3, CO2 and the variety of
substances, lower organic acids, aldehyde, and ketone.
Factors influence the Ammonification:
 Well aerated and drained soil
 High amount of organic matter, rich in protein
Factors affecting the Ammonification:
 Acid soil - Fungi are more active than bacteria, but the general
microbial activity is relatively less and hence there is less of NH3
production.
 If there is ready supply of energy rich carbohydrates, the
microorganisms do not attack the complex N2 compounds.
Other steps in Ammonification:
1. Besides proteins, other N2 containing organic substances are also attack by microorganism to
yield NH3. Many organisms utilize urea to liberate NH3 by urease enzyme production.
NH2-CO-NH2
+ H2O
+
2H2O
UREASE
(NH4)2CO3
UREASE
CO2 + 2NH3
Microorganis involved: Bacillus, Proteus, Micrococcus, Sarcina, Aerobacter etc.,
2. Few bacteria are known to utilize amines as carbon and nitrogen source for their growth.
Ex: Mycobacterium, Protoaminobacter, Pseudomonas etc., are known to possess capacity to
utilize methylamine, ethylamine, prophylamine etc., these are the byproducts of microbial
breakdown of proteins. The enzyme system involved in this process is amino oxidase.
NH2.RCH2NH2
NH2.RCH=NH
NH2RCH=NH
NH2.R.CHO + NH3
NH2.R.COOH
CO2 + NH3
NH2.R.CH2OH
CH3.CHO + NH3
3. Plants and animal residues contain nucleic acids in small proportion, when they reach soil,
they also can attacked by soil microorganism and are broke down into urea, ammonia, CO2 and
some organic acids.
Nitrification: S. Winograsky (1888 to 1891) brought out a important characteristic of bacteria
namely their capacity to oxidize NH3 to obtain energy which is similar to photosynthesis in
plants. Based on this principle, here isolated the bacterium which oxidize NH3 to nitrite and
called it is Nitrosomonas. Another bacteria convert nitrite to nitrate and named it as Nitrobacter.
The nitrification process-taking place in soil through microorganism is indicated by the following
chemical reaction.
(NH3)2 + O3
NH4NO2 + H2O2 + 4O2
NH4NO2
+ H2O2
NH4NO3
+H2O
The chemical reaction of the biological oxidation is
NH2
+ ½ O2
HO-NH2
HYDROXYLAMINE
9
½ HO-N
||
N
HO-N=O
NITRITE
OH
HO-N=O
H2O
OH
H2-
HO - N
OH
O
HO - N
O
NITRATE
Factors affecting nitrification
1. Ammonia is believed to be converted to nitrate in the presence
of catalyst at high temperature.
2. Application of nitrate fertilizers reduces their activity but
phosphate and other mineral enhance the activity.
3. Well aerated soil, a temperature below 300C, nearly neutral pH,
absence of large quantities of organic matter – favors
nitrification.
DENITRIFICATION:
This is the reverse process of nitrification that is nitrate is reduced to nitrite and
than to N2 gar and NH3. Denitrification process favored in anaerobic soil and there is
considerable decomposition (or) petrifaction of organic matter takes place. Anaerobic bacteria
like Clostridium may reduce nitrate to NH3 directly.
HNO3 + 4H2
NH3 + 3H2O
HCOOH + HNO3
CO2 + HNO2 + H2O
2HNO3
2HNO2 + O2
2HNO2
NH2 + 1 ½ O2 + H2O
HNO3 + H2
NH3 + 2O2
Some aerobic organisms such as Pseudomonas denitrificans also seems to
reduce nitrate under certain condition. Others include Thiobacillus, Serretia, Corynebacterium.
The presence of large quantities of organic matter is known to encourage the liberation of N2 into
the atmosphere.
Factors influencing Denitrification:
1. Addition of carbonaceous material
2. Temperature 25 C optimum temperature and above up to 600C to 650C
3. Addition of sulphur to soil stimulates the population of Thiobacillus
denitrificans.
Biological nitrogen fixation:
Introduction: The phenomenon of fixation of atmospheric N2 by biological means is known as
diazotroph (or) biological nitrogen fixation and these prokaryotes as diazotrophes (or) nitrogen
fixers. The diazotrophes may be free living (or) symbiotic forms. Biological nitrogen fixation is
a reductive process; the nitrogenase system transfers a total 8(H) to N2 and release 2(H) as
molecular H2. Ammonia is the first identifiable product of this enzyme reaction:
N=N + 8(H)
HN=NH
H2N-NH2
2NH3 +
H2
DIIMIDE
HYDROZINE
(INTERMEDIATE PRODUCT)
NITROGEN FIXATION BY SYMBIOTIC BACTERIA: (Section B)
There are some microorganisms which establish symbiotic relationship with different
plants and may develop (or) may not develop special structure as the site of N2 fixation.
10
MICROORGANISM
SYMBIOTIC STRUCTURE
Bacteria
No special structure develops
intimately associated with roots.
Azotobacter paspali
Azospirillum amazonense
Rhizobium sp.,
Beijerinkia sp.
Derxia sp.,
Frankia sp., (Actinomycetes)
No special stricture develops
Develop root nodule
No special structure develops
No special structure develops
Develop root nodule
Cyanobacteria
Anabaena, Nostoc
Anabaena azollae
Lichens
No special structure
Root nodule formation: Establishment of Rhizobium inside the host root and development of
nodules are complex process are follow many events such as recognition and infection of host
root, differentiation of nodules, proliferation of bacteria and conversion into bacteriods in
nodules.
Stages in root nodule formation:
1. The bacteria enter into young root hairs form the soil. Initial contact between the partners
is followed by recognition.
2.
3.
4.
5.
6.
Leguminous plants contain lectin.
These are glycoprotein that binds specific
polysaccharides. Lectins are ubiquitously distributed in nature and probable have general
recognition function. The interaction between the lectins on the outer wall of the young
root hairs and the polysaccharides on the outer cell wall of Rhizobium have been
investigates in the case of R. trifolium infecting white clover.
Binding occurs only between compatible partners; not Rhizobium can bind any
leguminous plants (or) vice versa.
When binding has occurred, the tip of root hairs bends and the bacteria penetrate and
from in the form of an infection thread (or) tube which is surrounded by the root cells
with a cellulose membrane towards the base that is upwards and infects further cell of the
root epidermis.
Growth hormones are produced and the root epidermal cells undergo multiplication. The
root nodule is the result of this tissue proliferation induced by the Rhizobia via growth
promoters probably cytokines. The rapidly dividing bacteria grow in the deformed cells the bacteriods which can have more than 10 times the volume of the Rhizobium. The
bacteriods are singly (or) in groups, surrounded by a peri bacteriod membrane, inhabit the
cytoplasm of the plant cells. The tissues containing the bacteriods are red because it
contains leghaemoglobin.
The nodules turned green during aging due to the breakdown of the leg hemoglobin to
green bile pigments (biliverdin). When the nodule dies, stationary phase Rhizobia, which
are still present in considerable number, are released and can multiply by using the
degradation product of nodules as substrate.
11
Function of Bacteriods:





The Bacteriods fix nitrogen
During the nitrogen fixing phase, they are supplied with C4 –
dicarboxylic acid by the plant cell.
In contrast to the free living Rhizobia, the bacteriods unable to
utilize sugars.
The secrete NH3 ions which are apparently incorporate into
organic compounds by glutamine synthatase present in the surrounding plant cell.
The relationship between the plant and Rhizobium is there
mutual symbiosis.
Function of Leghaemoglobin:

The formation of Leghaemoglobin is the specific effect of the
symbiosis. The prosthetic group proto-haeme is synthesized by bacteriods, while the
synthesis of protein part involves the plant cell.

Leghaemoglobin resembles myoglobin it is present in the
nodules predominantly in the FeII oxy form and has a very high affinity for oxygen.
12

The pigment is localized in the cytoplasm of plant cell and not
on the peribacteriod space. It is assume that the Leghaemoglobin facilitate the
transport of oxygen, through the plant cell to the bacteriod. Therefore that increases
the rate of oxygen transport. The presence of Leghaemoglobin seems to provide full
protection against oxygen damage for the nitrogen fixing enzymes.
Nitrogen Fixers:
The bacteria responsible for the formation of root nodules of leguminous plants belong to
the Genus Rhizobium. They occur as free-living, strictly aerobic, G-ve rods in soil and grow on
organic nutrients.
There are some strains (Bradyrhizobium), which are able to grow
autotrophically with hydrogen. Three groups can be distinguished according to their host
specificity and their growth, which have been given a number of generic names. Sub-groups of
leguminous nodule forming bacteria:
Genus and Species
Rhizobium leguminosarum
Rhizobium melitoli
Rhizobium trifolii
Rhizobium phaseoli
Rhizobuim lupine
Bradyrhizobium japnicum
A. caulinodmas
Host
Peas
Lucerne
Colver
Beans
Lupins
Soy beans
sesbania
Group I – Rhizobium includes the fastest growing nodule bacteria of the indigenous cultivated
bacteria.
Group II – Bradyrhizobium japonicum is the group of slow growing symbiont of soybeans.
Group III – Azorhizobium caulinodens is the bacterium that forms stem nodules.
Hydrogenase: Many diazotorphes evolved h2 during N2 fixation which in turn inhibits N2
fixation. To get protection from inhibition by hydrogen many diazotrophs possess an enzyme
hydrogenase to recycle H2 produced by nitrogenase.
