LECTURE NO.

1 INTRODUCTION AND HISTORICAL DEVELOPMENTS IN SOIL

MICROBIOLOGY. CONTRIBUTIONS OF BEIJERINCK, HELLRIEGEL, WILFARTH,
FRANK, WINOGRADSKY, FLEMING, WAKSMAN, DOBERIENER AND MOSSE
Introduction
Soil represents a medium or substrate in which numerous microorganisms live and bring about a
great variety of processes which are responsible for continuation of the cycle of life in nature. Soil
microbiology is a branch of science concerned with soil inhabiting microorganisms and their
activities. Agricultural chemists are also interested in the soil processes that result from the activities
of microorganisms. General bacteriologist, zoologist, botanist were interested in certain special group
of organisms found in soil. Recently, soil microbiology has expanded to include the study of the role
of soil microorganisms in genetic engineering, in the biological control of pests and diseases, the
degradation of pollutants, production and destruction of radioactive gases and its transfer. Thus
microbial participation in several important processes emphasizes that soil microbiology has become
a global science.
Soil microbiology
 Deals with the microscopic organisms of the soil
 Their population and activities
 Role of soil microbes in various nutrient transformations taking place in the soil and
 Their importance in plant nutrition and crop production
Distinct phases of soil microbiology
1. Ecological phase
It concerns with the study of the quantitative and qualitative composition of the microscopic and
ultramicroscopic soil population.
2. Experimental or physiological phase
It encompasses studies on the physiology and biochemistry of the organisms, role of soil
microorganisms in the cycle of life in nature and their utilization for the formation of valuable
metabolic products.
3. Agronomical phase
It deals with application of microbiological activities to improve soil fertility and crop production.
4. Pedological phase
This deals with studies to assess the importance of microorganisms in soil formation and soil
structure.
History and development of soil microbiology
Antony van leeuwenhoek, a linen draper from Holland (1632-1723) is credited with having made the
first authenticated drawings of microorganisms which had its roots in the age of Aristotle (384-322
B.C) and which was revoked by the so-called experimental evidence of John Needham (1713-1781)
was scattered by the conclusive experimental findings of Louis Pasteur (1822-1895). This was followed
by Robert Koch’s Koch postulates (1843-1910) and the pure culture technique of Joseph Lister (1878).
Soil microbiology emerged a distinct branch of soil science, only in 1838 after the french agricultural
chemists and farmer, J.B. Boussingault showed that legumes can obtain nitrogen from air when
grown in soil which was not heated. Since then several developments made in soil microbiology is in
the following paragraphs.
History and significant development of soil microbiology
1838 J.B. Boussingault showed that Legumes can obtain nitrogen from air when
grown in soil which was not heated
1858, 1866 In 1858 Leishman & 1866 Woronin showed that Nodules are formed on the
roots of leguminous plants and bacteria are responsible for forming nodules
1879 Frank explained that Nodules on the roots of the legume plants are formed as a
result of inoculation with microorganisms which contributes for the fertility .
1886 Hillriegel and Wilfarth
Growth of non-legume plant was directly proportional to the amount of nitrogen
supplied
Bacteria in the root nodules of legumes accumulate atmospheric nitrogen and
made it available to plants.
A mutually beneficial association exists between bacteria (Rhizobia) and legume
root; legumes could utilize atmospheric nitrogen (1888)
Legumes took Nitrogen from air through the agency of bacteria existing in the
nodules of the roots
1888 Beijerinck, Isolated the root nodule bacteria in pure culture
1856 Liebig - showed that nitrates were formed in soil due to addition of nitrogenous
fertilizers in soil i.e., Nitrification
1890 & 1903 S. N. Winogradsky in 1890 and Omeliansky in 1903 found that anaerobic
bacteria degrade cellulose
1903 Lipman and Brown
Ammonification of organic nitrogenous substances by soil microorganisms and
developed the tumbler / beaker method for studying different types of
transformations in soil
1904 Hiltner coined the term "Rhizosphere" to denote that region of soil which is
subjected to the influence of plant roots. Rhizosphere is the region where soil and
plant roots make contact
1929 Starkey
1940 Lochhead
1946 Katznelson
1956 Rovira
1961 Matcura & associates
Studied the microorganisms in the Rhizosphere
1909 Russel and Hutchinson
Studied the importance of Protozoa in controlling bacterial population and
activity in soil
1918 Conn developed the technique for Direct soil examination using microscope
1927 & 1930 1927, Rossi & Cholodny in 1930 developed contact slide technique to study soil
microorganisms
Frank 1885 Coined the term mycorrhiza and found ectomycorrhizae in pine plants
1921- 27 Rayner & Melin -
Harley (UK )
Gerdemann
(Germany ) Studied on Mycorrhizal Fungi
Marx, Trappe &
Hacskaylo (USA )
Browen (Australia )
Mosse
1929 Alexander Fleming Discovered Penicillin and received Nobel prize in 1945
1931 Van Niel Studied soil bacteria and bacterial photosynthesis
1932 Fred & Associates Worked on nodule bacteria
1936 Garret Made the ecological classification of soil fungi
1940 Allen and Allen Studies soil bacteria in general and root nodule bacteria
1944 Waksman Discovered Streptomycin and received nobel prize in
1952
1945 Starkey Studied Iron bacteria
1947 Umbreit Studies the Problems of Autotrophy
1956 Ruinen Developed Phyllosphere Concept
Contributions of some important scientists

S.N. Winogradsky (1856-1953)

 Russian microbiologist who is often called the “Father of Soil Microbiology”

 In 1885, at the University of Strasbourg, he began his investigations on Beggiatoa, sulphur
oxidizing bacterium. Beggiatoa is a genus of Gammaproteobacteria belonging to the order
Thiotrichales and Pseudomonadota phylum. This genus was one of the first bacteria discovered
by Ukrainian botanist Sergei Winogradsky.
 1887, he worked at the University of Zurich. His investigations were concentrated on the
oxidation of nitrogen in the soil.
 Demonstrated the role of bacteria in nitrification process in 1890. Isolated two groups of
nitrifying bacteria
 He proved that the oxidation was a two step process: NH3+ → NO2- → NO3-
 and that each step was performed by different organisms
 1891 - Established the role of microorganisms in N transformation process.
 Discovered the autotrophic mode of life among bacteria and established the microbial
transformation of N and S.
 Winogradsky took up the problem of non symbiotic nitrogen fixation.
 He discovered and isolated a nitrogen-fixing obligate anaerobe growing below the surface in the
N free medium and identified the organism as Clostridium pasteurianum (1890).
 In 1895, took on the problem of retting flax and hemp & showed that retting could be done with
pure cultures of anaerobic bacteria, away from a stream
 Between 1926 and 1929, he took up a detailed study of aerobic organisms involved in cellulose
decomposition
 He was the first person to describe the fusiform, cellulose-degrading cells in the genus
Cytophaga
 By his studies on nitrification and sulfur oxidation the concept of microbial autotrophy, wherein
inorganic substrates are used as a source of energy for growth by microorganisms has been
established
 The first microbiologist to investigate the organisms found in complex biofilm communities &
developed the Winogradsky column
 He is pioneer in selective enrichment techniques
 To isolate organisms from nature ,he made a miniature model pond cross section which has
since been called as Winogradsky column
 Winogradsky column is a self-contained ecosystem ,where in which he studied the sulfur cycle
 Winogradsky also studied the consumption of hydrogen sulfide gas by sulfur-oxidizing bacteria
directly in their natural habitat
 Winogradsky also investigated microbial oxidation of ferrous iron, Fe 2+, the reduced from of iron
to ferric iron, Fe3+ an essential component of rust
Winogradsky and Beijerinck, 1890
 Developed the technique of enrichment culture making use of the principle of natural selection. It
is a technique in which environmental (including nutritional) conditions are controlled to favour
the development of a specific organisms or group of organisms.
 It involves the successive transfer of micro organisms in desired substrate for the isolation of
sparsely occurring unusual types of microorganism.

Martinus Willam Beijerinck was a Dutch microbiologist and botanist who was one of the founders
of virology and environmental microbiology
 The genus Spirillum was first reported by Beijerinck (1925), and decades later reclassified as
Azospirillum, because of its ability to fix atmospheric nitrogen (N 2), discovered and reported by
the group of Dr. Johanna Döbereiner in Brazil, in the 1970s

 1858- He was the first to isolate and describe Azotobacter and obtained the pure culture of
Azotobacter chroococcum and A.agilis.
 1888-obtained the pure culture of root nodule bacterium Rhizobium. Obtained the pure culture of
Thiobacillus thioparus, T.denitrificans and sulfur oxidizing bacteria
 1893 - He established the concept of microbial transformation of nitrogen.
Alexander Fleming (England)
 1929 - He discovered the Antibiotic Penicillin which is the important milestone in medical
microbiology and received Nobel prize for the same along with Howard Florey and Ernst Chain
in 1945 for physiology and medicine
 He found that natural substances / natural products are having antimicrobial activity. He reported
that Nasal mucous, saliva are having antimicrobial property due to the action of lysozyme.
 He worked with Staphylococcus aureus and observed the inhibition of growth of S. aureus in the
plate due to the growth of Penicillium notatum.
 Florey and chain latter isolated Penicillium sp producing antibiotic in pure culture.
Selman A. Waksman
Selman Abraham Waksman (July 22, 1888 – August 16, 1973) was a Jewish Russian-born American
inventor, Nobel Prize laureate, biochemist and microbiologist. 

 1922-Isolated Thiobacillus thioxidans

 1927- He published a book on "Principles of Soil Microbiology".
 1939- Identified the soil organism producing antibiotics
 1942 , he showed the importance of soil as the source of antagonistic organisms.
 Discovered the antibiotic streptomycin (1944) for tuberculosis caused by Mycobacterium
tuberculosis and received Nobel prize for the same in 1952
 He discovered Streptomycin, Neomycin, Actinomycin antibiotics.
 Studied variety of biochemical reactions carried out by soil microorganisms while decomposing
organic matter.

LECTURE #2. SOIL MICROORGANISMS AND THEIR ROLE IN SOIL FERTILITY AND
CROP PRODUCTION

Soil microorganisms include bacteria, actinobacteria, fungi, protozoa, algae, viruses and sub viral
particles. Among these organisms bacteria dominates in soil ecosystem, followed by fungi,
actinobacteria, algae and protozoa. Soil bacteria, fungi and actinobacteria are involved in
biogeochemical cycling of nutrients in soil.
Soil microorganisms can be classified broadly into two groups based on their ecology
Ecological classification of soil microorganisms (Winogradsky)
1. Autochthonous or indigenous population,
2. Allochthonous or zymogenous or introduced microorganisms.
Based on their nutritional requirement, soil microorganism’s particularly bacteria are classified
further as given below.
Nutritional classification of bacteria
Inorganic source Organic source
Carbon Autotroph (CO2 , carbonate & Heterotroph (Glucose, sucrose,
bicarbonates) disaccharides, polysaccharides)
Reducing Lithotroph (H2O, H2S, S etc) Organotroph (any organic compounds)
power
Energy Chemotroph (deriving energy by Phototroph (deriving energy from
oxidation of reduced inorganic light)
compounds)

Distribution, importance of soil microorganisms and factors influencing the activities of

microorganisms in soil
Soil contain five major groups of microorganisms. Bacteria, Actinobacteria, fungi, algae and
protozoa. The soil ecosystem includes these microbial groups as well as the inorganic and organic
constituents of a given site. The collections of cells represented in the community are considered as
distinct populations. All the inhabitants of the particular locality make up the community.
I.Bacteria are the most dominant group of M.O in soil and more numerous than the other four
combined. They present in all types of soil but their population decreases as the depth of soil
increases (Horizon A > B > C). The number of cells of bacteria in the soil is always great, but the
individuals are small, (µm in length). Because of the minute size of the bacteria and large cells or
filaments of the other 4 groups, the bacteria probably account for appreciably less than halt of the
total microbial cell mass. In aerated soil, B, will be dominating, alone are responsible for all the
activities in environment lacking O2 or little O2. In transformation process Bacteria stand first, due to
their rapid growth and capacity of vigorous decomposition of variety of substrates.
Soil microbiological population has been divided into two broad groups. Ecological classification of
microorganisms (Winogradsky’s classification) 1. a. Autohcthonous (Indigenous) and b.
Zymogenous or fermentative group of microorganisms (Allochthonous or introduced).
a. Autochthonous or native microbes. Indigenous, which are characteristic of the particular soil and
which many be expected always to be found there.eg.Arthrobacter
b. Zymogenous, or fermentative organisms require an external source of and their normal
population in soil is low (Pseudomonas, Bacillus). When specific substrates are added to soil, the
 population is increased . Then gradually declines when the added substrate is exhausted
eg.cellulose decomposers. N utilizing bacteria,nitrifiers etc.,
Bergey's marginal; of determinative bacteriology is universally used for the classification of Bacteria.
Nutritional classification of microorganisms
Based on carbon requirement
1. Autotrophs – Inorganic carbon utilizing microorganisms
2. Heterotrophs – Organic form of carbon utilizing microorganisms
Based on energy requirement
1. Phototrophs - derive energy from sun light
2. Chemoautotrophs - energy by oxidation of inorganic chemicals
Based on source of reducing power requirement
1. Lithotroph –Inorganic source of reducing power
2. Organotroph –Organic compound as a source of reducing power
Nitrogen (Nitrobacter and Nitrosomonas), sulphur (Acidithiobacillus sp) and iron transformation
(Ferrobacillus ferroxidans) of soil depend on the chemoautotrophic organisms
b) Based on the O2 requirement
Aerobic - Need 20% (100 % atmospheric O2) for growth
Anaerobic - Complete absence of O2 for growth
Facultative anaerobic- Basically aerobic: but could growth in the absence of oxygen
Facultative aerobic- Basically anaerobic; but could growth in the presence of some amount of oxygen
c) Based on cell anatomy
Bacilli - Rod shaped
Cocci - Spherical shaped
Spirillum - Spiral shaped
Vibrio - less than one twist
Pelomorphic – definite shapeless oraganisms
Abundance of Bacteria in extreme environmental conditions
In Arctic regions - frozen 9-10 m/year, bacterial counts of 10 6/ g observed even when temperature
remains below freezing point. Such organism are in a state of dormancy awaiting the spring to
recover. Desert soil is another extreme. Spore forming bacilli are often predominant in oven dry state.
II. Actinobacteria
These are gram positive filamentous bacteria. They share the characteristics of both bacteria and
fungi.
Fungal like characteristics
1. Produce mycelium with extensive branching. Like fungi many actinobacteria form aerial mycelium
and conidia
2. Growth in liquid medium do not resemble (which bacteria is turbid), but form clumps and pellets
like fungi.
3. They show unique colony morphology of small powdery growth
Bacterial characteristics
1. Resemble in all respects both morphologically and physiologically including susceptibility to virus
attack.
2. Actinobacteria differ from fungi that they do not have chitin and cellulose which are commonly
found in the cell walls of fungi
3. The colony characters are not similar to bacteria. Some species have flagella that resemble those of
true bacteria. The cell wall is similar to in cell wall composition and sensitivity to antibacterial
compounds.
Common genera in soils
Streptomyces (70 %), Nocardia, Micromonaspora, Frankia. Actinobacteria are sensitive to
antibacterial compounds and not to antifungal compounds
Streptomycetes
Musty odour they elaborate; an odour reminiscent of freshly turned soil, the metabolite of
Streptomyces formed geosmin and other volatile products elaborated by Streptomyces are responsible
for the characteristic earth odor/ smell.
Distribution
Actinobacteria are numerous and widely distributed not only in soil but in a variety of other habitats
including composts, river muds and lake bottoms. Present in surface soil and also in lower horizons
to considerable depth. In abundance they are second only to the Bacteria and the viable counts almost
equal to both. Saprophytic existence, but a few species can cause diseases of plants, domestic animals
and even humans.
Isolation
Population is 105 to 108 / g in temperate zone, but lower counts in waterlogged soils, acid peat,
arctic, Tundra regions. In alkaline areas, especially when dry, the relative abundance is high.
Importance
 Decomposition of resistant components of plant and animal tissue.
 Transformations at high temperature particularly in the manure and compost pits – thermophiles
dominant
 Cause certain soil borne diseases of plants, potato scab of apple, sweet potato pox.
 cause infection to humans, animals.
 Importance in microbial antagonism by production of antibiotics and production of enzymes.
 Antibiotic Industry
III. Fungi
As the important constituent group of the soil population, they are widely distributed in most well –
cultivated soils. Fungi account for a large part of the total microbial population. Though they are not
the major inhabitants, they do infact makeup the significant part of he biomass because of the large
diameter and extensive network of the filaments. Fungi exist in soil in the form of vegetative
mycelium and spores. Characteristically the fungi possess a filamentous mycelium network of
individual hyphal stands. The hypha itself is rather broad and has a diameter appreciably greater than
that found in the common actinomycetes. In nature consider or asexual spores are abundant and
widespread, the sexual spores relatively uncommon. In contrast with bacteria the high can be
effectively differentiated into Genera and species on the basis of morphology.

Distribution and Abundance

Estimates of microbial density reveal the presence in soil is ranging a few as 20,000 to as many as
1,000,000 fungal propagules per gram, the propagule being considered as any spare, or hyphal
filament that is capable at giving rise to a colony. The length of the fungal mycelium has been
reported to range from 10 to 100m per g surface soil, but various up to 500 and sometimes in excess
of 1000mt have also been obtained. Assuming the dilanent has an average diameter of 5µm and a
specific gravity of 1.2 and taking the range of 10 to 100 m per gram. It would appear that the weight
of fungi ranges from 500 to 5000 kg per ha of surface soil. Thus the filaments make up a significant
part of the soil mass. Individual species and genera have been recorded in diverse and highly
dissimilar habits. They are the inhabitants of peats, flooded soils planted to rice, regions with low and
extremely high salt contents, locations in many deserts, sits in Antartica, as well as in the tundra.
Importance
 Degrade complex molecules-cellulose, hemicellulose, pectin, starch, lignin
 Improve soil structure-aggregate formation
 Pathogenecity in plants,animals and humans
 Predator against soil protozoa-Hyphal penetration reduces mobility,total cessation of movement
 Trapping nematodes-Arthrobotrys,Dacylaria,Dactylella,Harposporium
 Beneficial association with plants-Mycorrhizal fungi

IV. Yeast
Non filamentous fungi, their presence maybe demonstrated in most soils. Soil yeasts include
Candida, Debaryomyces, Rhodotorula, Torulopsis. Yeasts have been found in comparable numbers
in soils of Antarctica, grasslands, cultivated fields, and forests.
Function and activity
 Involved in the degradation of complex molecules. They can utilize and degrade the major plant
constituents – cellulose, hazicellulose, pertins, starch and lignin.
 Participate in humus formation from fresh organic residues.
 Carry out inorganic transformations (T.V) and influences the formation of stable aggregates by
means of hyphal penetration and the mechanical binding of the particles.
 Pathogenecity is common character associated with several soil borne Fungi Fusarium,
Helminthosporium.
 Cause disease among humants, animals
 Predator against protozoa. The hyphae penetrates the protozoan with a resulting decrease in
motility of the animal and an eventual total cessation of movement. The F slowly digests the
cellular components and assimilates the substances released.
 Nematodes are also trapped by the F by hyphal extensions. Eg: Arthropotrys, Dactylaria,
Dactylella, Harposporium
 Involved in beneficial association with higher pls. – Mycorrhiza – nmt. Mobilisation.
IV. Algae
They occur in small numbers in soil. They are photosynthetic organism. Abundant in habitats where
in moisture is adequate and light is accessible. Isolation can be done in liquid media and by planting
with agar media. Because of their similarities to bacteria, the BGA are sometimes classified together i
the bacteria.
Photoautotrophic nutrition
In the absence of light heterotrophy occur in some species of chlorophyta, cyanophyta and diatoms.
These species use the oxidation of organic carbon to replace the light in supplying energy for
anabolic process. Occurrence of this photosynthetic microorganism has been recorded in soils
throughout the world. Algae are moderately adaptable to environmental changes; persist in
unfavorable circumstances such as in alkaline and desert soils. Some species colonies the zone under
the surface crusts of limestone and sard store rocks in the desert, liking at sites where the heredity is
retained and where sufficient light patently.
 Enumeration by MPN
 100 to 50,000 / g soil
 biomass – 7 to 300 kg / ha (Chlorophyll ext).
 flooded pady field is the environment algae could have a great agronomic significance. The
microbial action may be associated with the utilization of aton, N, the release of O 2 or the
excertion of products stimulating plant developments. Under water logged rill soils, an algal
film farms at the liquid surface, make up an appreciable mass.
Common inhabitants : Anabaena, Calotheix, Noster, Oscillatonia.
Importance
 Involved in CO 2 fixation
 Colonizes the barren surface and corrode and weather rocks-Contribute to soil formation
 Algal layer covers the rocks ,on death it favors secondary colonizers
 Surface bloom reduces erosion losses probably by binding together with soil particles
 Improve soil structure, texture and add fertility to soil after decay
 Photosynthesis liberates 02 , provides 02 to the roots and other m.o
 N2 fixation and ammonia excretion
 N 2 is released after decomposing of algal biomass
 Production of metabolites
 SCP Production ,B carotene
V. Protozoa
The subterranean faith a contains protozoa, earthworms, nematodes, insects. Most abundant one is
protozoa, the simple from of animal life. Primitive, unicullular organic majority of are dependent on
preformed O.M. as aprobic feedero obtaiing their nutrition from soluble organic and inorganic
substances, or by a phagotrophic ntrution (direct feeding upon microbial cells). They maintain the
equlibrium by enguliting.
 Regulate the bacterial community
 Maintain the biological equilibrium in soil
 Participate in the decomposition of plant remains
LECTURE # 3. ASSESSMENT OF MICROBIAL DIVERSITY. FACTORS INFLUENCING THE
ACTIVITIES OF SOIL MICROORGANISMS. ROLE OF SOIL ENZYMES IN NUTRIENT
TRANSFORMATION

Microbial diversity indices

Measures of diversity are frequently seen as indicators of the well-being of any ecosystem. They also
serve as a measure of the species diversity in the ecosystem. The following indices are normally used
to assess and compare the diversity and distribution of different microorganisms at different
locations.
After assessing the microbial density, the diversity is calculated using various indices. The alpha
diversity indices viz., richness, evenness and dominance are calculated using the following standard
formulae
Richness indices
It can be calculated by assessing any one of the three indices namely a. Species number; b.
Margalef’s D and c. Shannon diversity index
a). Species number (Magurran, 1987)
This represents the total species number in each sample
b).Margalef’s D (Clifford and Stephenson, 1975)
Margalef’s D has been favourite index for many years. It is calculated as the species number minus
one divided by the logarithm of the total number of individuals. This program uses the natural
logarithm.
Dmg = (S – 1)/ ln N
Where, S = the number of species recorded; N = the total number of individuals summed overall S
species
c).Shannon diversity index (Batten, 1976)
This presents the Shannon-Wiener (also known as Weaver) diversity index for each sample and is
defined as : H’ = åpiln pi
Where, pi = the proportion of individuals in the ith species
This program calculates the index using the natural logarithm.
Dominance indices
a) Simpson’s index (Simpson, 1949)
Simpson’s index describes the probability that a second individual drawn from a population should
be of the same species as the first.
D = å[Ni (Ni – 1)] / [NT (NT – 1)]
Where, Ni = the number of individuals in the ith species and NT = the total individuals in the sample
So the larger the value of Simpson index, the greater the diversity. The statistic 1 – C gives a
measure of the probability of the next encounter (by the collector or any animal moving at random)
being from another species (Hurlbert, 1971).
b) Berger parker diversity index (Berger Parker, 1979 and May, 1975)
A simple dominance measure is the Berger Parker index. The index expresses the proportional
importance of the most abundant species.
D = Nmax / N
Where, Nmax = the number of individuals in the most abundant species
N = the total number of individuals in the sample
This simple index was considered by May (1975) to be one of the best indices. It is a simple measure
of the numerical importance of the most abundant species.
c) McIntosh index (McIntosh, 1967)
This index is calculated using the following formula Proposed by McIntosh (1967) as
D = N – U / N – Ö(N)
U = Ö (åni2)
Where, N = the total number of individuals in the sample
U is given by the expression
U = Öå [n (i)]
Where,n (i) is the number of individuals in the i th species and the summation is undertaken over all
the species.
Evenness indices
Evenness (E), is a measure of how similar the abundances of different species or categories are in a
community, when all species in a community are equally abundant. The evenness index should be
greater and decrease towards zero as the relative abundance of the species diverges away from
evenness.
a) Shannon’s equitability (Pielou, 1969)
Equitability or evenness refers to the pattern of distribution of the individuals among the species.
This measure of equitability compares the observed Shannon-Wiener index against that distribution
of individuals between the observed species which would maximize diversity. If H ’ is the observed
Shannon-Wiener index, the maximum value is log (S), where S is the total number of species in the
habitat. Equitability assumes a value between 0 and 1 with one being complete evenness. Therefore,
the index is
H’ H’
EH =------- = -------
Hmax InS