Reuse of H2 by Hydrogenase
N2
NITROGENASE
NH3
H2
HYDROGENASE
Reutilization of hydrogen produces more ATP and improves the efficiency of N2
fixation. The hydrogenase of purple bacteria is membrane bound (or) membrane associated. It is
cold liable and very sensitive to O2. The purified enzyme with a molecular weight of about 6500
and most probably consist of single polypeptide chain with 4 iron atoms and 4 acid labile sulfur
atoms / molecules.
N2 fixation in aerobic respiring cell: All the bacteria possess a membrane bound hydrogenase,
indicated that H2 might have a protective function. Therefore that it might be a protective gas for
13
the O2 sensitive nitrogenase (detoxifying). Hydrogenase combines to H2 and reduces into
members of a substrate (SH2) to form (or) liberate H2 from the reduced compounds as below:
H2 + S
SH2
H2 production and N2 fixation has shown a close relationship were both
the processes are catalyses by the same enzyme (or) enzyme complex.
Ex: Azotobacter venilandii
Nitrogenase: Nitrogenase has been isolated from the following genera of free living nitrogen
fixing microorganism. Clostridium, Bacillus, Anabaena, Azotobacter. All the diazotrophs
possess an enzyme nitrogenase which helps to conversion of N2 to NH3.
Structure of Nitrogenase: It consist of 2 brown metalo proteins of which joined action is
essential for reduction of N2 to NH3. Component I: Mo-Fe protein which is also known as
nitrogenase has a molecular weight of about 2.2x105 Dolton. Nitrogenase contains molybdenum
( 2 atoms / molecule), iron (32 atom / molecule). It is a larger unit than component II and nonsensitive to cold but loose the activity at 00C.
Component II: On the other hand, nitrogenase reductase is a smaller unit. It contains Ferro –
Protein ( Fe-Protein) and has a molecular weight of about 5 X 10 4 Dolton, It contains iron ( 4
atom / molecule) and Sulfur ( 4 atom / molecule) and is less stable than the component I.
Thus, nitrogenase is an equilibrium mixture of Mo – Fe protein and Fe protein in
the ratio 1:2
Mo – Fe protein
+ 2(Fe – Protein)
Nitrogenase complex
+
The specific need of these components for the organisms is probably due to the presence
of the elements in 2 protein components of nitrogenase. All diazotrophs contain similar protein
and suggesting there by a similar non-identical sequence. This is why the molecular weight of
these 2 components in different microbial cells differs. In addition to nitrogenase the N2
reducing system requires Mg-ATP as a source of energy and a reductant, (e- donor) to catalyze
the reduction of substrates. Here, ATP function as carrier of energy.
N2 fixation by nitrogenase complex:
Energy released in metabolic oxidation of carbohydrates is utilized in
phosphorylation of ADP in the presence of inorganic phosphate (Pi) to reduce the energy ATP
rich. Whenever energy is needed ATP undergoes enzymatic hydrolyses to form ADP + Pi.
Mg++ functions as catalyst.
N2 + Nitrogenase complex
2NH3
2 (Fe + Protein). Mo.Fe.Protein
N2
ATP
Mg++
Ferrodoxins
6H
2(Fe + Protein). Mo.Fe.Protein
14
+
NH3
Biochemistry of N2 fixation :
1. A characteristic feature of the healthy nodule of leguminous plants is the presence of a
special red pigment like Hemoglobin known as Leghaemoglobin (LHb).
2. LHb has character similar to myoglobin (or) varieties of hemoglobin found in animal.
3. It is red in color due to presence of Fe. Fro the first time LHb was isolated and crystallized
from soybean root nodule.
LHb is found only in healthy nodules. The unhealthy plants (or) white nodules do not develop
LHb ; therefore N2 fixation does not takes place in such nodules. LHb is present outside the
bacterial membrane (peri bacterial space). But in their close contact LHb regulates O2
concentration as bacteriods are aerobic and consume O2. LHb is found only in root nodule of
legumes. It is not found in Actinorrhyzic nodules – nodules formed by Frankia in roots of Nonleguminous plants. Therefore, presence of O2 buffering has not been reported so for, in
Actinorrhyzic nodules.
LHb facilitates is mediated by an enzyme system, the nitrogenase system, which has two
components: Nitrogenase and nitrogen reductase. Both these associated components are located
in the cytoplasm and are extremely sensitive to O2. Both the metalo proteins, nitrogenase
(Mo.Fe-protein) and nitrogenous reductase
( Fe protein) are essential for nitrogenase activity.
Fe protein interacts which ATP and MG2+ and Mo-Fe-protein catalyses the reduction of N2 to
NH3, H+ to H2 and Acetylene to ethylene.
The reduced ferrodoxin (or) Flavodoxin serves as a source of reduced for e- transfer
during N2 fixation. From reduced form of ferrodoxin (Fe-red) E-s flow to Fe protein which
reduced to Mo-Fe-protein with subsequent release of inorganic phosphate. This enzyme complex
gets energy from Mg-ATP which in turn is produced after bacterial respiration through
carbohydrate synthesis.
Finally, Mo-Fe-Protein passes on the electron to reducible substrate that N2 (or) other
substance like H2 and ethylene. The equation of N2 fixation in nodules of legumes may be
written as
Mg++
N2 + 16ATP + 8e- + 10H+
2NH4 + H2+ + 16ATP + 16 Pi
It is obvious that NH3 is the first stable product of N2 fixation. But it is not clear whether
neutral NH3 (or) cationic NH3 (NH4+) is formed. Son after function it is transferred through 3
layered bacterial membranes (2of bacteria and 3rd of host origin) to host cells, where it is
enzymatically converted into many products.
Free living nitrogen fixers: Nitrogen fixation in soil by free living microorganism is known for
over a century now. In 1855 J.B.Lawer and J.H. Gilbert found that all plants except legumes
need ammonia (or) nitrate as addition to the soil. M. Berthelot in 1988 observed that in
unsterilized soil there was a definite increase in N2 after some months, but not in sterile soil. In
1894 – 95 S. Winogradsky was the first to isolated into pure cultures and an anaerobic bacterium
which could grow in a N2 free medium and he named it is
Clostridium pasteurianum.
In 1901, Beijerink isolated two aerobic free living N2 fixing bacteria and named
them as Azotobacter chrococcum and Azotobacter agills. Inspired by these reports during the
next two (or) decades several workers demote their attention to the practical applicability of these
results, the prevalence of other such bacterial species and the populations in various soil types.
The free living bacterial having the ability to fix molecular N2 can be distinguished into obligate
aerobic, facultative aerobic, anaerobic organism.
Obligate aerobic bacteria belong to the genus Azotobacter, Beijerinkia, Derxia,
Achromobacter, Mycobacterium, Arthrobacter and Bacillus. Among the facultative anaerobic
bacteria are the genera Aerobacter, Klebsiella and Pseudomonas. Anaerobic N2 fixing bacteria
15
are represent by the genera Clostridium, Chlorobium, Phromatium, Rhodomicrobium,
Rhodopseudomonas, Rhodospirillum, Desulfovibrio and Methanobacterium. In some of these
genera N2 fixation takes place in a photo autotrophic manner by virtue of the presence in them of
photosynthetic pigments. Ex : Rhodopseudomonas. On the other hand, the genus Desulfovibrio
fixes N2 in the presence of reducing sulphates.
Detail account on Microbial interactions? (Section C)
Introduction:
Microrganisms have evolved over billions of years to adopt themselves to the presence
of other organisms living in the environment.
The association may be with plants, animals or other microbes. The term symbiosis [
Greek : sym – together , bios- life] is used to describe an intimate association between organisms
of different species.
The German botanist Heinrich, Anton, De Dary first used symbiosis in 1879 in
describing the close relationship that he observed between an algae and a fungus.
Later, it was observed as mutualism by the other scientists, the term symbiosis also
includes,
Parasitism: (Section A)
A relationship in which one organism, the parasite is benefited and the other organism
the host is harmed.
Commensalism: (Section A)
One organism is benefited and other is unaffected.
Categories of symbiosis:
Symbiosis may be divided into 2 groups based on the closeness of the association.
 Endosymbiosis
Here, the microorganisms grows within the host cell.
 Ectosymbiosis
The microorganisms may be attached to the host cells but it remains
outside.
Synergism:[ Protocooperation] (Section A)
Introduction:
A relationship between 2 microbial populations , where both the population are
beneficial, but unlike mutualism [ symbiosis]. It is not an obligatory relationship.
On synergism both the population remains in their natural habitat and carryout their
microbial activities. Sometimes synergism confuse since it sometimes resembles either
 Commensalism
 Neutralism
But anyhow speaking 2 microbial population combine together to do a work, which
increase the effectiveness than the individual performance.
Example:
Synthesis of a product that neither population would perform alone.
Ex: completion of a metabolic pathway by synergistic effect of both population that otherwise
could not be completed.
Ex: this term is applied to the interactions of two or more microbial populations that supply each
other nutritional needs.
Compound A
[ by population 1]
Compound B
16
[ by population 2]
Compound C
[ by population 1&2]
Energy + End product
Population 2 : cannot utilize compound A
Population 1: cannot go beyond compound B
This both population to combine compounds. The compound C is acted upon by the
population 1 and population 2 to produce needed energy and end products.
Ex:2
E.coli
Arginine
Strep.faecalis
Agmaline
E.coli
Ornithine
Putrescine
Neither organisms,alone convert arginine to putrescine. But if once putrescine is
produced both the E.coli and S.faecalis can use it.
Thus synergism occurs between these two organism.
Ex:3
Syntrophism also exists that based on the ability of one population to supply growth
factors for another population.
Ex:4
Cyclohexane
Nocardia
Release the end products
Supplied to
Biotin plus growth factors
Neither population alone can’t degrade cyclohexane.