Where, H = Shannon Wiener index

b) McIntosh equitability (Pielou, 1969)
McIntosh equitability (E) is calculated using the following formula
N–U
E = -------------
N – N/ÖS
Where,N = The total number of individuals in the sample
U is given by the expression
U = Öå [n(i)]
S = The total number of species
Role of microbial diversity in soil health and plant nutrition
In nature, plants live in a microbe-rich environment and must interact with a myriad of pathogenic,
commensal, and beneficial microbes. How plants harness the beneficial functions provided by
microbes and, at the same time, combat microbial pathogens was the question for years together.
Extensive molecular studies conducted since the early 1980s have revealed a number of basic
principles underlying plant-microbe interactions. Among them are discovery of (1) signals from
microbes that are perceived by cognate plant immune receptors to initiate defense or symbiotic
responses, (2) microbial DNA and/or protein secretion systems that transport effector molecules into
the plant cell to modulate host cell functions , (3) microbial and plant developmental programs that
orchestrate the formation of specialized nutrient-exchanging/producing organs (e.g., nodules and
galls) during symbiotic and pathogenic interactions, and (4) binary and community-level antagonistic
warfare in plant-microbiota interactions.
Microbiome density and diversity
In the rhizosphere, the microbial density is typically higher than in bulk soil and ranges from 10 8 to
109 bacteria per gram. However, soil microbial communities are considered to hold the most diverse
microbial communities in the world, with up to 104 bacterial species per gram. Root microbiomes of
plants grown in the same soil differ between plant species and between genotypes within a species.
However, the existence of classifiable ‘rhizotypes’ has not yet been reported.
Effects on plant nutrition and health
1. Aids in nutrient uptake: Rhizobia and mycorrhiza assist plants with the uptake of nitrogen
and phosphorus, respectively. Furthermore, the rhizosphere microbiome assists in the
weathering of minerals and the degradation of recalcitrant organic matter. In return, the
microbiome is provided with carbon in root exudates and other rhizodeposits
2. Prevents colonization by pathogens : Mechanisms include competition for (micro) nutrients,
production of antibiotic compounds or lytic enzymes and consumption of pathogen
stimulatory compounds
3. Modulates host immunity: Many beneficial soil-borne microorganisms have been found to
boost systemically the defensive capacity of the plant. This induced systemic resistance (ISR)
is a state in which the plant immune system is primed for accelerated activation of defense
4. Provides tolerance against biotic and abiotic stresses
5. Distinguishes friend from foe: Symbionts and pathogens express similar molecular patterns
that are recognized by the innate immune system, and it is largely unknown how plants
distinguish friend from foe. Both pathogens and beneficials are also known to suppress plant
immune response to promote their own colonization through secretion of effector molecules.

Factors affecting microbial activities in soil

Several environmental conditions affect the density and composition of the microflora and
frequently alter their activities in soil. Primary factors include moisture, aeration, temperature, pH,
organic matter, inorganic fertilizers. Lesser variables are the secondary factors which include crop
rotation season, soil depth, cultivation practices etc.
I. Primary factors
Soil moisture
Soil moisture is one of the important factors influencing the microbial population. Water is the major
component of protoplasm, an adequate supply must be available for vegetative development. But,
when it becomes excessive, proliferation is suppressed due to limitation in gaseous exchange and
lowers the availability of O2 supply creating anaerobic environment. Most of the organisms prefer a
moisture percentage between 20 and 6 per cent. Many bacteria and fungi are able to adjust
themselves to different moisture conditions. Under dry conditions, the bacteria may form spores
which can resist the drought conditions. The fungi may sporulate or form chlamydospores to tide
over adverse conditions. The protozoans also may form cysts and can survive under dry conditions.
Actinobacteria are the chief group of organisms that prefer dry conditions.
At moisture, it is believed that the concentration of nutrients are diluted and also the aeration is very
much limited and hence only the anaerobic and microaerophilic organisms can develop better.
Soil air : It is directly linked up the moisture level of the soil. Most of the forms are active in aerated
soil and under waterlogged soils anaerobic and microaerophilic forms develop.
Soil temperature : exhibit considerable influence on the microbial population. Though
microorganisms have been found to exist under extreme temperature conditions, such as –60°C and
+60°C the soil temperature usually does not reach such extremes. Microbial population varies both
quantitatively and qualitatively under extreme conditions. In tropical and subtropical regions,
temperatures vary widely in summer and winter and the population may also be varied. In
temperature regions, not much variation in temperatures of summer and winter and not much
variation in the soil population. Soil temperature influences the temperature of air, water and solid
phase of the soil. Soil water and temperature excert a combined influence on the microbial
population.
Soil organic matter : Community size is related to the organic matter content, so that humus rich
localities have the largest biological numbers. Organic matter content varied with soil types from less
than 0.5 per cent in desert soils to 40 per cent in peaty soils. 0. M being the chief source of energy
and food for most soil organisms, it has great influence on the population. Nature of 0.M is
responsible for the differential stimulation of the population. There are several indirect effects of the
organic matter on soil microflora. It influences the structure and texture of soil, besides enriching
with nutrients for plants and microorganisms. Such influences on the soil also greatly affect the
activity of the soil microorganisms.
Soil pH : It is a key factor influencing the microflora of soil. It influences enzyme system and thus
plays a important role in microbial activity. In general fungi thrice better then bacteria and
actinomycetes in acid soils. Bacteria flourish well in neutral and alkaline soils. The saline and
alkaline soils have different microflora. Salinity is due to excess salts, alkalinity is due to high H-ion
concentration. Several direct and indirect effects of H-ion and salt concentration in soil are exerted on
microbial population.
Fertilizer application : Application of fertilizers, to the soil improves the microbial activity because
of the availability of more readily obtainable nutrients. Some fertilizers, may however have inhibitory
effect on specific bacterial types. Continuous application of ammoniacal fertilizers favour the growth
of fungi due to the formation of nitric acid and which inhibits the growth of bacteria and
actinobacteria. Addition of nitrate inhibits the activity of free living N2 fixing bacteria like
Azotobacter. Some of the autotrophs are encouraged by the addition of fertilizers.
Cropping and vegetation : Two kind of effects are exhibited by the crop plants. One is through root
exudates, which may have different compounds with reference to the crops grown vegetation have
selective stimulation over population. Continuous cultivation leads to more microbial activity than
the uncultivated land.
II. Secondary factors
 Crop rotation : Crop rotation with different species like legumes, graminaceous plants, etc.
brings about different stimulatory effect on the microflora. Some crops have deeper roots than
others and some are more fibrous. Such variations bring about physical changes in soil which
in turn may have direct and indirect stimulatory effects on the soil microflora.
 Cultural practices : Various cultural practices, such as tillage operations and irrigation have
several physical and chemical changes in soil which are reflected on the soil microflora.
Through subsoil ploughing deeper layers may get better aeration and there may be quicker
multiplication of aerobic organisms. Weeding, irrigation etc. may influence the microbial
populations in the soil.
 Soil depth : Most of the organisms are almost in top layers, largely in upper few centimeters
and decline with greater depth, more active at few cm down and less active at deeper layers.
Low O2 and less sunlight in deeper layers reduce population.
 Season : Cell number are greatest during the spring and autumn and a decline occur during
hot, dry and winter, the cells remain in a state of dormancy for biochemical inactivity. The
influence is due to mainly the alterations in temperature, moisture as well as availability of
organic matter.
III. Specific influences
 Sunlight favours algae and other autotrophs.
 Herbicide application had devasting effect on algae.
 Attach by neighbours – protozoa, nematode, earthworms consume algae.
 Presence of parasites and predators – eg. viruses eat on specific bacteria
 Increase in bacterial population increases protozoans also.
IV. Other factors
 Burning of top soil : Leads to partial sterilization of the top soil and may kill protozoan
population, which may lead to the increase in bacterial population. This condition may affect
biological equilibrium in soil.
 Application of nematicide, fungicide and bactericide may exhibit partial sterilization.
Thus every change in crop production direct or indirect, alters the soil microflora, still many
more are not clearly understood.
Soil Enzymes
Soil enzymes are a group of enzymes whose usual inhabitants are the soil and are
continuously playing an important role in maintaining soil ecology, physical and chemical properties,
fertility, and soil health. These enzymes play key biochemical functions in the overall process of
organic matter decomposition in the soil system. These enzymes may include
1. Amylase
2. Arylsulphatases
3. β-glucosidase
4. Cellulase
5. Chitinase
6. Dehydrogenase
7. Phosphatase
8. Protease and
9. Urease
These enzymes may be constitutive or inducible. Constitutive enzymes are always present in nearly
constant amounts in a cell eg. Pyrophosphatase. Inducible are present only in trace amounts or not at
all, but quickly increases in concentration when its substrate is present eg. Cellulases. Both types of
enzymes are present in the soil
Soil Enzymes as Indicators of Soil Health
Enzymes are the direct mediators for biological catabolism of soil organic and mineral
components. Thus, these catalysts provide a meaningful assessment of reaction rates for important
soil processes. Soil enzyme activities (1) are often closely related to soil organic matter, soil physical
properties and microbial activity or biomass, (2) changes much sooner than other parameters, thus
providing early indications of changes in soil health. In addition, soil enzyme activities can be used as
measures of microbial activity, soil productivity, and inhibiting effects of pollutants. These include
dehydrogenase, glucosidases, urease, amidases, phosphatases, arylsulphatase, cellulases, and phenol
oxidases.
Soil Enzymes as Indicators of Soil Health
Soil enzyme Enzyme reaction Indicator of microbial activity
Dehydrogenase Electron transport system C-cycling
β-glucosidase Cellobiose hydrolysis C-cycling
Cellulase Cellulose hydrolysis C-cycling
Phenol oxidase Lignin hydrolysis C-cycling
Urease Urea hydrolysis N-cycling
Amidase N-mineralization N-cycling
 Phosphatase Release of PO4 P-cycling
Arylsulphatase Release of SO4 S-cycling
Soil enzymes Hydrolysis General organic matter degradative
enzyme activities

Soil dehydrogenase
The dehydrogenase enzyme activity is commonly used as an indicator of biological activity in soils.
It is an intra cellular enzyme and does not accumulate extracellularly in the soil. This enzyme is
known to oxidize soil organic matter by transferring protons and electrons from substrates to
acceptors.
 LECTURE NO.4 : CARBON CYCLE. ROLE OF SOIL MICROORGANISMS IN THE
DECOMPOSITION OF ORGANIC MATTER IN OXYGENIC AND ANOXYGENIC
ENVIRONMENTS; HUMUS FORMATION

Carbon cycle
The term soil generally refers to the loose material of the earth surface and is the region that supports
the plant life. It consists of five major components such as mineral matter, water, air, organic matter
and living organisms. The proportion of these components, varies with soil type and other soil
conditions. To maintain the level of these components it is essential that they undergo a regular
process of recycling. This process of recycling through various transformation is brought about by
different microorganism
 Soil organic
matter

Plant and algal Aerobic

Photosynthesis decomposition

Carbon dioxide
Aerobic condition

Methanotroph Carbon dioxide

Anaerobic condition
Methane
Organic acids
Anareobic
through decomposition
Fermentation
Bactreial Photosynthesis
Soil organic
matter

The most important element in the biological realm and substance that serve as the cornerstone of the
cell structure is carbon. It constituents about 40-50% of all living organisms, yet the ultimate source
is the CO2 that exist in a perennially short supply, only 0.03% of the earth’s atmosphere, which
undergo a cyclic change from an oxidized to reduced state. Carbon (CO 2) is constantly (reduced into
organic carbon compounds) being fixed into organic form by photosynthetic organisms
(photosynthesis). Once bound, the carbon becomes unavailable for use in generation of new plant
life. It is thus essential for the carbonaceous materials to be decomposed and returned to the
atmosphere. It is estimated that 1.3x1014 kg CO2 is fixed annually in the biosphere. To the lesser
extent CO2 is also fixed through the agency of photosynthetic bacteria and other chemolithotrophs
with the convertion of so much of the plant available carbon to organic form each year and the
limited supply in the air, it is apparent that the major plant nutrient element would become exhausted
in the absence of microbial transformation.
The carbon cycle revolves about CO2 and its fixation and regeneration. The green plants utilize CO2
as their sole carbon source, and the carbonaceous matter synthesized serves to supply carbon to other
heterotrophic organisms and animals. Upon the death of plants and animals, microbes assume a
dominant role in carbon cycle. The dead tissues are degraded and transformed into microbial cells
and humus or soil organic fraction. Further decomposition of these materials leads to the production
of CO2 and once again it is recycled.
Organic matter decomposition (Aerobic decay)
Soil organic matter
Organic matter subjected to microbial decay in soil comes from several sources. Plant residues, forest
litter, incorporation of plant tissues, animal tissues, excretory products. The chemistry of organic
matter is clearly very complex, and investigations of the transformations and the responsible
organisms have therefore been extremely interesting. The organic constituents of the plants are
commonly divided into six categories.
a) Cellulose - Most abundant 15-60% of the dry weight
b) hemicellulose - 10-30% of the plant dry weight
c) lignin - 5 to 30 % of the plant dry weight
d) Water soluble fraction - 5-30%, included simple sugar, a. acids,
e) ether and alcohol soluble constituents, a fraction containing fat, oils, waxes, resins and a number of
pigments
f) proteins.
As the plant ages, the content of water soluble constituents, proteins and minerals decreases and the
% of abundance of cellulose, hemicellulose and lignin rises.
Soil organic matter comprises residues of plant and animals and these compounds occur in soil in
close combination with inorganic substances. Animals and plant residues are made up of complex
carbohydrates, simple sugars, starch, cellulose, hemicellulose, pectins, gums, mucilage, proteins fats,
oils, waxes, resins, alcohols, aldehydes , ketones, organic acids, lignin, phenols, tannins,
hydrocarbons, alkaloids, pigments etc.
 The soil microorganisms play important role in the decomposition of soil organic matter.
 Bacteria are the dominant group – mostly heterotrophic organisms (use energy from organic
sources such as sugars, starch, cellulose and protein) – are involved. Autotrophic organism which
occupy a small portion of the biomass in soil (and use inorganic sources such as Fe and S) are not
directly involved in organic matter decomposition.
 Actinobacteria grow on complex substances such as keratin, chitin and other complex
polysaccharides and play active role in humus formation.
 Soil fungi are mostly heterotrophos and use organic residues easily
 Soil algae contribute a small amount of organic matter through their biomass, but they do not have
any active role in organic matter decomposition.
Organic matter decomposition serves two important functions
a) Provide energy for growth
b) Supply carbon for the formation of new cell materials
Hence only heterotrophs are actively involved in the process of decomposition. The relationship
between organic matter and plant growth may be direct or indirect.
 Organic matter is a natural substrate for saprophytic micro organism and provides nutrition to
plants indirectly through the activity of soil microorganisms
 It is essential for the formation of soil aggregates and hence soil structure which ultimately
determines the soil aeration and rooting habit of plants
 Organic matter helps in the conservation of soil nutrients by preventing erosion and surface
run off of nutrients.
Carbon assimilation
The process of converting substrate to protoplasmic carbon is known as assimilation. Under aerobic
conditions 20-40% of the substrate carbon is assimilated, the remainder is released as CO 2. Fungi are
more efficient, in their metabolism, since they convert carbon into cell carbon as filaments and
releases less CO2. Around, 30-40% carbon s used to form new mycelium during the decomposition.
Compared to fungi, bacteria are less efficient. Aerobic bacteria are less efficient than anaerobic
bacteria.
C Mineralization
It is the process of conversion of organic carbon substance to inorganic form of carbon.
Immobilization
Assimilation of nutrients and is the mechanism by which micro organism reduce the quantity of plant
available nutrient in soil. Mineralization is considered good than immobilization.
During the decomposition of organic matter three separate simultaneous processes can be
distinguished. The important changes during decomposition are:
1. Plant and animal tissues constituents disappear under the influence of enzymes
2. Synthesis of new microbial cells so that proteins, polysaccharides and nucleic acids typical of
bacteria and fungi appear.
3. Third, certain end products of the breakdown are excreted into surroundings there to
accumulate or to be further metabolized.
Importance of organic matter decomposition
1. Important function is the breakdown of organic matter by which CO 2 available for
photosynthesis is replenished
2. Any compound that is synthesized biologically is subject to destruction by soil inhabitants,
otherwise the compounds would have accumulated in vast amounts on the earth’s surface
3. Since, organic matter degradation is a property of all heterotrophs, it is commonly used to
indicate the level of microbial activity.
Methods to evaluate the decomposition rate
a) Measurement of CO2 evolution or O2 uptake
b) Determination of decrease in organic matter either chemically or by weight loss
c) Observations on disappearance of specific constituents such as cellulose, hemicellulose or
lignin.
Changes during organic matter decomposition
As a result of development of mixed flora on chemically complex natural products, some components
quickly disappear while others are less susceptible to microbial enzymes and persist. The water
soluble fraction contains the least resistant plant components and is thus the first to be metabolized.
Cellulose and hemicellulose on the other hand disappear not as quickly as water soluble substances,
but their persistence usually is not too great. The lignin’s are highly resistant and consequently
become relatively more abundant in the residual, decaying organic matter.
 Under aerobic conditions when carbonaceous substrates are incorporated into soil, immediate
drop in O2 and an increase in CO2 content of soil air occurs.
 Change in (O) (H) oxidation reduction potential (Ev) – it is shifted to a more reduced
condition (fall in oxidation reduction potential).
The end products of decomposition are humus, CO2, H2O, NO3, SO4, CH4, NH4, H2S and
microorgansisms depending on the availability of air.
Factors influencing the organic matter decomposition
Organic matter level of the soil
Cultivation
Temperature
Moisture
pH
Depth
Aeration
Nature and abundance of micro organic involved.
The extent of availability of C, N, P and K presence of inhibitory substance.
C:N ratio
 Nitrogen is a key nutrient substance for microbial growth
 If N content of the substrate is high it is readily utilized and decomposition is faster
 If N is poor decomposition is slower, needs additional N
 Protein rich substrates are readily decomposed
 Low N or wide C:N ratio results slow decay
 Optimum level of C: ratio for maximum decomposition is 20-25(1.4-1.7% N)
  Less than this range, more microbial cells, faster mineralization and it likely exceeds
immobilization
 Wider the ratio, lesser microbial cells, slower the immobilization and mineralization
increases gradually, resulting in accumulation of Ammonia and Nitrates
 Microbes scavenge the soil solution to obtain enough N
 At optimum level, there must be an equilibrium between Mineralization and Immobilization
 Soil N level constrains the maintenance of C:N (organic carbon /soil o.m)
 To make sound soil management
 Arable surface -10:1
o Sub soil -lower
Anaerobic decay / decomposition
The main products of aerobic carbon mineralization are CO2, water, cells and humus components. In
the absence of O2 organic carbon is incompletely metabolized, intermediary substances accumulate,
and abundant quantities of CH4 and smaller amounts of H2 are evolved. Energy yield during
anaerobic fermentation is low, resulting in fewer microbial cells per unit of organic carbon degraded.
Consequently, organic matter breakdown is consistently slower under total anaerobiosis than in
environments containing adequate O2. The rate in water logged soils is intermediate between the two
extremes.
When a soil is water logged or flooded there is a shift from aerobic to anaerobic transformation.
Formation and accumulation of organic acids viz., acetic, formic, butyric, lactic and succinic acids
appear too, these are frequently detrimental to root development. Organic acids accumulate because
of the fermentative character of the microflora of wet soils. The an aerobic carbon transformation are
thus characterized by the formation of organic acids, alcohols, CH4 and CO2 as major end products.
Under anaerobic conditions, decomposition of organic residues takes place by the activity of both
mesophilic, thermophilic microorganism resulting in the production of CO 2, H, ethyl alcohol, and
organic acids. Among mesoophilis, bacteria are more active than fungi or actinomycetes in
cellulolytic activity. They belong to genus Clostridium which are numerous in manure pits but rarely
encountered in cultivated arable soils. In compost pits both meso and thermopholic (bacterial and
actinomycetes) are important in the breakdown of cellulose substances.
The primary microbial colonizers initially breakdown the complex carbohydrates and proteins into
organic acids and alcohols. At a later stage, the methane bacteria which are strict anaerobes begin to
act upon the secondary substrate chiefly lactic and butyric acids and ferment them into CH4 and CO2.
Humus
 A dark coloured and fairly stable soil organic matter with known and unknown physical and
chemical properties
  It is an integral part of the organic matter complex in soil
 Humus can be defined as lingo protein complex containing approximately
o 45 % lignin compounds
o 35% amino acids
o 11% carbohydrates
o 4% cellulose
o 7% Hemicellulose
o 3% fats, wax, resins
o 6% other miscellaneous substances, including plant growth substances and inhibitors.
 Age and composition of the humus are dependent on its origin and environment.
 Bacterial and algal protoplasm contribute a good deal to the nutritive value of humus
 Soil micro organism take part in humus formation. Some fungi such as Penicillium,
Aspergillus and actinomycetes produce dark humus like substances which serve as structural
units for the synthesis of humic substances.
benefits of humic substances
 Improved seed germination, root growth, uptake of minerals by plants and other physiological
effects on plant growth
 Increases the enzyme activities involved in plant metabolism. Since humic acid serves as
hydrogen acceptors.
 Increases the cytochrome oxidase activity in root systems results in growth stimulatory effect
(on roots)
 Chelating effect – on trace elements Fe uptake by roots
 Vigour and yield of plants enhanced
 Humic acid known to influence the grown and proliferation of micro organism
 Aspergillus niger, Pencillium, Bacillus sp., Azotobacter are enhanced
The organic fraction of soil, often termed humus. It is a product of synthetic and decomposing
activities of the microflora. Since it contains the organic C and N needed for microbial development,
it is the dominant food reservoir. Because humus is both a product of microbial metabolism and an
important food source, the organic fraction is of special interest.
Humus formation
 Once the plant or animal remains fall on or are incorporated into the soil, they are subjected
to decomposition
 From the original residues, a variety of products are formed
  As the original materials and the initial products undergo further decomposition, they are
converted to brown or black organic complexes
 At this stage any trace of the original remains no longer remains
 The native organic fraction originates from two sources: the original plant debris entering the
soil and the micro organism with in the soil body. The micro organism in soil body work
upon the former and synthesize microbial protoplasm and new compounds that become part
of the organic fraction.
 Humus exist in a dynamic state
 Chemistry of humus is complex
 It has been pointed out that the organic fraction is derived from
a) Plant constituents that are modified by the microflora.
b) Constituents of microbial cells and products of microbial metabolism that are
relatively resistant to decay and therefore persist for sometime after death of organism.
In terms of specific elements
The organic fraction contains compounds of C, H, O, N, P, S and small amounts of other elements.
Only a small portion of the total is soluble in water, but much can be brought into solution by alkali.
In terms of type of compounds
Humus contains a number of polymerized substances, aromatic, molecules, polysaccharides, ascorbic
acids, polymers of uronic acids and P containing compounds. No definite composition can be
assigned. It should be considered as a portion of the soil that is composed of a heterogenous group of
substances, most having an unknown parentage and an unknown chemical structure.
Lignin and lignin derived molecules have long been considered to be of significance in the formation
of humus. It is possible either that simple aromatics released in the microbial attack on lignin
polymerize to yield constituents of the soil organic fraction or that partially altered lignin itself give
rise to humus constituents. The monomeric portions of humus are similar to the constituents of lignin.
Degradation processes
(1) Cellulose is a CH2O composed of glucose units bound by β-linkage at carbon 1 and 4 of the sugar
molecule. The cellulose concentration of higher plants is never fixed and the concentration. It is a
polymer of glucose and is might abundant organic material in nature changes with age and type of
plants. Woody materials have more cellulose and succulent tissues had poor, but the concentration
increases as the plant matures. Cellulose breakdown in soil is influenced by several environmental
factors.
Aerobic organisms convert cellulose to 2 major products : CO 2 + cell substance, but certain group
releases small amount of organic acids. It is however resistant to microbial decomposition. When
cellulose is associated with pentosans (xylan, mannans) it undergoes rapid decomposition. When
associated with lignin, the decomposition rate is very low. Degradation is by the enzymes (cellulase
complex) that convert cellulose into glucose. Celluases include exoglucanase, endoglucanase and β1-
4 glucosidase.
Exo glucanase
Native cellulose Amorphous cellulose + cellobiose
Endoglucanase Endogluconase

β-glucosidases
D- Glucose Cellobiose
(cellobiase)

Most cellulolytic bacteria do not excrete significant amounts of cellulases but fungi are found to
excrete these enzymes. The soluble sugars released by enzymatic hydrolysis are later utilized by the
same or other microorganism for biosynthetic purpose.
(2) Hemicellulose
Polymer of simple sugars such as pentose, hexose and uronic acids. May be either homo or hetero
polymers. When they are added to soil, degradation takes place at faster rate in initial stages. The
hemicelluloses such as mannans are decomposed rapidly while galactons (polymer of galactose) are
decomposed slowly. Many soil micro organism utilizes hemicellulose in both aerobic as well as
anaerobic conditions. The microbial degradation occurs through the agency of extracellular enzymes
called hemicellulases.
(3) Lignin
Third most abundant costituent of plants. It consists of heterocyclic aromatic organic molecules
containing C, H and O. The degradation is very slow and rate of decomposition depends on the
presence of other compounds such as cellulose and hemicellulose acid. Lignin is highly resistant to
microbial degradation. Degradation is a complex process.
Lignin --à coniferyl ether --à coniferyl alcohol --à coniferyl aldehyde --à vanillin --à
vannillic acid --à protocatechuic acid --à ring cleavage