Pseudomonas growth
EX:5
In the presence of light + hydrogen sulphide+ carbon di oxide
Cholorobium
Produces organic matter
Utilized Chlorobium
Produces hydrogen sulphide and CO2
17
Sulphur & Formate
Utilized by spirulina
Thus both the population are benefited . single organism alone can’t get their nutrition.
EX:6
Light
Chlorobium
Water and CO2
Organic carbon , sulphur
Desulfovibrio
Cycling of carbon and sulphur from oxidized to reduced state. Allow both organism to
metabolise vigorously in habitats, where as on their own they would be subjected to substrate
limitation and product inhibition.
Ex:7
Similarly synergisgtic relation ship occurs between bacteria and BGA.
Anabaena oscillatoroides [ heterocysts]
Secrete organic compounds
Attracted by heterotrophic Pseudomonas
Forms a dense aggregate around heterocysts
A part of Pseudomonas oxidize the excreted organic and also stimulate nitrogenase activity
Stimulation is due to lowering of oxygen concentration
Ex:8
Synergistic relationship between algae and epipohytic bacteria on cardon and oxygen
cycle. When grown individually both the population grows and maintain a constant level.
Paramecium caudatum
Paramecium aurelia
+ when grown
When both mixed at a particular habitat P. caudatum was overcome by P.Aurelia after 12
–16 days.
This competition does not involves the production of toxic substances P.Aurelia grows
fastly than p. caudatum and overcome P. caudatum
In contrast
P. caudatum
P. bursaria
occupies different regions of culture flask
18
When mixed population are grown in a single habitat, they grows well. Both the
population occupy different habitat in a particular niche [ occupy different region of the culture
flask].
Thus competition is minimized and prevents the extraction of one of the species.
Microbial population with highest intrinsic growth rate overcome the population with the lowest
intrinsic growth rate.
Intrinsic growth rate of the organism vary under different environmental conditions.
For example: in marinehabitats
Psychrophiles --- survive and exclude psychrotrophic under low temperature.
Psychrotrophic microorganisms – dominate under high temperature
Thus according to environmental conditions, microbial population are replaced by
another.
Continuous light + Chromatium vinosum
CV overcome CW
Chromatium weissei
Chromatium vinosum
Chromatium weissei
Intermittent
Illumination
Balanced growth appears
During continuous illumination C.vinosum grows well utilizing sulfide, C.weissei during
dark period, sulfide accumulates and upon illumination C.weissei oxidizes greater proportion of
the accumulated sulfide.
Thus the alteration of dark and light period balance the two population, allowing them to
co-exist. Similarly seasonal variations in environmental condition of the habitat led to temporal
oscillation in the success of displacement of competing population.
Studies with algae:
Asterionella formosa
Under phosphate limiting conditions
Cyclotella meneghiniana
Asterionella Formosa
During silicate limiting conditions
Cyclotella meneghiniana
Abiotic factors like temperature, pH and oxygen greatly influence the intrinsic growth
rate of microbial populations.
Ex:
Spirulina
E.coli
19
Dominate at low
Dominate at higher
substance concentrations
substance concentrations
Thus based in nutrient availability one population [ dominant ] replace the sensitive
population.
When sewage water mixes with fresh water microbial population of sewage was replaced
by the indigenous force of river ,since the organic matter is diluted in fresh water.
Competitive inhibition need not be rarely based on the ability to utilize a substance more
rapid but also tolerance to environmental stress conditions forms an important factor in
determining the outcome of competition.
For instance, under conditions of drought, population that are tolerant to dessication can
displace less tolerant populations. Similarly under environmental stress conditions like high
temperature, high salt concentrations, and the population with greatest tolerance to that factor
may succeed in the competition.
During growth conditions competitions advantage returns to the microbial population
with the high growth rate.
Ammensalism [Antagonism] : (Section A)
Introduction:
Microorganisms that produce chemical substances, which suppress or inhibit the growth
of other microorganism. This relation is called as Ammensalism or antagonism.
Fungistasis
Virucidal
Bactericidal
comes under ammensalism
Ammensalism helps one microbial population to colonize at particular habitat. Once it
colonize, it prevents other population from surviving and establishing in that habitat.
EX: 1
Production of lactic acid and Lactobacillus sp
EX:2
Anaerobic heterotrophic microbial population in human
Volatile fatty acids
Preclude the growth of E.coli
EX:3
Fatty acid production by normal microbial flora of the skin
Preclude the colonization of akin by other microbial population
EX:4
Acids in vaginal tract produced by normal microbial flora
Prevent the infection by pathogens such as Candida albicans
EX:5
20
Oxidation of sulfur by thiobacillus thioxidans
Produces sulfuric acids
Inhibits other bacterial population
EX:6
Sulfuric acid produced in acid mine drainage pH[1-2] by T.thiooxidans
Consumption or production of oxygen may alter the habitat in such a way that it inhibit sensitive
population.
EX:1
Production of oxygen by legal populations
Inhibits obligate anaerobes
EX:2
Production of ammonium during protein degradation
Inhibit certain bacteria [nitrate oxidizing population of Nitrobacter sp]
EX:3
Some microbial population produce alcohol, that is inhibitory to many organisms.
EX:4
Production of ethanol by Zymomonas mobilis & Sacchromyces cerevisiae. But certain
bacteria can tolerate ethanol to certain environment
EXAMPLE:
Acetobacter
Ethanol
Acetic acid
[ under aerobic conditions]
It is inhibitory to many bacterial populations.
Presence of lactic acid, propionic acid in cheese and acetic acid in vinegar
Inhibits the growth of spoilage causing microbial populations in such products inhibitory
substances produced by certain microorganisms can also act as a preservative agent
Production of organic acids by microorganism during cellulose degradation prevents
further breakdown of cellulose metabolites in subsurface soil
Some microorganisms produces antibiotics, though it is successful in laboratory
condition, it is a debate under natural conditions like soil and water. Under invitro conditions
21
antibiotics produced by one population inhibits another population. The action may be either
germicidal or germistatic.
Antibiotics are secondary metabolites, produced after maturation when excess substrate
concentration are available. In natural environmental like soil and water [ aquatic ] habitats,
limited substrate are available. Even if produced they not tend to accumulate in soil.
In aquatic environment antibiotics get diluted and become ineffective. In soil environment
antibiotics binds to clay minerals or other particular and this become inactivated. Inspite of these
factor antibiotic resistant strains were also prevailing.
EX:
Zymogenous population
Grows at high concentration of substrate
Produces antibiotics
Inhibit growth of competitive microbes and colonize in such microhabitats.
Other example:
Microorganisms& plants Cephalsporium gramineum
Survive in dead tissues of wheat
Cephalsporium gramineum secretes antifungal popualtion in dead wheat tissues
EX:2
Types of strains preventing among cephalosporium gramineum
Cephalsporium gramineum
Anti fungal + ve
Anti fungal –ve
Those strains that lack the ability to produce Antifungal substance are less able to prevent
colonization of dead wheat tissues by other fungal population.
Another interesting example is the interaction between Trichophyton mentagrophytes and
Staphylococcus aureus in Newzeland hedgehog.
Trichophyton mentogrophytes
Produces penicilllin
Staphylococcus
aureus
coexist
Penicillinase +ve
Penicillinase -ve
Commensal relationship
Organic compounds
During photosynthesis [light]
Algae
22
Epiphytic bacteria
Utilization by algae
Co2 +Vitamins
Ex: 9
Some synergism relationship is based the ability of a second population to accelerate the
growth rate of the first rate.
Pseudomonas
Orcinol
Extracted organic materials
Brevibacterium Curtobacterium
Pseudomonas shows higher affinity to orcinol substrate and grows more rapidly in the
presence of other bacterial population.
Brevibacterium & Cartobacterium can’t utilize intact orcinol.
They utilize the organic secretions of Pseudomonas. If excreted organic matter was not remove
properly. Otherwise would act through a negative feed back mechanism to represent catabolic
activity.
Some synergistic relation ship allows microorganisms to produce enzymes that are not
produced by either population.
Ex:10
Production of lecithinase by population of closely related pseudomonas sp, where as it
cannot produce lecithinase alone.
Ex:11
Degradation of agricultural pesticides. Two soil fungi are involved in this process namely.
Penicillium pisacarium
Geotrichum candidum
Herbicide[ propanil]]
Propionic acid
3,4 dichloroaniline
Used by P.pisacarium as carbon & energy source
Toxic to P. pisacarium
G. candidium unable to attack propanol but can degrade 3,4 dichloroaniline
3,3,4,4 tetra chloroazobenzyl
Thus the end [product become less toxic to both fungi such synergistic relationship occurs only
in the presence of herbicides. In the absence of herbicide these 2 fungi compete with each other
for the available nutrients
Ex:12
Arthrobacter & Streptomyces
Degrade organophosphorus insecticides
Ex: Diazinon [ carbon & energy source]. Either alone can’t degrade or mineralize pyrimidinyl
ring of Diazinon.
Ex:13
Pseudomonas stutzeri
Convert parathion [ organophosphorus insecticide]
23
Pnitrophenol & diethyl phosphate
Ps.aeruginosa
excretory prdts
Can mineralize Para nitrophenyl [ not intact parathion]
utilized by Ps.stutzeri
Explain Competition?
Introduction:
It is a negative interaction in contrast to positive interactions. Competition occurs
between microbial population for shelter, food etc., sometimes certain microbial population
secrte chemical substances and inhibit the growth of other population, which is termed as
Ammensalism or Antagonism.
Sometimes competition tends to bring about, ecological seperation of 2 closely related
populations. This is known as competition exclusion principle. When 2 microbial population
occupies the same, one will win the competition and other will be eliminated.