Genera of microorganisms capable of utilizing different components of organic mater as

reported by several workers: F-Fungi; B-Bacteria; A-Actinomycetes
Nature of substrate in
Genera of microorganisms
organic matter
Cellulose F Alternaria, Aspergillus, Chaetomium, Coprinus, Fomes,
Fusarium, Myrothecium, Penicillum, Polyporus,
 Rhizoctonia, Rhizopus, Trametes, Trichoderma,
Trichothecium, Verticillium, Zygorynchus
B Achrombacter, Angiococcus, Bacillus, Cellfalcicula,
Cellulomonas, Cellvibrio, Clostrium, Cytophaga,
Polyangium, Pseudomonas, Sorangium, Sporocytophaga,
Vibrio
A Micromonospora, Nocardia, Streptomyces,
Streptosporangium
Hemicellulose F Alternaria, Fusarium, Trichothecium, Aspergillus,
Rhizopous, Zygorynchus, Chateomium, Helminthosporium,
Penicillium, Coriolus, Fomes, Polyporus
B Bacillus, Achromobacter, Pseudomonas, Cytophaga,
Sporocytophaga, Lactobacillus, Vibrio
A Streptomyces
Lignin F Clavaria, Clitocybe, Collybia, Flammula Hypholoma,
Lepiota, Mycena, Pholiota, Arthrobotrys, Cephalosporium,
Humicola
B Pseudomonas, Flavobacterium

LECTURE NO.: 5 NITROGEN CYCLE – MICROBIOLOGY AND BIOCHEMISTRY OF

MINERALIZATION, AMMONIFICATION, NITRIFICATION AND DENITRIFICATION
Nitrogen Cycle
Biological availability of N, P and K is of considerable economic importance, since they are the
major plant nutrients derived from the soil. Of the three, N stands out as the most susceptible one to
microbial transformations. This element is the key building block of the protein molecule upon
which all life is based, it is an indispensable component of the protoplasm of plants, animals and
micro organism.
Molecular N2 constitutes about 78% of the earth’s atmosphere but it is chemically inert and cannot be
utilized by more living organism, plant animals and micro organism therefore depend on a source of
combined N such as ammonia, nitrate or organic N compounds for their growth.
Nitrogen undergoes a number of transformations that involve organic, inorganic and volatile forms of
nitrogen. A small part of the large reservoir of N2 in the atmosphere is converted to organic
compounds by certain free living micro organism or by plant microbe association, that makes the
element available to plant growth. The nitrogen present in the proteins or nucleic acids of plant tissue
is used by animals. In the animal body, the N is converted to other simple and complex compounds.
Upon the death, plants and animals undergo microbial decay and organic N is released as ammonium,
which is then utilized by vegetation or is oxidized to nitrate by microorganisms. The nitrate from of
N is mostly used by the plants or may be lost by bacteria reduced to gaseous N 2, which escapes to
atmosphere, there by completing the cycle. The Nitrogen cycle mainly includes transformations such
as
1. Nitrogen mineralization : In which N containing organic complexes are decomposed and
converted into inorganic compounds for use by plants
2. N immobilization : In which N containing inorganic compounds are assimilated
N2 is acted on by certain micro organism sometimes in symbiosis with a higher plant, which can use
it is as a N source for growth. This process nitrogen fixation, results in the accumulation of new
organic compounds in the cells of responsible micro organisms. The N 2 thus fixed reenters general
circulation when the newly formed cells are inturn mineralized.
By means of these reactions the subterranean microflora regulates the supply and governs the
availability and chemical nature of N in soil.
 Biogeochemical Cycling of Nitrogen

Free living Nitrogen Soil Ammonification

organic
fixation
matter
Endophytic nitrogen Animals
fixation

Atmospheri
Ammonium
c nitrogen Plants
(NH4+)
(78% N2)

Assimilation

Denitrification Nitrification
Nitrate
(No3-)

I. Mineralization (Ammonification)

Organic nitrogenous compounds are incorporated into soil as organic matter after death of animals
and plants. Organic forms include proteins, nucleic acids and energy rich compounds. Among these
sources, protein is the profound compound in soil organic matter. Protein first undergoes
decarboxylation, followed by deamination to form ammonia.

Ammonification by heterotrophic microoganisms

Proteins
Peptides Aminoacids
(Polypeptides)

Ammonia

a. Ammonification
It is the process of mineralization in which proteins, nucleic acids and other organic components are
degraded by micro organism with the eventual liberation of ammonia. This is called ammonification.
A part of the liberated ammonia is assimilated by the micro organism themselves. The first step in
ammonication process is the hydrolysis of proteins, nucleic acids and other organic nitrogenous
compounds into amino acids (proteolysis). The amino compounds are then deaminated to yield
ammonia. Ammonification usually occurs under aerobic conditions while under anerobic conditions
protein decomposition leads to conversion of ammonia into amines and related compounds (eg)
clostridium. The anaerobic decomposition of protein called as putrefaction. These amines are
subsequently oxidized in the presence of O2 to release ammonia.Break down of nitrogenous
substance is brought about by the activity of a multitude of microbial species.Almost all bacteria,
actinomycetes and fungi can bring about proteolysis and the amino acids produced are utilized for
the growth of these organisms.
(b) Nitrification: The biological oxidation of ammonium salts (in soil) to nitrites and the subsequent
oxidation of nitrites to nitrates is called as nitrification. i.e. the biological convention of N in soil
from a reduced to a more oxidized state, called nitrification.
Nitrification occurs in two steps;
First ammonia is oxidized to nitrite.
2 NH3 + 1½ H2O2 → NO2- + 2H+H2O-Nitrosofication
This change is brought about by chemoautotrophic bacteria of the genera Nitrosomonas,
Nitrosolobus, Nitrosococus, Nitrosospira. These bacteria obtain their energy requirement by the
oxidation of NH4+ to NO-2. Among the nitrifiers Nitrosomonas are most important in soils.
Some heteotrophs involved: Streptomyces, Nocardia
Second step
Nitrite is further oxidized to nitrate
HNO2 + ½ O2 → HNO3.
Organisms : Nitrobacter, Aspergillus, Penicillium, Cephalosporium.
Factors influencing the growth of nitrifying bacteria in soil
Levels of ammonia and nitrite, aeration, moisture, temperature, pH and organic matter. In acid soils –
nitrification is poor. In Waterlogged soils – deficient in O2 – not congenial for nitrification.
3. Denitrification
The convention of nitrate and nitrite into molecular N2 or nitrous oxide through microbial processes
is known as denitrification. Certain bacteria are capable of using nitrate as the terminal electron
acceptor under anaerobic conditions. This is called nitrate respiration. As a consequence of nitrate
respiration, NO3 is reduced to N2 gas or nitrous oxide. Denitirifcation leads to the loss of N from the
soil. It depletes N, and therefore it is not a desirable reaction.
The escape of molecular N into the atmosphere is also known as volatalization.
Denitirfication occur mostly in waterlogged anaerobic soils with a high organic matter contents.
Denitrification of bound nitrogen to gaseous N is mediated by numerous species of bacteria, which
normally use O2 as hydrogen acceptor (aerobically) and, also use nitrates and nitrites (anerobically).
Anaerobic conversion of nitrate into molecular nitrogen is known as nitrate respiration.

Bacterial genera which bring about denitirfication Pseudomonas, Achromobacter, Bacillus,

Micrococcus
2NO-3 +10 H → N2 + 4H2O+ 2OH- (or)
2NO-2 +6 H → N2 +2H2O +2OH- (or)
N2O + 2H → N2 + H2O
Since nitrates are used as a source of electron acceptor, there is a net loss of N from soil. This
process is termed also as dissimilatory nitrate reduction. Many soil bacteria like.
Acidithiobacillus denitrificans
Oxidize S (chemoautotrophically) and also reduce nitrate to nitrogen
5S + 6 KNO3 + 2 H2O → 3N2 + K2SO4 + 4KHSO4 (or)
5 K2S2O3 + 8 KNO3 + H2O → 4N2 + 9 K2SO4 + H2SO4
General pathway of denitrification:
Nitrate is first reduced to nitrite, which is then transformed to nitrous oxide (NO). The nitrous oxide
is converted to N2 with N2O as an intermediate.

1 2 3 4
2 HNO3 → 2HNO2 → 2 NO → N2O → N2
The enzymes involved
1. Nitrate reductase
2. Nitrite reductase
3. Nitric oxide reductase
4. Nitrous oxide reductase
 Fallow soils flooded with water are more congenial for denitrification than well drained and
continuously cropped soils.
 Though it is a undesirable reaction in point of view of plant nutrition, but have ecological
importance. Because without denitrification the supply of N on the earth world have got
depleted and NO3 would have accumulated.
 High concentration of NO3 are toxic, denitrification is a mechanism by which some of the N
is released back to the atmosphere.
5. Nitrate reduction
The reverse of nitrification process is nitrate reduction. That is the reduction of nitrate to nitrite and
then ammonia.
HNO3 + 4H2 → NH3 + 3H2O
Since organisms are able to obtain cellular nitrogen that is ammonia assimilation, the process is
called as assimilatory nitrate reduction

II. Nitrogen immobilization

The process of microbial assimilation of inorganic nitrogen is referred as immobilization. In contrast
to mineralization, microbial immobilization leads to the biosynthesis of the complex molecules of
microbial protoplasm from ammonium and nitrate. Immobilization results in a marked depression of
nitrogen uptake by the plant.
The mineralization of organic N and the microbial assimilation of inorganic ions proceeds
simultaneously. Both mineralization and immobilization takes place regardless of the % of N in the
organic N in organic matter. On the death of micro organism, the immobilized N is however released
through mineralization. It is also a loss of nitrogen. NO3 when accumulated in microbial protoplasm
it is referred as assimilatory NO3 reduction.
Biological N2 fixation (BNF)
Fixation of elemental nitrogen in the atmosphere by the micro organism through a reductive process
into ammonia is called as BNF. A variety of prokaryotic organism have the ability to reduce the
atmosphere N2 BNF accounts for about 70% of the total N fixed in the biosphere. The ability to
reduce atmosphere N is restricted only to bacteria, which are belonging to the diverse groups. The
root nodule associations were the first to be recognized for their ability to fix atmosphere N 2.
Rhizobia are the first group of organism realized for its potential of nitrogen fixation.
Nitrogen fixing bacteria
Nitrogen fixing bacteria are classified according to their mode of fixation.
1. Free living N fixers – capable of fixing mol. N 2 to cellular N independently of other living
organism.
2. Associative N fixers
3. Endophytic N fixers
4. Symbiotic N fixers
Rhizobium is predominant symbiotic N2 fixing bacterium. Boussingault showed that leguminous
plant can fix atmosphere N2. Then hellriegel and wilfarth – proved that N2 is fixed by certain
bacteria living in root nodules of leguminous plants. Latter isolated in pure culture by Beijerinck.
Winogradsky isolated clostridium pasteurianum. Which is an anaerobic N2 fixer. Beijerinck isolated
Azotobacter as a free living aerobic N2 fixing organisms.
Cross inoculation groups of rhizobium (CIG)
It (CIG) refers the groups of leguminous plants that will develop effective nodules when inoculated
with the rhizobia obtained from the nodules from any member of that legume group.
I. Rhizobium
1. Rhizobium Cross Host
leguminosarum inoculation
grooup
bv. viceae Pea Peas,lenfils, vicia
bv. phaseoli Bean Phaseolus spp
bv. trifoli Clover Trifolium spp
2. R. meliloti Alfalfa Alfalfa, clover, fenugreek
3. R. loti Lotus Trifoli, lupine,
4. R. fredii Soybean Soybean
5. R. spp Cowpea group Vigna, Arachis, Cajanus, Dolichus,
Sesbania, Acacia, Prosopis, green gram
and blackgram
6. R. sp Chickpea group Chickpea
II. Bradyrhizobium
B. japonicum Soybean Soybean
B. spp Cowpea group Cowpea group plants
III. Azorhizobium - Stem nodulating – one. Nodulates Sesbania rostrata.
IV. Photorhizobium - Nodulates aeschynomene sp.
V. Sinorhizobium - fast growing soybean nodulator
I. Biological nitrogen fixation
Free living nitrogen fixers
Azotobacter - Aerobic
Beijerinckia
Clostridium – Anaerobic
Cyanobacteria (Blue green algae) etc.,
II. Associative symbiotic nitrogen fixer : Azospirillum, Herbaspirillum
III. Endophytic nitrogen fixer: Gluconacetobacter diazotrophicus
IV. Symbiotic nitrogen fixers
Rhizobium (Rhizobium – legume association)
Bradyrhizobium (Bradyrhizobium – soybean association)
Azorhizobium (Azorhizobium- Sesbania rostrata association)
 Anabaena azollae (Azolla – Anabaena association)
Frankia (Frankia – Casuarina association)
Species of Azospirillum
A. lipoferum, A. brasilense, A. amazonense, A. halopareferans, and A. irakense
Species of Azotobacter
A. chroococcum, A. vinelandii, A. Beijerinkii, A. Paspali, A. Agilis, A. Insignis and A.
macrocytogens
Important genera of blue green algae
Anabaena, Nostoc, Cylindrospermum, Rivularia, Oscillatoria, Plectonema, Aphanothece, Lyngbya,
Scytonema, Calotrhix etc.,
Species of Azolla
A. pinnata, A. filiculoides, A. microphylla, A. caroliniana, A. Mexicana and A. nilotica
Nitrogen fixation
Process of N2 fixation
The process of N2 fixation is mediated by the enzyme, called nitrogenase (which mediates the
reduction of N2 to ammonia) first, this enzyme was extracted from the anaerobic di nitrogen fixer
Clostridum pasteurianum. Latter, this has been isolated form most other N 2 fixing bacteria. The
mechanism of N2 fixation appears to be quite similar in most N 2 fixing prokaryotes. he enzyme has
been fairly well characterized and the enzymes from these different systems share common properties
allowing a unified single discription of nitrogenase.
Nitrogenase
Nitrogeanse is a functional enzyme which reduces N2 to ammonia and depends on energy source
from ATP. The nitrogenase has two components one containing Mo-Fe, designated as Mo – Fe
protein and the other Fe protein . Two components are necessary for the nitrogenase activity.
Mo-Fe protein
Consists of 4 subunits and having the molecular not of 22,0000 or 270,000 daltons and it is the big
component.
Fe-protein
Smaller component, contains 2 subunits, molecular weight 60,000 daltons.
Ammonia is the end product of N2 fixation. The over all reaction is as follows.
ATP
N2 + 3H2 2 NH3