Classical example for competition exclusion:
Paramecium caudatum [ alone]
When grown isolated both
the organisms
[ population ] grow well
Paramecium Aurelia [ alone]
Parasitism:
Introduction:
In the relationship of parasitism 2 microbial populations are involved.
Parasite - Benefited
Host
- Affected
Among the 2 microbial populations one is affected and the another is benefited host
parasite relationship is a long time or long period of contact, brought about by physical or
metabolic means.
Parasites are of 2 types
Ectoparasite
Endoparasite
Ectoparasite:
Ectoparasite remains outside of host cells.
Endoparasite:
Endoparasite penetrate the host cells.
Usually parasites are smaller than host, certain exceptional; cases may be there. Host
parasites relationship is specific, mostly specificity depends on the surface properties of the
parasite to the host cells. Ex: viruses.
Viruses are obligate intracellular parasites viruses that infect bacteria are called as
bacteriophages. They undergo either lytic or lysogenic cycle.
EX: are T4 and lambda phages respectively. Viruses can infect bacteria, fungi, algae and also
protozoan. In many environments such as viruses are responsible for the decline and
disappearance of bacterial populations.
Ex: disappearance of faecal organism in sewage that enters aquatic habitats.
Interaction between bacteriophages and their host depends on the environmental factors.
For instances, if host E.coli cells get adsorbed to the clay particle it can’t be easily parasitized by
phage. This condition occurs in higher salinity zones bath E.coli and bacteriophages are desorbed
from the particle and lysis of host cells by bacteriophages takes place.
Thus protection of host population by adsorption on to suitable particular mineral,
provides an important mechanisms for escpe form parasitism. Like bacteriophage, bacterium
24
Bdellovibrio is parasitic on gram bacteria. Bdellovibrio is highly mobile [ speed 100 cell
length/second], where as for E.coli is only 10 cells length /ond. No chemotaxis was known for
the attraction of Bdellovibrio towards the host. Small percentage of cells contacts results in the
permanent attachement.
Parasitic Bdellovibrio attaches to the outer cell membrane of the gram negative bacteria
Enter the periplasmic space[ but not the cell proper] since it is an ectoparasite
Losses its flagella
During 1 hr of interaction , it modifies the cell envelope of the host from the original shapes to
spherical called as bdelloplast
Yet cell contains retained for the parasite.
Cell contents of host are particularly degraded by the parasites and then utilized by Bdellovibrio
with higher efficiency.
When it enter periplasmic space it losses it flagella
Grows into thin filaments without cell division
When the cell contents exhausted , filaments divide into individual cells, which develop flagella
Bdelloplast burst and releases the progeny
Burst size:
Burst size is the number of progeny released per host cells. Bdellovibrio though
obligate intracellular parasite they have catabolic, anabolic and energy yielding enzymes.
Interaction between Bdellovibrio a nd E.coli was partially inhibited by the presence of clays.
A clay particle appear to form a coat around host cell and inhibits the ability of the
parasitic Bdellovibrio to reach the host cells. Other ectoparasite microorganism can lyse host
cells without direct contact [ eg: myxobacteria can cause the lysis of host with the aid of
exoenzymes and derive the nutrition from the lysed host cells. Soil myxobacteria are gram
negative and they lyse other gram negative and gram positive bacterial population.
Some Myxobacteria like Cytophaga produce enzymes that lyse susceptible algae. Some
bacterial population produces chitinase, celulase or laminarinase that attack fungi and certain
algal populations.
In all above cause, after the lysis of host cells parasitised utilise lysed host cell contents
as nutrients. Some microbial population are more resistance to lysis eg: spores and cyst formers.
They are resistance to lytic activities of ectoparasite than vegetative cells. Algae, fungus and
bacteria parasitise many protozoans. For eg: Legionella pneumophila parasitising protozoa.
Algae too attacked fungi, a special group[ called as Chitrids.
Chitrids are of 2 types:
CHITRIDS
UNIFLAGELLATE CHITRIDS
CHITRIDS
BIFLAGELLATE
25
ECTOPARASITE
ENDOPARASITE
Chitrids infects fresh water algae and decrease the population. Some fungal population
parasitised by other fungal population.
Agaricus
Trichoderma
Host
Parasite
This interaction creates difficulties andloss in the cultivation of mushrooms.
Microorganisms that is themselves parasites serves as a host for other parasites. This
phenomenon is known as hyperparasitism.
Parasitic interactions provide a maximum for population control.
Parasites
Depends on host
Depends on nutrients in the environment
If environment nutrients get exhausted
Host populations decreased
Sometimes extinction of parasitic populations occur.
Explain about Predation? (Section B)
Introduction:
In microbial would the distinction between parasitism and predation and susceptible
bacteria are considered as parasitism by some people, by other as parasitism. The relation
predation consists of two populations namely predator and prey. In predation 2 microbial
population are involved.
Predator- benefited
Prey - affected
Predator capturre and engulf the prey completely. Always prey is smaller than the
predator. Predator prey interactions occurs for a short time duration. Interaction between prey
and predator lead to regular cyclic fluctuations of these populations.
Predatory population is directly proportional to the amount of prey population. If prey
population increases predator population also increase. If once prey populations decrease,
predatory population is also decreased. The prey population increased due to very low predatory
population. Then again predator population rises. Then again repetition of the some cycles
occurs.
A scientist namely lotka – voltera devised certain derivation to calculated prey-predator
relationship [ theoretically]. But experimental needs however rarelymodel. Gause[1934], showd
that
26
Predator--- Didinium nasutum
Prey ---- Paramecium caudatum
Didinum nasutum preys on Paramecium populations extinct. A very few members of P.caudatum
are also to hide and escape freom predation of Didinum. Such escaped P.caudatum population
can recover the following the extinction of predator Gause[ 1984].
P.bursaria—predator
Saccharomyces pombe—Prey
Same condition occurs as above. The above experiment indicates that intenssse predator –
prey relationship may lead to the extinction of both the predator and prey populations. Although
predation process destroy individuals prey mortality of prey is compensated by increase in
Phytoplankton growth rate due to nitrogen regeneration by predaceaous zooplanktons. If either
predator or prey population is completely eliminated, the population of other would be
deleteriously affected. But in nature stable relationship occurs between prey and predator.
Protozoa—Tetrahymena pyriformis[ predator]
Bacterium – Klebsiella pneumoniae [ prey]
Stable prey- predator relationship occurs between prey predator. Niche diversification [
i.e] the ability of 2 different populations to occupy separate niches. It may permit persistent
population oscillation and coexistences. Similarly physical protection in the environment allows
prey to escape from predator populations.eg: clay materials
Protozoa--- vexillifera [predator]
Bacterium – E.coli
E.coli was protected by clay material such protective mechanism in the natural
environmental lessens the preserve of predation on prey population, allowing coexistences.
Population of ciliates, flagellates and amoeboids feeds on bacterium prey. Feeding of bacterium
by protozoans is called as ‘grazing’. Protozoans engulf bacteria by phagocytosis process
protozoa has lytic enzymes to digest prey. Grazing activity of protozoan on bacterium retains
carbon within the food web.
Paramecium
Vorticella
protozoan feed on Enterobacter aerogenes
Stentor
These protozoans doesn’t waste their energy by chasing small, low calorie prey. Their carryout
filter-feeding mechanism.They stop filter feeding mechanism of bacterial population is highly
diluted i.e 10-5 to 10-6 cells/ml. Thus they conserve their energy. Some microbial structures are
resistances to predation.
EG:1
Soil amoeba
Bacillus sp
[ predator]
[ prey]
if Bacillus from spores, it is less susceptible to predator.
EG:2
Entodinium caudatum [prey]
Entodinium volax [predator]
Spined cells
spineless cells
Spined cells of E.caudatum are resistance to predation.
EG:3 Eupoltes octocarinatus [prey]: Lembladion lucens [predator]
E.octocarinatus often changes its shapes as to escape predation. In nature this defense would
include predator to engulf alternate prey species.
27
Positive Interactions:
Commensalism
Introduction
In this interactions one population is benefited while the other remains unaffected. The
terms commensalism is derived from the latin word where “Mensa” means table. It described a
relationship in which the organism lives off the “scraps” of another one. It is not an obligatory
relationship and is common among microbial populations.
Commensalism is a unidirectional relationship, the recipient population is benefited.
Number of physical of commensalism.
Donar (unaffected population) modifies the habit in such a way that another population
get the benefit, at the same time that habitat is more suitable for its growth.
For ex:
In a habitat faculatative aerobes exists
Utilises oxygen
Reduces oxygen tension
So anaerobes thrive
Production of growth factor forms the basis for many commensalisms relationship [[ bell et.al
.1974]. some microorganisms extracellular growth factor which utilizes by other microbial
population.
EX:
Flavobacterium pneumophila[ cysteine]
Utilized by Legionella pneumophila
Production of growth factors and the secretions into environment allows fastidious organsism to
develop in natural habitats.
Transformation of complex insoluble substance to soluble one and the
conversion of soluble substance to gaseous compounds forms a basis for commercial
relationship.
Ex:1
Desulfovibrio[ during anaerobic respiration & fermentation]
Acetate + hydrogen
Utilized by Methanobacterium to reduce carbondioxide to methane.
EX:2
Production of hydrogen sulfide in buried sediment layers[ decomposers]
Utilized by phototrophic sulfur bacteria as sulfur sources.
This occurs in both terrestial an aquatic environment.
Ex:3
Soil bound ammonia is converted to nitrate by certain microorgansism
Nitrosomonas
Ammonia-----------Nitrate
28
This simple form of nitrate reached into the soil and utilized by microorganisms in soil. Thus in
mutualism relationship activities of one microbial population helps another microbial population
to get a compound available in simple form that can be readily uptaken.
Ex:
For the conversion of complex organic molecules to simple substances.
Trichoderma viride
Cellulose-----------------simple sugars
Cometabolism:
Co-metabolism is the another basis for commensalisms. A microbial population
growing on one substrate, gratituously oxidise the second substrate that cannot be utilized as a
nutrient and energy source for it and it forms a food for other population.
Second substrate is not assimilated by the microbial population but the
oxidation products available for another population.
Propane ------------------
Energy + CO2+ H2O
Mycobacterium vaccae
Cyclohexanor
Pseudomonas
Cyclohexanone
Pseudomonas
Energy +CO2+H2O
Cometabolism is an assimilatory metabolism yielding energy. These Pseudomonas cannot
metabolise intact cyclohexane.
Detoxification or neutralization of toxins also form a commensal relationship.
Ex:
Oxidation of H2S by Beggiatoa
So that H2S sensitive aerobic microbial population get benefited
Ex:
Precipitation of heavy metals by certain organism
Detoxify the environment. So that heavy metals sensitive organisms are benefited
Ex:
Some microbial population detoxify compounds by immobilization.
Leptothrix reduces manganese concentration in certain habitats. If not, increased of
manganese is toxic to other microorganisms.
Some cases microorganisms themselves provide suitable habitat that benefit a commensal
partner.
Ex:
29
Bacteria o algal surface. Bacteria on algal surface gets benefited from the metabolic
activities of algar.
Explain in detain about Mutualism? (Section B)
Microbe- Microbe interaction:
Introduction:
A lichen is an associated of a fungus and algae in which the 2 organism are so intercoined
as to form a single thallus.
The fungus component of the lichen is called mycobiont and the algal component is
called phycobiont. Lichen thallus represents a mutualism symbiosis form both organism benefit.
The fungus lives on the algal cells both parasites as well as saprophytically; while the
algae survives because of it association with the fungal plectanchyma formed in the lichen
thallus.
The fungus provides water, and nutrients to algae and also protects it from high light
intensity.
Lichens are formed by the interaction of fungal spores and free algal cells.
Occurrence and importance:
Lichens are ubiquitous in distribution occur in a as wide variety of habitats.
Lichens can be classified into different types upon their occurrence.
A] saxiculous lichens:
They grow on rocks. They are important initiating soil formation, either chemically by
selecting lichen compounds that weather the rocks on which they live, mechanically by the
destruction of the rock directly by physical action of the lichen thallus.
B] Cyanophycophillus lichens:
They contain blue-green algal phycobionts. They are important in certain ecosystems
where they provide majority of the fixed nitrogen.
Importance:
Cladonia rangifera and similar sps are important in winter food for certain animals like
Reindeers.
A number of commercial product such as dyes, ;litmus essential oils for furfume
manufacture, where obtained for lichens.
Uronic acid produced by a number of lichens is an antimicrobial substance. The most
important role of lichens is an indicators of air pollution.
In centers of heavy industrial pollution no lichens can be found.
Anatomy and morphology:
There are 2 general types of lichen thalli,based in the distribution of algal cells among
the fungal tissue.
A] Homomerous:[homios- same & meros-part]
The algae are more are less evenly distributed through out the thallus.
B]. Heteromerous-[ hetero-diff]
The algal cells formed a distinct layer with the thallus.
Three layers are present namely, algal layer, the medulla and cortex.
The cortex can be further divided into and upper and lower cortex depending on the
configuration of the thallus.
Growth forms:
The 4 major growth forms, that has been recognized in the lichens are,
The foliose thallus is leaf like
The upper cortex is a gelatinized mycelial layer which is protective in nature.
Below this is the algal layer consisting of algal cells enveloped by hyphae.
In many sps, they may be penetrated by fungal haustoria.
30
The medulla occupies the major portion of the thallus and is situsted just below the algal
layer.it consist of hyphae interwoven into loose parenchyma.
The lower cortex.if present,is situated at the bottom of the thallus and resembles the upper
cortex in structure.
It is often covered with rhizoidal hyphae or hairs.
Crutose:
These lichens closely adhere to their substrate.
Squmulose:
This is scale like composed of many small lobe.
Fructicose:
It is erect, cylindrical or branched with finger like projections.
Classification on the basis of occurrence of Phycobionts:
Chlorophycophilus:
Presence of green algal phycobionts.
Cyanophycophillus:
Presence of blue green algal phycobionts.
Diphycophillus:
Presence of both green and blue green algal phycobionts.
Physiology:
The physiological reaction between the phycobiont and mycobiont in a lichen thallus is
not well understood.
The phycobiont supplies the carbohydrate. it fixes the nitrogen and release it into the
thallus.
Water is adsorbed from the environment by the hyphae and is transported to the algal
layer.
The fungal hyphae is usually in intimate contact with the algal cells by means of
appressoria and often through haustoria.
The phycobionts of most lichens is the green algae Trebouxia [ name of green algae].
Reproduction:
The lichen thallus forms probacules consisting of both symbionts.
Reproduction of mycobionts:
Lichen fungi produce either ascospores or basidiospores that are discharged from the
lichen thallus and germinmate to form mycelium.
These will then combined with free algal cells under favourable conditions to synthesize a
new lichen thalus.
The ascospores may be one celled or septate. They vary in size form 1-500 micrometer.
Reproduction of phycobiont:
The green algal symbionts multiplied by vegetative cell division and by the formation of
aplanospores.
BG phycobionta may multiply by cell division haterocyst , akinetes.
Lichen probacules: [diasospores]
The lichen thallus reproduces itself asexually by various efficient means.
Portions of thallus cut off and those that land in favourable environment may grow into
thalli.
2 types of more differentiated diasospores [greek- diasore- dispersal ] are also produced.
Isidia:
They are minute more or less columna structures consisting of both fungal hyphae and
algal cells that break off and are distributed by wind, animals, raindrops etc.,
Soredia:
31
They are micrscopic powdery masses of algal cells enveloped by fungal hyphae produced
in soredia[ Pustules of various types on the thallus]. They are also distributed by wind,
animals, raindrops etc.,
In diphycophillus lichens that have 2 phycobionts , a green& BG algae. The BG are
segregated in special internal or external swelling called sephalode.
Microbe- plant interaction:
Leaf nodule bacteria:
The occurrence of leaf nodule is confined to 2 families namely,
Rubiaceae
Myrsinaceae
Many genera of the family Rubiacea formed leaf nodule, they are
Pavatta, Chomenia, Pschotria
The genius Psychotria has received considerable attention form many workers.
Leaf nodules & Nitrogen fixation:
Role of leaf nodule but in nitrogen fixation was not known for a while.
Earlier reports claiming nitrogen fixation have later been disproved by experiments with
nodulated leaves form plants grown in media free of combined nitrogen.
The tests employed were
Acetylene reduction technique for nitrogenase activity estimation
Exposure of the leaves to an atmosphere containing 15N labeled nitrogen gas determine the
extent of nitrogen fixation.
Bacteria present in the leaf nodule:
Mycobacterium rubidearum, Mycoplana rubra, Flavobacterium sp, Phyllobacterium
rubiacearum & Klebsiella rubiacearum
Production of phytohormones:
Evidences have shown that cytokines may be produced in leaf nodules.
Bioassay for cytokinin activity:
Retention of Chlorophyll by leaves is a characteristics feature commonly used in the
bioassay for cytokinins. The naturally occurring scencent and yellow leaves of Psychotria
retain Chlorophyll around leaf nodules indicating the presence of cytokinins.
Drascorea macroura, member of the family ioshoreaceae possess leaf glands at the
abscission of leaves which are inhabited by bacteria who’s identity is not known.
The nitrogen content of leaf tissue containing the glands is higher than the remaining of
the leaves. This indicates the ability of the symbiotic association to fix molecular
nitrogen, though test with 15 N have not been carried out yet.
Endosymbionts of protozoa:
It is an interesting mutualistic relationship exists between population of algae and
protozoa.
EX: Paramecium
Chlorella
Algae [ Chlorella]
Protozoa [ Paramaceium]
Supply protozoa with
growth factor
organic carbon and oxygen
provides algae with protection motility co2 and perhaps other
32
Chlorella cells present
Within the ciliates of protozoa Chlorella allows Protozoa to move in anaerobic habitats as long as
there is sufficient light
Forminifera & Pyrophycophyta or Chrysophycopjhyta:
 Algae supply protozoa with food and oxygen protozoa protect algae form grazer. This
type of mutualistic relationship exists to normal conditions. But during environmental
stress conditions i.e prolonged absence of light, protozoa digest the algae population.
 Many other relationship occurs between algae and protozoa. Algae of fresh water
environment that has contact with protozoa algae belonging to chlorophycophyta are
called as Zoochlorella [ fresh water algae found with protozoa].
 Algae found within marine protozoa are called zooxanthallae [most dinoflagellate of
group pyrophycophyta are involved.
 Endozoic cyanobacteria which occurs both in fresh water and marine protozoa are called
cyanellae. Endosymbiotic of protozoan has been established by electron microscopic
studies some multiplies with in the nucleus of protozoan and some within cytoplasm.
 Certain flagellated protozoa Blasocrithidia & Crithidia exhibits mutualistic relationship
with bacterial endosymbionts.
 Bacteria: Caedibacter[ Endosymbiont]
 Protozoa : Paramecium Aurelia
 Cardibacter formerly called as kappa particle
Paramecium Aurelia
Killer strains
Contain endosymbionts
sensitive strains
lack endosymbionts
Cardiabacter has plasmid [ contain R body ] that codes for the production of toxin. Cardibacter
derive its nutrients
Form P. Aurelia and in turn Protozoa escape form enemies by the production of toxin by the
endosymbiont.