Biochemistry of Nitrogen Fixation

The process of conversion atmospheric nitrogen into ammonium in soil or plant system is termed as
biological nitrogen fixation. Organisms involved are termed as biological nitrogen fixers or
diazotrophs.
Nitrogenase
N2 + 8H++ 8e- + 16 Mg ATP NH3 + H2 + 16 Mg ADP+Pi
Anaerobic condition

Nitrogenase is an anaerobic enzyme. It is made up of 2 units (Fe and Fe Mo). Besides normal Mo
containing nitrogenases, there are alternate nitrogenases with vanadium and iron.
Nitrogenase protection mechansims
1. Leghaemoglobin scavenges O2 to protect nitrogenase in legume rhizobium symbiosis
2. Confirmatory protection in Azotobacter as well as the higher respiratory rate.
3. Thick walls of Heterocyst protect O2 in BGA, since Nitrogenase are present in the heterocyst.
4. Microaerophilic nature in Azospirillum
Free living nitrogen fixers: Azotobacter, Clostridium, Beijerinckia, Derixia etc.

Symbiotic nitrogen fixers: Rhizobium – legume, Frankia-actinorhizal plants, Lichens

Endophytes: Herbaspirillum, Gluconoacetobacter

Factors affecting N2 fixation

1. Presence of nitrate or ammonium : More N2, No, N2 fixation
2. Presence of certain inorganic substances
Ca, Co, Mo – influence N2 fixation along with P
3. Availability of energy source – addn. of C source increase N2 fixation
4. pH : Neutral – favours Azotobacter – Acidic- Beijerinkia
5. Soil moisture : Adequate is good for fixation
6. Temperature : Mesophilic – 30°C.
The energy requirement for BNF is very high and it is a major factor determines the amount of N 2
fixed. In, Azotobacter the rate depends on amount of available carbon. In symbiotic N 2 fixers since
photosynthesis is the ultimate source of energy the rate of N 2 fixation is influenced by the factors
that effect photosynthesis and rate of translocating photosynthates to the N2 fixing system.
Losses of N by non biological ways
Leaching
20 to 50%of fertilizer N. The most striking loss of N in rice soils where more than half of the
fertilizer N applied get lost through leaching.
Volatalization
Another factor is the volatalizaiotn of ammonia in soil 5-20%.
Fixation of ammonium in soils is the minor contributory factor to overall loss of N 2 available for
plant growth. Such losses of N by physical causes and by nitrification and denitirfication process can
be controlled by the application of certain chemicals. Some chemicals have been designed to control
the rate of release of nutrient from nitrogenous fertilizers, while others retard nitrification in soil by
controlling the activity of nitrifying bacteria.
a. Controlled release fertilizers
Urea from
Fertilizers, sparingly soluble in water can
isobutyeldene diurea
regulate the release of N from fertilizers
Crotonilidene diurea
S coated urea
b. Nitrification inhibitors
These are substituted with pyridines, pyrimidines, anilines and isothiocyanates,
Examples
1. 2 chloro 6 (tricholormethyl) – pyridine – (N serve )
2. 2 amino 4 chloro 6 methyl pyridine –( AM.)
N serve inhibits the growth of Nitrosomonas europea and N. agilis.
The seeds of neem conain lipid associates act as nitrification inhibitors and there by increases the
efficiency of urea fertilizers.
Ammonia assimilation
N2 fixation results in NH4 formation which reacts with organic acids and form amino acids which is
mediated by ammonia assimilating enzyme.
GS – Glutamine synthetase
GOGAT – Glutamate synthese
GDH – Glutamate dehydrogenase
Genetics
Nif genes are responsible for N2 fixation.
Nif genes are 22, which are located in 7 or 8 clusters.
 LECTURE NO. 6: BIOLOGICAL NITROGEN FIXATION – FREE LIVING,
ASSOCIATIVE, ENDOPHYTIC, EPIPHYTIC AND SYMBIOTIC DIAZOTROPHIC
MICROORGANISMS. NODULATION IN RHIZOBIUM- LEGUME AND FRANKIA –
ACTINORHIZAL SYMBIOSES (PRACTICAL AND LECTURE NO17)

Rhizobium
It is a gram negative, aerobic, soil dwelling heterotrophic bacterium. It forms symbiotic association
with leguminous plants excepting a non leguminous plant-Parasponia. There are numerous nodule
forming bacterial genera besides Rhizobium which are given below.
Classification

Signalling between Rhizobium –legume symbiosis and nodule formation

The legume-rhizobial symbiosis starts with a signal exchange between the host plant and its
microsymbiont. Recognition of compatible bacteria by the host induces cortical cell divisions to form
root nodule primordia, and simultaneously initiates an infection process to deliver the bacteria into
the nodule cells. Infection of most legumes involves the development of plant-made infection threads
that initiate in the root hair. The infection threads harboring bacteria grow through the epidermal cell
layer into the nodule cells, where the bacteria are released and internalized in an endocytosis-like
process. In nodule cells, individual bacteria are enclosed by a membrane of plant origin, forming an
organelle-like structure called the symbiosome, within which the bacteria further differentiate into
nitrogen-fixing bacteroids.
 Nodulation process during legume - Rhizobium symbiosis

Frankia- Actinorhizal symbiosis

Actinorhizal symbioses result from the interaction between actinobacteria of the genus Frankia and
plants belonging to eight dicots plant families collectively called actinorhizal plants. While legume
nodules have a stem-like morphology with their peripheral vascular system and infected cells in the
central tissues, all actinorhizal nodules are structurally and developmentally related to lateral roots.
Frankia is a gram-positive nitrogen-fixing actinobacterium that forms a symbiotic association with
actinorhizal plants. It is a filamentous free-living bacterium found in root nodules or in soil. The
genus Frankia has been classified in the order of Actinomycetales on the basis of morphology, cell
chemistry, and 16S rRNA sequences.

Phylogenetic group of Plant family Plant Genera

Frankia
Group 1 strains Coriariaceae Coriaria

Datiscaceae Datisca
Rosaceae Cercocarpus, Chamaebatia, Dryas,
Cowania, Purshia
Rhamnaceae Ceanothus
Group II Betulaceae Alnus
Casuarinaceae Casuarina, Allocasuarina,
Ceuthostoma, Gymnostoma
Myriaceae Myirica, Comptonia
Group III Elaeagnaceae Elaeagnus, Hippophae , Shepherdia
Rhamnaceae Calletia, Discaria, Kentrothamnus,
Retanilla, Talguenea, Trevoa
Group III strains are more promiscuous and can occasionally inhabit root nodules of Rosaceae,
Coriariaceae, and Ceanothus
Host-Frankia Specificity
Successful actinobacterial symbiosis requires a compatibility between the host plant and its symbiont.
Phylogenetic analyses reveal that Frankiae form a coherent clade within the actinobacteria, and that
strains generally fall into three major groups or clusters. 1. Clade belonging to Frankia infecting
plants of Fagales - they have the most specific host range and are only able to interact with plants
belonging to this clade. 2. A subgroup of cluster I infects
the family Casuarinaceae, and appears to have evolved even higher levels of specificity, as some
Frankia are only able to nodulate Casuarina and Allocasuarina in natural conditions. Strains
belonging to cluster III have a wider host range and can interact with plants belonging to five families
within the two distant plant orders Rosales and Fagales.
Frankia signals
The initiation of the actinorhizal symbiotic relationship involves a molecular dialog between the plant
and its bacterial partner. Some undescribed factors of bacterial origin have been found to be involved
in Root hair deformation (RHD) in the host and they are termed as RHD factors.
Plant signals
Involvement of flavonoid-like compounds to influence nodulation of actinorhizal plants by Frankia.
In Alnus sp., nodulation was enhanced by the addition of flavonones or flavonols compounds.
Steps involved nodule formation
1. Root hair deformation and infection
2. Pre nodule and nodular primordium formation
3. Nodule development

Azospirllum, Azoarcus, Glucnoacetobacter, Herbaspirillum - host interactions and nitrogen

fixation
The classical endophytic plant growth promoting bacteria include Azospirllum, Azoarcus,
Glucnoacetobacter and Herbaspirillum .
Azospirillum  is a gram negative, spiral shaped, microaerophilic and a associative symbiotic nitrogen
fixing bacterium. It has the capacity to synthesize phytohormones, in particular indole-3-acetic acid.
Recently, many studies attributed an important role of Azospirillum in conferring to plants tolerance
of abiotic and biotic stresses, which may be mediated by phytohormones acting as signalling
molecules. Tolerance of biotic stresses is controlled by mechanisms of induced systemic resistance,
mediated by increased levels of phytohormones in the jasmonic acid/ethylene pathway, independent
of salicylic acid (SA), whereas in the systemic acquired resistance—a mechanism previously studied
with phytopathogens—it is controlled by intermediate levels of SA. Both mechanisms are related to
the NPR1 protein, acting as a co-activator in the induction of defense genes. Azospirillum can also
promote plant growth by mechanisms of tolerance of abiotic stresses, named as induced systemic
tolerance, mediated by antioxidants, osmotic adjustment, production of phytohormones, and defense
strategies such as the expression of pathogenesis-related genes. There are 17 species of Azospirillum
which are given hereunder.

Azospirillum mediated plant growth

Azoarcus
Associative nitrogen fixing species of the genus Azoarcus was first found in association with Kallar
grass (Leptochloa fusca (L.) Kunth). This grass is an undomesticated C4 plant tolerant to soil salinity,
alkalinity and water-logged conditions cultivated in Pakistan.
Later, this organism was also isolated from field-grown rice cultivated in Nepal and from resting
stages of plant-associated fungi found in rice field soils of Pakistan. There are three species like A.
indigens, A. communis and A. sp. BH72 . They have been found inside roots or on the root surface of
Gramineae and have never been isolated from root-free soil, except for members of A. communis
originating from a petroleum refinery oily sludge in France or from a compost biofilter in Canada.
This is in contrast to the soil-borne strains of the genus, such as A. tolulyticus, A. toluvorans, A.
toluclasticus, A. evansii, A. buckelii or A. anaerobius, which do not originate from living plants but
mostly from soil and sediments. Nitrogen fixation was studied in more detail in Azoarcus sp. BH72.
Compared to Azospirillum spp., Azoarcus is more tolerant to oxygen. Similar to Azospirillum, the
expression of nitrogenase genes is transcriptionally regulated in response to oxygen (fully repressed
at 4% O2 in the headspace) or combined N (repressed by 0.5 mM NH4+ or nitrate). A particular trait
of N2 fixation in this strain is the formation of so-called ‘diazosomes’. This novel intracytoplasmic
membrane stack is formed when cells are shifted down to extremely low O2 concentrations (30nM)
and is associated with augmented rates and efficiency of N2 fixation, called ‘hyperinduction’.
Gluconoacetobacter
Gluconacetobacter diazotrophicus is an endophytic bacterium first isolated from the sugarcane
growing regions of Brazil (Cavalcante and Dobereiner, 1988). After its first discovery, it was
reported from variety of crops viz, coffee, ragi , pineapple , beet root , radish, and a wild rice and a
salt tolerant Pokali rice variety. G. diazotrophicus can grow in a wide range of conditions, such as
low pH, high sucrose concentrations and high salt conditions. G. diazotrophicus is found to enter the
plant system through multiple sites such as roots, stems and leaves. It is also found in the vascular
system. It promotes plant growth by fixing nitrogen and also by the production of plant hormones.
This organism has recently been found to protect sugarcane from pathogen attack by inducing
systemic resistance.
Herbaspirillum
Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of
grasses such as rice and sugar cane. It was isolated from the stems of wild and cultivated rice on a
modified Rennie medium and reported for the first time in 1986. It is an aerobic, phototrophic,
endophytic nitrogen-fixing, plant-growth promoting bacterium, of the Betaproteobacteria. It is also
found inside tissues of important crops such as corn, wheat and sorghum without causing disease to
the plant partner.
The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity
to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct
terminal oxidases. The genome contains a multitude of protein secretion systems, including type I,
type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential
to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the
four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved
in modulating the associated plant ethylene-signaling pathway, is also present. Genes for
hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion.
These features may endow H. seropedicae with the ability to establish an endophytic life-style in a
large number of plant species.
LECTURE NO. 7: BIOCHEMISTRY AND MOLECULAR BIOLOGY OF NITROGEN
FIXATION IN DIFFERENT TYPES OF DIAZOTROPHS (REFER PPT.)
 LECTURE NO.8 PHOSPHORUS CYCLE AND MICROBIAL TRANSFORMATION OF
PHOSPHORUS – MINERALIZATION, PHOSPHATE SOLUBILIZATION AND
TRANSLOCATION (Also Refer ppt)