Cardibacter
P.Aurelia
Has R body codes for toxin production & help protozoa form enemies supply food for the
bacterium
Certain Methanogens have interesting mutualistic relationship with other organism.
Ex:
Methanobacterium omelianskii
sulfur utilizing organisms
[ use e-s to render CO2 –CH4]
Methane
Reduction
utilizes acetate and supplied electrons [ hydrogen
thus mutualism occurs between these ions ] 2 microbial partners.
In a 3 membered syntrophic association involved in the anaerobic digestion of a whey effluent to
methane and Co2.
33
Acetolastic methanogens
Ethanol ------oxidation------- acetate ------------------ Methane & Co2
Methanosarcina barkeri
Desulfovibrio vulgaris
HCO3- bicarbonate---------------- formate---------------methane -------Desulfovibrio vulgaris ------ ethanol –acetate+ bicarbonate to formate
Methanosarcina barkari --acetate -----methane + CO2
Methanobacterium formicicium----- formate --------methane.
Explain about the interactions among Microbial population?
Introduction:
Even under natural conditions microorganisms does not exist lonely. For instances in a
petriiplate containing agar [ agar plates ] microbes multiply to form a colony [ lone to a single
cell].
The environment of microorganisms is called habitat where population is similar group of
individuals in a particular habitat. The term community refers to different population inhabit ting
a particular locality.
Interaction may occur between 2 microorganisms of a population or community.
Interactions refers to the relationship between 2 organisms or more. Interactions may be positive
or negative in nature. Positive increases the growth rate of the organisms. On the other hand
negative interactions decrease the growth rate of the organisms. Alles et al [ 1949 say that both
positive and negative interactions may occur within a single microbial population. The total
interactions between population maintains the ecological balance of the community [ Bull &
Stater 1982]
Alles principle , also explains the density dependent interactions
Commonly 2 types exists
 Cooperation
 Competition
Cooperation:
It is the positive interaction that occurs to low-density population.
Competition:
It is the negative interaction that occurs in high-density population.
Positive interactions:
The positive interactions occurring within the population is called Cooperation.
Ex: 1
Long lag phase is needed in the cultivation of fastidious organisms. Generally if
population is high log phase is less.
Ex:2
Minimal infectious dose:
It requires a thousand of microorganisms [ pathogens ] to cause a disease. A single
organisms rarely able to overcome host defense mechanism.
Ex:3
Semi permeable membrane:
In many cases semi permeable cell membrane of microorganisms tend to leak low
molecular weight metabolic intermediates that are essential for the biosynthesis of growth.
If population is higher this can be overcome by excess amount of extra cellular substances
in medium. Sometimes to overcome this difficulty sterile filtrate of spent enrichment culture
medium added in new medium.
Ex:4
34
Even motile bacteria associate to form colonies for efficient uptake of nutrient sources
from the medium [ soil] and to seek new sources of nutrients.
Motility & Nutrition:
Ex: slime mold –Dicytostelinum
Ex:5
Protective mechanisms:
Cooperation is a population function as a protective mechanism against host
environmental factors.
Metabolic inhibitors is effective, only if cells were in low concentration, where as it is not
effective if the cells were in higher concentration.
Microbial films were sensitive to Antimicrobial agents if the population density is less
and vice versa.
Genetic interactions:
Genetic interaction is another cooperation interaction among the microbial population.
 Resistance to heavy metals’
 Ability to use unusual substances
 Resistance to antibiotics
Negative interactions:
Negative interaction occurs within a microbial population is celled s competition.
Competition exists among the microbial population for food substrate shelter available in the
environment. A place with low nutritive substance inhabited by a high population of
microorganism competition occurs. Competition occurs within predatory microbial population
for the available prey.
Competition occurs within microbial population of parasites for the available host.
Nagative interaction also occurs due to the accumulation of toxins and by the production of
antibiotics. If a microbial population at a particular site is very high ,metabolic products
accumulates at a higher level that may inhibit further growth of the organism.
Accumulation of some metabolic products such as hydrogen sulfide and fatty acid
constitute a negative back mechanism. Such accumulation inhibits further microbial growth even
in the presence of substance.
Ex:1
Excessive accumulation of hydrogen sulfide can limit further sulfate redu8ction.
Ex:2
Accumulation of more amount of ethanol in the medium, stop further fermentation by
Saccharomyces.
Ex:4
Accumulation of fatty acids during hydrocarbon degradation blocks further microbial
metabolism of hydrocarbon substrates.
Ex:5
Accumulation of dichloroaniline form propanil stop further growth of Penicillium
piscarium.
Neutralism:
The concept of neutralism implies the lack of interaction between 2 microbial population.
Neutralism cannot occur between populations having the same functional roles within a
community.
It is more likely to occur between microbial population having extremely different
metabolic capabilities than with similar capabilities. Neutralism is likely to occur at low
microbial population densities. Where one population does not sense the presence of other. Ex:
neutralism certain areas of marine habitat and oligotrophic lake habitats [ low nutrients], where
microbial population is extremely low.
35
Neutralism also occur between 2 microbial population when both populations are outside
their natural habitats. For ex microorganism in air [ atmosphere], here microbial population is
low and all microorganism are allochthonus [ non= indigenous ]. Neutralism can occur when
environmental conditions do not permit a active microbial growth.
EX:
Microorganism in frozen ice matrix.
Microorganism in polar sea ice.
,, ,, ,,, Frozen fish
, ,,, ,, ,,In frozen fresh water
resting stages of microorganisms are more likely to exhibit a relation ship of neutralism with
other microbial populations. Some environmental stress like scarce nutrients, excess heat allows
microbial population to enter resting stages and performs a relationship of neutralism.
Some microbial population produces enzymes that can degrade the resting stage of other
microbial populations,. But such instances are rare. In fact endospores have thick coat with high
amount of Melanin, DPA and calcium, which gives resistance to such negative interaction.
Ruminant symbiosis:
Ruminant are a group of herbivorous mammals like cattle, goat, sheep. Camel, giraffes
etc., Ruminant gut provides more obvious example of mutualism. They are plant cellulose as the
major carbohydrate source of their diet. However their normal gut cannot digest cellulose.
Digestive tract of ruminants contain not less than successive stomach. They have
developed a special region for cellulose digestion. This region in the human -that is essential –
incubation chamber associating with bacteria and protozoa.
In cow this resembles a large fermentation vat, about 100 lts , into which masticated plant
materials enter for further digestion by a large number of anaerobic bacteria and protozoa.
The mutualistic microbes hydrolyze cellulose and other complex plant polysaccharides to
their component fatty acids [ acetic , propionic acid & butyric acid] and gases [ methane, carbon
dioxide]
The fatty acids are absorbed through the wall of human into the blood stream for use as
carbon and enrgy source. The gases are passed out of rumen at frequent intervals.
The microbial population of the rumen grows rapidly. These microbial cells pass out the
rumen along with undigested plant materials into the stomach. These cells are destroyed and
digested in the stomach through protease [ the rumen produces no digestive enzyme] in normal
way to provide essential aminoacids and vitamins etc required for the growth of animals.
The cellulose digesting bacteria of rumen are all strict anaerobes. The species include
Bacteroides succinogenes
Ruminococcus flavofaciens
Ruminococcus albus
Botryvibrio fibriosolvens
The great bulk of bacterial population however is non- cellulolytic. Many of the rumen
bacteria including some in the cellulytic species are capable of digesting starch proteins and
lipids.
Only lignin of the ingested plant materials escape digestion. The products of digestion of
polysaccharides, proteins and lipids are fermented by the rumen of bacteria. During these
processes , hydrogen gas combines with carbondioxide to form methane by Methanobacterium
ruminantium.
36
BIOPESTICIDES & BIIOINSECTICIDES: (Section C)
VIRAL INSECTICIDES
Insect virus are classified as Inclusion viruses and non inclusion viruses, certain viruses
like Baculoviruses have inclusion bodies in form of polyhedra and capsules. The virus with
polyhedra are of 2 kinds.
1. Nuclear Polyhedrosis virus
2. Cytoplasmic polyhedrosis virus
Apart from this, capsules as inclusion bodies in some viruses known as Granulosis virus.
Insect viruses are classified under 7 important families.
Baculoviridiae
Reoviridiae
Iridoviridiae
Poxviridiae
Parvoviridiae
Picaviridae
Rhabdoviridae
Ture of Insect Viruses:
The infectious unit is virion, composed of condensed genetic material either DNA/RNA
surrounded by a coat or capsid of protenaceds subunit, the capsomer. These virus differ from
other mainly, this is occulusion of virus particles, itself inside protein crystals, granules or other
envelopes known as inclusion bodies.
One of the important characteristic viruses is the production crystal like bodies called
polyhedra in cells of tissues infected by viruses. Some cause formation as refrigerent inclusion
vary size and shapes and other small granular inclusions.
Inclusion bodies are mainly proteinaceous contents with virus particle embedded inside.
If virus is enclosed by the inclusion body it is called occuladed and it is not enclosed it is
nonocculuded.
Nuclear Poly Hedrosis Virus (NPV): (Section A)
NPV is a polyhedresis virus. Polyhedrosis are characterized by form of polyhedrose
shaped inclusion bodies in infected tissues and, mostly asosciated with Lepidoptera, insects.
37
NPV – Heliothus armigeria – Control Spodoptera Litura in tobacco, Sorghum, Caston, Cotton
and Sunflower.