Phosphorus cycle
Phosphorus is only second to N2 as an inorganic nutrient required by both plants and micro
organisms. Phosphate constitutes nearly 0.1% of the earth’s crust. They occur in soil in inorganic
and organic forms The inorganic forms are derived from parent rocks or through fertilizers
application and manuring with bone meal. They are soluble in water when present as phosphates of
Na, K, Ca, Mg etc.The organic phosphorus containing compounds are derived from plants and micro
organisms and are composed of nucleic acids, phospholipids, lecittin, phytin and related compounds.
 Phosphorus in phytin, phospholipids and nucleic acids is found as phosphates
 Phytin is the calcium – magnesium salt of phytic acid
 Phospholipids are compounds in which phosphate is combined with a lipid, contained 10% of
cell phosphorus.
 Inorganic polyphosphates are quite abundant in certain fungi
 In soil, from15-85% of the total P is organic. Soils rich in organic matter contain abundant
organic P.
 Ratios of organic C to P of 100 to 300:1 N: organic P = 5 to 20: 1
In cultivated soil P present in abundant about 1100 kg/ha but most of them as not available to plants;
only about 1% of the total P is in available form. Microorganisms bring about a number of
transformations of the element.
 1. Altering the solubility of inorganic compounds of P
2. Mineralization of organic compounds with the release of inorganic phosphate
3. Converting the inorganic, available anion into cell components, an immobilization process
(analogous to that occurring with N)
4. Bringing about an oxidation or reduction of inorganic P compounds
Particularly, important to P cycle are the microbial mineralization and immobilization reactions.
Solubilization of inorganic phosphorus
Insoluble inorganic compounds of P are largely unavailable to plants, but many micro organism can
bring the PO4 into solution. P solubilizing are 105 to 107 / g soil.
Eg: Pseudomonas striata, Microoccus Bacillus sp., Fusarium, pergillus sp, solubilise calcium salts,
iron, aluminum, and magnesium phosphate.
 P is solubilized by the production of organic acids. The acids convert Ca3 (PO4)2 to di and
monobasic phosphates and releases primary ortho phosphate to plants.
 Solubilization of phosphates by plant roots & micro organism is dependent on soil pH. In
neutrals and alkaline soils having a content of calcium, precipitation of CaPO 4 takes place.
Micro organism and plant root readily dissolve such PO4 and make them available to plants.
 On contrary, acid soils are generally poor in Ca ions and phosphates and precipitated in the
form of ferric or aluminum compounds which are not soluble. There, it is solubilized by the
addition of PO4 solublizing micro organism.
 Phosphorus exists mainly as apatides, with the basic formula M10 (PO4)6 X2. Commonly the
mineral (M) is Ca, less often Al or Fe. The anion (X) is either F -, Cl-, OH- or CO2-3. Diverse
combinations of M and X results in 200 forms of P.
Mineralization of organic phosphorus
Organic form of P is the larger reservoir of P in soil. By the action of bacteria, fungi and
actinomycetes, bound element in remains of the vegetation and in soil organic matter is made
available to succeeding generations of plants. Among the organic phosphours compounds, lecithin,
nucleic acids and phytin occupy a prominent place. Lecithin contains 9.39 % P 2O5, 1.6% N and
65.36% C.
It is a process of convention of organic forms of phosphorus into inorganic available forms of P a
highly significant correlation is observed between the rates of N and P convention to inorganic forms.
- Mineralization is favoured by warm temperature, with the thermophilic range being more
favourable than mesophilic range.
- Neutral pH increases PO4 release, which favours microbial metabolism
- Quantity of substrate ie presence of organic P. If more P, more of mineralization
 - Mineralization is mediated by the enzymes called phosphatases. These enzymes cleave
phosphorus from more frequently encountered organic substrates.
- Phytases liberates PO4 from phytic acid or its Ca-Mg, Salt, Phytin. They remove PO 4-s, one
at a time, yield penta – tetra, di- and mono PO4 and then finally free inositol.
- Bacillus, Pseudomonas, Aspergillus, Penicillium, Rhizopus can synthesize this enzyme.
Mycorrhizal (fungi) are also able to mineralize the organic forms of P and increases P uptake
by the plants.
(3) Immobilization
Process of assimilation of P into microbial nucleic acids, phospholipids or other protoplasmic
substances is called immobilization. It leads to the accumulation of non utilizable forms of the
element. P accounts for 0.5-1.0% of fungus mycelium and 1.0 to 3.0% of the dry weight of the
bacteria and actinomycetes.

(4) Oxidation reduction reactions

Biological oxidation of reduced phosphorus compounds into oxidized state. Phosphite (HPO 3=) is
oxidized to phosphate. A number of hetertrophic (bacteria), (fungi) & (actinomycetes) utilize
phosphite as sole P source. Hypophosphites (HPO2=) can also be oxidized to phosphate by
heterotrophs.
HPO3= → HPO4=
HPO2= → HPO4=
Reductive process, reductive pathway has also been functioned. PO4 is reduced to phosphite and
hypophosphite.
H3PO4 → H3PO3 → H3PO2
Clostridium. butyricum, E. coli form phosphite and hypophosphite from orthophosphate. It is
biochemically analogue to the process of denitirification. Only little information is available about
this process.
P exist in an organic form in the protoplasm on the death of living organism, this (P) is changed to
inorganic phosphoric acid. This is soon converted into insoluble salts of Ca, Fe, Mg and Al.
Phosphorus thus alternates between organic and inorganic, and soluble and insoluble forms. In
soluble P is solubilized by various acids produced by micro organism.
Microbial activities involved in the cycling of C, N and P are absolutely essential for maintenance of
soil fertility.
LECTURE NO. 10: SULPHUR CYCLE - SULPHUR OXIDIZERS; MICROBIAL
TRANSFORMATION OF K, ZN AND SI.
(REFER PPT)
LECTURE NO. 11: ROLE OF MICROBES IN RECLAMATION OF PROBLEM SOILS.
MICROBES IN SOLID WASTE MANAGEMENT.
 LECTURE NO. 12: BIODEGRADATION OF AGRICULTURAL RESIDUES AND
CHEMICALS- PROCESSES INVOLVED IN REMEDIATION

Organic matter content of plant residue

Chemical composition of macromolecules

Starch
Hemicellulose
Lignin
Basic unit is phenyl propanoid moiety
Chitin

Bioconversion of wastes through composting

Wastes are the rich sources of plant nutrients, which increase the fertility of the soil. Organic matter,
chemically, is a combination of very complex organic compounds like carbohydrates, proteins, fats,
vitamins, and enzymes.
Microbial decomposition of organic matter results in the formation of humus and these, release many
plant nutrients. Microbes also produce sticky substance called polysaccharides that glue soil particles
together. Humus exerts a positive influence on the physical, chemical and biological properties of
soil and therefore is a major factor determining the soil fertility.

Composting can be defined as a method of solid waste management whereby the organic component
of the solid waste is biologically decomposed under controlled conditions to a state in which it can be
handled, stored and/or applied to the land without adversely affecting the environment.

Substrates
1. Crop residues: Wastes comprising stalks, trash, leaves of cereals, pulses and oilseeds.
2. Tree wastes: Leaf litter
3. Aquatic weeds: Water hyacinth
4. Green manures: Daincha, sunhemp, cluster beans, etc.
5. Livestock and human wastes: Cattle shed wastes comprising dung, urine, slaughterhouse
wastes and night soil.
6. Rural and urban wastes: Solid wastes, sewage and sludge
7. Agro-industrial wastes: Oil cakes and baggase, pressmud, fruits and vegetable industry
wastes, sericultural waste, cotton waste, etc.,
8. Marine waste: Fish meal, tank silt from tanks, ponds and reservoirs
Methods of composting
Although the major quantities of compost at present used in agriculture and prepared in village either
by traditional or improved (Indore or Bangalore methods) techniques these cannot be suitably used
for processing of large quantities of organic refuse of bigger cities.
The Indore method
This method was developed by Howad and Wad (1931) at Indore. This method requires a heap of
trapezoidal cross section. The heap is about 4 m to 5 m in length, 1 m in breadth and 1 m in height.
The heap is alternately layered with carbonaceous and nitrogenous wastes starting with 20 cm of
carbon rich and 10 cm of nitrogen rich material. Finally it is covered with soil or hay as thermal
insulator. Under these conditions, the rate of decomposition is very rapid and high temperatures
develop quickly. The process is accelerated by periodically turning the material. In this method,
losses of organic matter and nitrogen are very high, amounting to 50 to 60 percent of the initial
levels.
The Bangalore method
Acharya (1939) developed the Bangalore method to produce compost from city refuse and right soil
in pits. Pits of about 1 m depth, breadth and length are used. In this process, at first the refuse is
dumped into the trench and spread out with rakes to make a layer of 15 cm. Night soil is then
discharged and spread over the refuse in a layer of about 5 cm. This is than covered with a 15 cm
layer of refuse. The right soil and the refuse thus follow in alternate layers until the pit is filled to 15
cm above ground level, with a final layer of refuse on the top. This may be dome shaped and covered
with a thin layer of soil. The decomposition of dumped of air except in the surface layer. This
arabiotic decomposition is comparatively slow but markedly less wasteful.
Synthetic compost
In the preparation of synthetic compost, the nitrogen in the form of dung required by micro
organisms can be completely substituted with inorganic nitrogen compounds like ammonium
sulphate or urea which are utilized equally effectively for decomposition of carbonaceous materials
into compost. This facilitates utilization of large quantities of various organic waste materials where
supplies of dung are limited nor available at all as in mechanized farms. The manure becomes ready
for application in about 4 to 6 months.

High temperature compost

High temperature compost is prepared from night soil, urine, sewage and animal dung and chopped
plant residues at a ratio of 1:4. The materials are heaped in alternate layers starting with chopped
plant stalks and followed by human and animal wastes. Water is added to optimum amount.
The windrow composting
The window composting is a traditional and widely practised method of composting in USA. In this
process, the waste materials are piled in long rows of 2 to 4 m width and 1 to 2 height on a hard
surface and usually in the open area. Aeration of the windrow is by periodic turning using equipment
such as front end loader or by specially designed machinery. Occasionally forced aeration in
conjunction with turning has been applied to the windrow process.

The static pile composting

In static pile method of composting a mixture of solid wastes is stockpiled in the open air and turned
occasionally for aeration. This technology is considered to waste both ammonia and energy. But
these disadvantages have been substantially mitigated by mechanical forcing of air through
perforated pipes at the bottom of the static pile by i) continuous air suction; ii) continuous air
blowing; iii) alternate air blowing; iv) alternate blowing and sucking; v) intermittant air blowing to
keep the temperature below 60oC.

Accelerated composting and enrichment

The conventional method of composting takes a long time to produce quality compost. In order to
hasten the process and to improve the quality of the end product, the materials to be composted is
inoculated with microbes such as cellulolytic, lignolytic, nitrogen fixing and phosphate solubilising
organisms. Addition of sources of nitrogen and phosphorus may also be desirable when the materials
to be composted lack much of these elements. The best additive for a compost mix is to add mature
compost that will produce a suitable starting population throughout the composting mass and furnish
bio available minor elements essential to life.
Composting for bioremediation
Environmental contamination by synthetic polynuclear aromatic hydrocarbons (PAHs) has become a
major pollution problem. Plant tissues, lignin, soil humus constituents, some pesticides and
numerous commercial organic chemicals are also based upon aromatic building blocks. Many of
these molecules are potentially toxic and carcinogenic. Some PAH compounds occur naturally at a
low concentration in soils. On exposure to naturally occurring hydrocarbons has enabled bacteria to
evolve enzymes that degrade them.