PV – Polyhedra Composition: (Section A)
Polyhedra is composed of lipids, proteins iron, aminoacids, NH3, N2 etc. Polyhedra does
not dissolve in hot or cold water, alcohol, ether, Choloform and acetone, but dissolved in NaoH,
KOH, H2SO4, NH3, and acetic acid. They are don’t destroyed by bacterial pathogens. When
draied remain unchanged for 20 years under refrigeration.
Mode of Infection of Bacluovirus:
Food contaminated with occlusion body
Ingestion by larvae
Disaggrecation of occulusion body in alkaline contents of gut
Release of viral Particle
Entry by fusion to gut epithelial cell
Removal of coat
Replication of DNA in 6-10 hrs
Haemolysis occur
Transmission of Disease to other tissue
38
Death of Larvae
Cell death
Cell lyses
4-6 days accumulation body in Nucleus
Mass production of Occlusion body
Hymptomatology:
Larvae become sluggish and there is reduction in hepatile. Skin becomes oily and changes
colour. Skin becomes fragile and repture liberating liquid body contents consisting polyhedra.
Larvae in the last stage hang the head downward. Younger caterpillers are most sensitive.
Incubation period is 3-21 days.
Histopathology:
Muscles, nerve ganglia, nerce sheath, hypodermous fat body, trachial matrix and blood
cells and mid gut epithelial cells are infected.
Procedure and preparation of NPV:
 10 days larvae measuring 25-4 cm are ideal for culturing NPV
 collect the larvae and starve them over night.
 Prepare virus suspension (107 IB/ml in 250ml water, and few drops of teepel).
 Treat the leaves with virus suspension dry in shade for few minutes, (provide treated to
sterved larvae) provide viruses inoculated with infected leaf, followed by normal leafs.
 Due to virus infection, the skin on undersurface turns pinkish white due to polyhedra
accumulation. After death, the skin rupture and white liquified body contents ooze out.
 Grind the dead larvae and filtered through musclin cloth. Allow the virus to settle.
 After a weak pourout the supernatant the white sediment is mixed with water and teepol at
0.05% and sprayed in the evening.
Application of Viral Pathogens:
39
Virus suspension in 250 lt/bec
Soya oil – 0.1% as a sticker
Soya flour – 1.0% as a sticker
Applied as foliar spraying.
Cytoplasmic Ployhedrosis Virus: [CPV]: (Section A)
[SMITHA VIRUS]
The virus is sphericle and multiply in the cytoplasm of the cell of midgut. The polyhedra
inclusions are regular dodecahedra or icosahedral. They average 0.5 – 2.5 nm. It contain 1001000 virons composed of RNA. Polyhedra are does not dissolve in water, and dissolve in weak
alkalies. Viruses particle consist of a capsid made of 2 concentric cells and an inner core and the
capsid has 12 tubular projection.
Symptomatology:
1. Infected larvae lack behind in growth
2. Loss of apetite and smaller in size
3. Colour changes occur – ventral abdominal in midgut region may show chalky white patches
due to accumulation of inclusion bodies.
4. Blocking of alimentary canal.
5. Skin will not rupture
6. mid gut appear opague, pale yellow, or milky in appearance
Histopathology:
 sections made at the early stage of the disease show formation of polyhedra in midgut
epithelia cell.
 As infection spreads, polyhedra spread through the alimentary canal.
 CPV is present in every cell of fore, mid, and bind gut.
 As polyhedra are formed, the cytoplasm may swell extending into lumen.
 The cell wall breaks and polyhedra are liberated into gut lumen, the cell nucleus remain intact
until disintegration occurs.
Fungal Biological Control
This is the largest group of insect pathogen having more than 700 spp. The pest belonging
to insect orders. Lepidoptera, coleoptera, Homoptera, Hymenoptera and Diptera are susceptible
to different fungal pathogens.
40
Fungi belonging to Ascomycotina, Phycomyatina, Basidiomyeodina and
Deuteromycotina attack different Spp. Of insects.
Best Example: TRICHODERMA
General characteristic of Trichoderma. Sp
Mainly I. Vivide T.Harixanum used as a biocontrol agent. Their character listed below.
Character
T. Viride
T.Harizanum
Colony size
4.7 cm
9 cm
Optimum temperature
20 – 30 degree C
30 degree C
Ph
4.5
3.7 – 4.7
Hypae
short, downside long branches
same in (T.V)
with divergent
Philodes
2-4 number
more than 4
Conidia
freeze edged conidia
smooth oval conidia
Soil  clay loamy soil paddy field
Rhizosphera  Tabaco, Potato, Sugar Beat, Wheat, and Grasses
Roting tuber  cassava, pea seeds, grains of sorghum, sewage, composed house hold refuse
Active against – Plant Phathogens:

Rhizoctina Solani

Fusarium oxgsponcum

Mucor hiemalls

Phythium ultionum

Verticillum albo atrum

Collectrotrichum lini

Hermithosporium sp

Alternaria. Sp

Candida albicans

Aspergillus niger etc
41
Mass preparation of Trichoderma
Inoculum Preparation:
Trichoderma Sp Isolated from soil
Pure Culture
A loopful of spore transferred to SDA / PDA plate
Inculate room temp for 4-5 day
That culture was transferd to 50ml medium in 250 ml flask
Incubate 4-5 day
Green colour sporosare appeared
Poured 300ml pure sterilized cooled water
Flask mix well
Filtered through sterile muslin cloth
Filtered solution is a stock of Inoculum
Product preparation Process:
42
Partially milled sorghum/paddy
Put in polythene bag 250 gms in 500 gms capacity bag
Mouth tightened with rubber bands
Autoclaving
Allowed to cool
Inoculate 5 ml of stock culture into the bag aseptically.
Spare load 107 spore / gm of substrate
Ready  for direct used.
Modified form
Talc formulated Trichoderma Production:
Trichoderma viride
Grown in mollasses yeast medium for 10 days
2.5 litres of inoculum, 50 litre sterlie medium in fermantor
today, 4-8 hrs aeration
43
From this  harvested broth mixed with 100 kg of supper white talk and 500g CMC
Mixed well
Dried in shade for 72 hrs.
Quantity 300 * 106 CFU / gm at the time of use
Ready to use
Field application
1. Seed coating
2. Foliar spray
3. Soil amenments
4. Liquid suspension treatment 10ml suspension / kg seed.
5. Seed dipping
Direct coating 4 gms of biocontrol / Kg of seed.
INSECT PARASITOIDES
The term “Parasitoids” is used to refer an insect parasite. Insect parasite is an insect much
smaller than its best insect. The parasitoid may irritate, weaken or killing the best insects. Based
on their time of pathogenic to plant pathogenic insect, the parasitoides classified as
 Ecto and Endo parasitoids Eg: Ealophidae – External parasitoid of stem borer and leaf mines
of Hemptera Bracondiae Endo parasitoids.
 Larval purpat parasitoids: Eg: Chalcididae – primary / secondary parasites of purparia of
Diptera insects.
 Egg Larval parasitoids: Eg: Trichogramma – Oophagous/ Egg. Parasitoids.
Advantages of Biopesticides:
The viruses of biocontrol of insect pests with biopesticides lie in not having the dangers
involved in using chemical insecticides.
44
1. Bio control is safe, free from environmental pollution, toxic resistance and durable
2. Comparatively cheaper than chemical pesticides
3. Do not need any special equipment’s for application
4. No danger to any one in field.
5. Continuous and sustained control pests
6. Relative high specificity tends to protect beneficial insects.
7. Requirement of low dosage.
Disadvantages:
1. Correct timing of application with respect to incubation period of disease.
2. The narrow specificity of pathogens narrow spectrum of effectiveness.
3. Necessity to maintain in a viable condition
4. Difficulty in culturing in large quantities.
5. Require favourable climatic conditions for best efficiency
AZOSPIRILLUM INOCULANT
Beijerinck in 1925 reported N2 fixing bacteria. Under the name spirillum lipoferum. The
ability to fix N2 in pure culture was not determined later, N2 fixation by S. Lipoferum was
conformed by several workers by definitive methods as acetyleme reduction technique, and the
isotopes enrichment method involving 15N2 . Tarrand et.al., (1978) renamed this bacterium as
Azospirillum.
Dobereiner and Day (1976) observed that Azospirillum could be isolated from tropical
grassess like Digitaria, Panicum, Maize, Sorghum, Wheat and rye.
Characterization
Azospirillum are gram-negative, mobile, vibroide in shape, and contains PHB granules.
On semi solid malate medium white dense and undulating tine pellicles is characteristics of
Azospirillum. mIcroscopic examination shows pleomorphic rods, shows lacteral flagella in
addition to single polar flagellum.
Azospirillum grows well on salts of organic acids like maltose, succinate, pyruvate, or
lactate. Azospirillum can be classified into two groups as group I strain of A.brasilienge differing
from group II a. lipoferum by their inability to use glucose as sole carbon source, for growth in
nitrogen free medium by their requirement for biotin, formation of wider longer. ‘S; shaped or
helical cells in semisolid - N2 free malate medium.
A. Amazonense – pink colonies appear on potato agar, lacteral, flagella absent, uses
sucrose as carbon source.
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A. Sero pedicae have 1 to 3 flagella at poles, small, moist, green centered colonies on N2
free bromothymol (NFB) medium.
Isolation of Azospirillum:
 Collected root washed with water to remove adhering soil
 Cut the roots to small bits 1cm size with help of razer.
 The root bits are surgface sterilized by 1% chloranium ‘T’ solution ofr 2 – 5 times or 0.1%.
HgCl2 for 1 minute, follwed by washing with sterile water and po4 buffer (Pt 1.70)
 The root bits are inocculated into a semisolid malic medium (5ml – sterile medium)
 Incubates the tubes at room temperature for 3 to 5 days.
 Observe development of pellicles and colour changed of medium from yellow to blue.
Identification
Malic acid medium prepared with 1.5% agar and pur into sterile petridishes. Then streak a
loopful of culture from tubes over solidified medium. Incubate the plate 3 to 5 days.
Azospirillum change the colour of the medium from light yellow to blue, and further
confirmation is done by its active special movement when viewed through microscope.
Carriers for Azospirillum:
Soil and farmyard mannure (FYM) in ratio 1:1 used as carrier. The carrier is sterilized for
3 hrs at 121 degree C consecutively 3 days. It is best carrier for Azospirillum. The bacterium was
able to survive upto 6 months and gave about 106 cells/gm of carrier.
Mass production of Azospirillum
Purified strains of Azospirillum
From Agar slant into large flask, ammonia containing medium
Incubate 3 – 10 days at 28 degree C
Starter culture trnasfer to production fermentor
Incubate 3-5 days
Obtain 2 * 109 cells/ ml of medium
Check purity
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Mixed with carrier
Adjusted moisture content to 40%
Packed in polythene bags expelling the air.
Application Techniques
 For murseries Azospirillum is to be mixed with water and seeds should be shown over night.
In field further 2 kg of Azospirillum mixed with 25 kg FYM + 25 kg soil and broad caster
over field before transplantation.
 Another method is to prepare slurry of 1 kg. Azospirillum in 4 litrs, water and dip roots of
rice stand for 15 to 30 minutes before seedling.
Crop Response
Sorghum, Pearl millet and finger millet appeared to the responsive to Azospirillum. The
seed inoculation with Azospirillum brasilens increase grain yield of sorghum, pearl millet and
ragi, respectively 17.9%, 15.4% and 17.7%. Azospirillum culture increase root biomass of rice
and wheat. It produce plant growth harmones in pure culture.
BIOCONTROL AGENTS
Introduction of Biological Control:
When man started cultivation, he had a little knowledge about plant disease. Many
control measures were adopted, time to time, which include cultural practices, and use of organic
and inorganic chemicals. In recent years the increasing information, on hazordous effect of
synthetic pesticides on plant and animal health, have alarmed the scientists to seek an alternative
method. That is biological control method which involved.
Disease control by some biological agents. (living Micro-or Macro – Organisms) other
then disease causing organisms.
Examples of Biocontrol Agents
Bacterial biological control:
Bacillus thruringiensis
B. Subtilis, B. Popillae
Pseudomonas fluorescens
Agrobacterium radiobacter
Fungal Agents:
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Trichoderma Viride
T. barizanum
Viral Agents
NPV – Virus – Heliothys armigen
Cytoplasmic polyhedrosis virus.
Parasitic Agents
Trichoderma-. Egg parasite
Insects used as Biocontrol agents
 Oecophylla Smaragclina  group at ant population control certain pest of citrus plant.
 Senecio Jacobae  Weels of watter areas  controlled by
Tyrea Jacobae
DE of Action:
 Antibiosis:
The biocontrol agent produce large quantities of antibiotics  lead cost.
 Plasmalemma retraction,
 Breakdown of organnelles,
 Cytoplasmic disintegration due to induce permiability of phospholipids.
Eg: B.Subtities  Iturin, Pengyein  Control Rhizoctina solani – xanthomonas compestris.
B.Fluorescense  Oomycine,
Tropolene
Pyconine
Trichoderma Viride  Trichodermin, Dermin, Trichouiridin.
 Enzymes:
Control agents produce some extracellular enzymes like chitinase, glucanase, that lead
out
degradation at cell walled pathogens.
 Antagonistic protein:
B.subtilus produce antogonistic protein pertaining to 37KD, 24KD, 10KD and 8KD,
they
are strongly inhibitory to seed borne fungi and bacteria.
 Competition:
Flueroscent pseudomenads compete with pathogens for iron. During low iron cone, they
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produce iron-seqnestring compound called siderophores, which decrease iron availability and
inhibit growth of pathogens.
Eg: Pseudomonas Flueroscense  Control cotton with.
Siderophose eg: Pseudobacteria, ferribacteria, ferrichrome, pyochelin  Ercoinia soft rot
disease.
 Parasitism and predation:
Mycoparasitism when one fungus is parasitized by another one the phenomenon is called as
mycoparasitism.
Hyper parasite & biocontrol
On hypo parasite  pathagen
Form coiling about host fungus.
Secreting cellwall lytic enzymes
Control the pathogen
Nematophagy:
This is the phenomenon eating upon nematodes by fungi (Nemata fhagus fungus – NF)
Eg: Fungus: Arthrobotrys, Dactylana, Phialosper control  Heterodera rastochiensis nematodes.
Sycophagy:
Feeding of fungi by amoebae
Eg: Amoebae: Arachula, Geococcus, Vampyrella Succamaebae  Control Fusonium
oxgysponum phylophthorasp of – fungus.
a. Attachment of parasite
Tropozoint of amoebae attached on fungal propagules
Fungal propagules engulbed
Causes Pertoration
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Digested the fungal cytoplasm completely
Mode of Action:
B.T. Strains is contained with a very large structural body called para sporal crystal which
is synthesized during sporulation. It is 20-30% of the dry cot of the sporulate culture. It consists
mainly. 95% protein and a small amount of carbohydrate 5%. The crystal aggregates of one kind
of protein that can be dissociated by mild alkali treatment into two subunits, each with a mio 250
Kda approximately 20 glucose and 10 mannose residues are associated with each unit.
The parasperal crystal is not the active form of the insecides, rather, it is a protoxin, a
precursor of the active toxin. When the parasporal body is ingested by target insect the protoxin
is activated within its gut by the combination of alkaline. Photosynthesis(7.5 to 8.0) and specific
digestive protease. Which coverts the protoxin into an active toxin with a mw (08k Da. In its
active form, the toxin insert into the membrane of the gut epithelial cells of the insect and creates
an ion channel through which there is believed to be an excessive loss of cellular are ATP. About
15 minutes after than ion channel formation cellular metabolism is ceased, the insect stop
feeding, becomes dehydrated and eventually dies.
BACTERIAL PESTICIDES
There are several bacterial pathogens of insects that currently are used as insecticides.
They are
Rickettsiella popiliae
Bacillus popiliae
B. Thuringiensis
B. Lentimorbus
B. Spharecus
B. Subtilis
Clostridium malacosome
Pseudomonas aeruginosa, fhioeveseens.
Xenorbabdus nematophilus and
Agrobacterium radiobacter.
Among them B.thruinigiensis is an important biological control of lepidopteran (target pest)
used. Since the late 1960’s. B.thuringiensis or BT as it is commonly known, is an important
biological control. Agent, it was first observed as the cause of the Sotto disease in Silkworm
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[Bombay mori} in 1901. The name B.Thuringiensis comes from the German province Thoringen
in Which it was discovered. In 1959 thuricide [a preparation of BT] was commercialized by
pacific yeast products as a biological control agent.
B.thuringiensis has many subspecies that differ in the number and types of plasmid they
contain. The genetic information coding for the insecticidal toxins of these strains is borne on
these plasmid.
Bacillus thuringensis
B.Thuriensis is a gram positive soil bacterium that grow by digestive organic matter
derived from dead organisation or by colonization within living insects. Initially isolated form
dust inside silk worm rearing house and soil around it.
Strains of B.thuringiensis are classified in five pathogenic types on the basis of their
insecticidal range.
B.thruiensis sub.sp krustaki kills lepidoptron larvae, including moths, butter files,
skippers, cabbage worm and spurce budworm.
B.thiruensis sub.sp instaeliensis kills muskitoes and black flies. B.thruiensis sub.sp
tenobrious agains colepder (bedttles) such potato beetle and the boll weevil.
Apart from this B. thiruensis var aizawai and no known toxicity strain. B. thiruensis var
dakota are used as biological control.
Mass production of Bacillus Thuringiensis:
Isolation of B.Thuringiensis strains from lepidepidron insects
Mother culture
In ordinary media – prepare Incoculum
Inoculum introduce into production termentar
72 hrs – Shake culture
Harvested (cell load 1011 cells /ml)
Finished product 1. Tale Formulation
2. Gypsum formulation
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3. Pecot formulation
packed in polythene bags.
Introduction of Bacillus Subtiles: A:13
 Prepare nutrient broth
 Inoculated with mother culture
 in large scale – 72 hrs for production time.
 Harvested : mixed with 250 kg stread sterile peat soil amounted with 37kg) CaCo3 for
(100litrs molecules)
 Packed, life time – 6 months.
Agrobacterium Radio Bacter – 84
This bacterium has been found to control the effectively Croron gall of stone fruit caused
by A.Fumifaciens.
Inoculation of seed and root in combination with A.Radiobacter strain 84, 98.8% control
of crown gall was achieved. They organ’s produce was coded by conjucative plasmid of the
antagonist.
Other bacterial pesticides:
Pseudomonas Fluorescence:
It is antagonistic to ercoinia Caratovora Causing of soft root of potato.
Mass production and formulation:
Mother culture
One loopful culture introduce into 100ml of king’s ‘B’ Broth
Room temp. 48 hrs.
1 liter mother culture
Inoculate 100 litrs. Of king’s ‘B’ medium
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72 hrs – 150 rpm – 80 degree C temp
Harvested( 9 * 10 degree CFU /Ml)
Total formulation / End products
400ml medium with ( 9 * 10 degree CFU /Ml)
1 kg super white talk + carboxymethyl cellulose (CMC) (Photosynthesis – 7.0 adjusted)
packed the product
Life Time : 120 days. 4 gms/kg seed for application.
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