Vermicomposting
Earthworms play a key role in soil biology as versatile natural bioreactors. They effectively harness
the beneficial social microflora, destroy soil pathogens and convert organic wastes on to vitamins,
enzymes, antibiotics growth hormones and protein rich products. Earthworms gut is an effective
tubular bioreactor with raw materials (feed) entering from one end and the product coming out
through the other end (casting). They promote growth of microorganisms in their gut by providing
favourable conditions; castings contain nutrients in a balanced proportion and are rich in vitamins,
enzymes, antibiotics and growth hormones. Vermiculture, is the rearing and utilization of earth
worms to sustain soil fertility, to reclaim waste land and to treat solid wastes and waste waters. As a
natural bioreactor, the earthworms stimulate biological activity important to the degradation of
organic residues.

Vermi Compost
Vermi composting refers to the use of earthworms for composting organic residues. Earthworms can
consume all kinds of organic matter. Chemical changes in the degradation of organic matter occur
through enzymatic digestion, enrichment by nitrogen excrements and transport of organic and
inorganic materials. There are three species of earthworms viz., Eisenia foetida, Eudrillu eugeniae
and Perionyx excavantus which are called measure worms and these can be cultured on animal dung,
poultry droppings and vegetables. The worms form the needed organic fertiliser which contain all
the nutrients in an available form. Disadvantage with this type of composting is that it requires
pretreated or partially decomposed waste material for the process.
Vermicomposting techniques
1. Vermicomposting of wastes in field pits
2. Vermicomposting of wastes in ground heaps
3. Vermicomposting of wastes in brick columns

Field Vermicomposting technique involves

1. Sorting out of wastes into degradable and non-degradable matter.
2. Pre-treatment of waste
3. Treatment of Vermicomposting area and beds with chemicals
4. Filling of beds with wastes
5. Provision of optimum eco factors such as bed moisture, temperature.
6. Monitoring the activity of natural enemies and earthworms.
7. Harvest of vermicompost and storage.
Prerequisites for Vermicomposting
1. Selection of site: It should be preferably black soil or other areas with less of termite and red
ant activity. pH should be between 6 and 8.
2. Collection of wastes and sorting: For field composting raw materials are needed in large
quantities. The waste available should be sorted into degradable and non degradable (be
rejected).
3. Pre-treatment of waste: For example
a) Lignin rich residues: Chopping and subjecting to lignin degrading fungi and later to
vermibeds.
 b) Crop stalks and stubbles: Dumping it in layers sandwiched with garden soil
followed by watering for 10 days to make the material soft and acceptable to worms.
c) Agro industrial wastes: Mixing with animal dung in 3:1 proportion and later
subjecting it for vermicomposting.
4. Insecticidal treatment to site: Treating the area as well as beds (in case of pit system) with
chlorpyriphos 20 Ec @ 3.0 ml/litre to reduce the problem of ants termites and ground beetles.
The following steps are involved in Vermicomposting irrespective of the method
of composting.
1. Select a levelled ground and spread soil uniformly over the ground to a width of three feet and
height of one foot. The length of the heap will vary according to the availability of cowdung.
Standard length is 150 feet, will yield sufficient vermicompost.
2. Moisture the soil (water stagnation should be avoided)
3. Spread a layer of cowdung (shade dried and powdered) and organic wastes (kitchen wastes,
dried leaves, etc.) over the soil to a height of six inch. Moisten this layer also.
4. Collect earthworms from the field locally and release them in the heap (Around 1000
earthworms/sq.m is required).
5. Earthworms will feed on the wastes and leave the excreta in the form of heaps. Remove the
heaps every week and dry them in the shade. It is very rich in nutrients and is known as
Vermicasting. With in 45 – 60 days, about 10 kg of castings will be produced per kg of
earthworms.
6. When the feed material disappears, replenish it.
7. Remove the vermicastings as before and store them aside in conical heaps. As the heaps get
dried up, the worms will move to the bottom of the cone.
8. Remove the upper ¾ layer of the heap and the separated worms can be returned to the bed.
9. The entire setup can be kept as such for six months alone. As the volume goes down during
composting the soil gets compacted and the worms will not be able to burrow through it.
10. To get continuous supply of vermicompost throughout the year, more beds should be laid out
at regular intervals.
 LECTURE NO. 13. IMPORTANCE OF SOIL AND PLANT MICROBIOMES–
RHIZOSPHERE, SPERMOSPHERE, PHYLLOSPHERE, EPIPHYTIC AND ENDOPHYTES.
PLANT GROWTH PROMOTING MICROBES-TYPES AND MECHANISM OF ACTION.

Rhizosphere and its importance

Rhizosphere
The term Rhizosphere was introduced by the German scientist Hiltner in 1904. It is region of the soil
which is subjected to the influence of plant roots. It is the unique environment under the influence of
plant roots. Rhizosphere is characterized by greater microbiological activity than the soil away from
plant roots.
The term rhizsophere is often devided into two general areas, the inner rhizosphere (very root
surface) and the outer rhizosphere (immediate adjacent soil). The microbial numbers are larger in the
inner zone, where the bio chemical interactions between micro organism and roots are most
pronounced.
Rhizosphere
The root surface and its adhering soil are termed rhizosplane. In rhizosphere and rhizosplane, higher
organism contributes excretory products and sloughed off tissue, and certain benefits are derived
from the microscopic organisms.
Microflora of the root zone
The rhizosphere is a highly favourable habitat for the proliferation and metabolism of numerous
microbial types. Microscopic examination reveals the presence of a vast microbial community
surrounding on the surface of roots and root hairs.
 Bacteria are quite abundant
 F and A are observed but not as frequency
 Protozoa are relatively conspicuous, particularly small flagellates and large ciliates.
Rhizosphere effect
It indicates the overall influence of plant roots on soil microorganisms. It can be put on a quantitative
basis by the use of R-S relationship. The rate of metabolic activity of the rhizosphere
microorganisms are different from those of non-rhizosphere soil.
R:S ratio
It is defined as the ratio of microbial numbers per unit weight of rhizosphere (R), to the numbers in a
unit weight of the adjacent non rhizosphere soil (S). The rhizosphere effect is consistently greater for
bacteria than for other microbe.
 Short, gram – negative rods, which almost invariantly make upto of larger % of the
rhizophere
 The % incidence of short gram positive rods, coccoid rods and spore forming (bacteria)
(Bacillus spp.) declines.
 No selective stimulation or inhibition of gram variable rods, cocci, non spore – formers etc.
 On a generic basis, root effect appears to be most pronounced Pseudomonas, Flavobacterium,
Alcaligenes are especially common. Some other species of Arthrobacter, Brevibacterium,
Cornebacterium, Micrococcus, Xanthomonas, Bacillus are present.
 Anaerobic (bacteria) are affected by the root, this may be attributed to the reduced O2 tension
resulting from root and microbial respiration.
 Bacterial density is 109/g rhizosphere soil. It may cover 4-10% of the root area
 Fast growers and metabolic active population are dominating in the rhizohsphere. This
suggest that the rhizosphere flora has a greater ability to effect rapid biochemical changes
than the organisms of fallow land.
 Preferential stimulation and enhancement of aminoacids requiring organisms and those
organism proliferate in the absence of preformed growth factors, is there in the rhizosphere.
 Qualitative changes / selective influence of he root system is exhibited
Eg: Preferential stimulation of G-ve, non spore forming short rods.
- Abundance of N2 fixing P solubilizing organic
- Preponderance of amino acid requiring (bacteria)
- Preferential stimulation of vitamin requiring (bacteria)
 In contrast to their effects on (bacteria), roots do not appreciably alter the total counts on the
other hand specific fungal genera are stimulated; that the influence is selective rather than the
total number. The spectrum of genera varies with plant species and age and kind of the soil.
 Actinomycetes and algae are not significantly benefited by their proximity to roots, and the
R:S ratio may rarely exceed 2 or 3:1.
Flora of the rhizosphere is affected by number of factors
 Proximity of the soil sample to the root-more counts in soil collected when it is closer to the
tissue surface.
 Depth of sampling-The frequency of organism declines with depth
 Plant species-Due to variation in rooting habitats, tissue composition and excretion products.
 Legumes engender a more pronounced rhizosphere effect than grasses and grain crops
  Biennials – Exert more prolonged stimulation than annuals because of their long growth
period.
 Individual plant species exert influence on specific flora
 The age of the plant – stimulation is detectable in very young seedlings.
 Soil treatment also have little influence on the total number of organism.
 Different plant species in the same field have widely divergent numbers of organism in their
rhizosphere than the same species (i.e.) the character of vegetation is more important than the
fertility level of the soil.
 Therefore the plant plays a greater role than the soil, and the composition of the root’s
excretion and the chemical constituents of the tissues probably determine to a large extent the
microbial composition of the environment.
Root exudates
 One of the most important factors responsible for rhizosphere effect is the great variety of
organic substances available at the root region by the way of exudates from roots, which
directly or indirectly influence the quality and quantity of the micro organism in the root
region.
The substances exuded by plant roots include amino acids, sugars, organic acids, vitamin, nucleotides
and many other unidentified substances
Organic compouns detected in plant root exudates
Sugars
Amino compounds
Organic acids
Fatty acids and sterols
Growth factors
Nucleotides, favonones, and enzymes
Miscellaneous
Influence of the rhizosphere microflora on plant growth
The rhizosphere community have either a favourable or a detrimental influence of plant development.
Because the microflora is so intimately related with the root system, partially covering its surface,
any beneficial or toxic substance produced can cause an immediate response.
 Vast microorganisms demands a variety of anions and cations for its own development and
immobilization may takes place
 Aerobic (bacteria) remove O2 from the environment and add CO 2 and either by lowering O2 or
increasing CO2 tension, may reduce root elongation and development and reduce the water
and nutrient uptake.
  It may however favour plant development by producing growth stimulating substances,
contributes to the formation of a stable soil structure, releasing elements in organic forms
through mineralization of organic complexes, and by entering into symbiotic root
associations.
Important role in nutrient transformation
PO4 Solubilization
Rhizosphere microorganism
PO4 Mineralization
 Rhizosphere community is mainly of non pathogenic micro organism but, the biological
interactions in rhizospehre may lead to harmful and beneficial …
 Resistance or often susceptibility of one variety may depend on the excretion through which
the roots of a substance that induce the development of a flora, competitive with or
antagonistic to the pathogen.
Hence the rhizosphere may be considerable as a microbiological buffer zone in which the
microflora serves to protect the plant from the attack of the pathogen. The mechanism of buffering
action is unknown. Antibiotic production by the root microflora is often cited.
Spermosphere Microorganisms
The spermosphere is the region of soil directly under the influence of seeds and the critical interface
between plants and microbes where beneficial and detrimental interactions occur. The spermosphere
has only a temporary existence during germination sensu stricto: before germination, there is no
interaction between the mature dry seed and the soil, while after the emergence of the radicle, the soil
environment surrounding the seed is defined as the rhizosphere.
Phyllosphere Microorganisms(Epiphytic)
As an ecosystem or ‘biome’ for microorganisms, leaves and other above-ground plant parts are
referred to as the ‘phyllosphere’, a term coined in the 1950s by Ruinen inspired by Hiltner’s
‘rhizosphere’. This supports large number of beneficial bacteria. Eg. PPFM (Pink pigmented
facultative methylotroph).
Endophytic microorganisms
The term endophyte was first introduced by De Bary (1866), and defined as any organism that grows
within plant tissue. Now, the term is precisely described in terms of their types (fungal and bacterial)
and relationships (obligate or facultative)33 with the host plant.
Endophytes are microorganisms, that is, bacteria or fungi or actinomycetes reside in the living tissues
of plants intercellularly and/or intracellularly without causing any harm and symptoms of disease.
According to Fesel and Zuccaro (2016), a comprehensive definition of endophytes does not specify
their functional relationship and apart from commensalistic symbionts, they can exist from latent
pathogens or saprotrophs to mutualistic associations. The mutualistic association by colonizing plant
tissues both intercellularly and/or intracellularly is a well-versed component of their lifestyle and
most of the modern research clearly shows that survival and health of plants are very much dependent
upon these microorganisms.
.
LECTURE No. : 14 SOIL MICROORGANISMS AND THEIR INTERACTIONS – POSITIVE
AND NEGATIVE INTERACTIONS. AN OVERVIEW OF INDUSTRIALLY IMPORTANT
MICROORGANISMS AND PRODUCTS

Microbial interactions
Within a biological community, various types of interactions can occur between diverse microbial
populations, between microbes, microbes and plants and between plant and animal populations. The
interaction between populations within a community is dependent on the environmental conditions of
the habitat, and under different environmental conditions the same populations can exhibit different
interpopulation relationships. Interactions between two different biological populations can be
classified according to whether both populations are unaffected by the interaction, one or both
benefited, or one or both populations are adversely affected.
The positive interactions between biological population enhance the ability of the interacting
populations to survive within the community of a particular habitat, sometimes permitting
populations to co-exist in a habitat where individually they cannot exist alone.
Negative interactions between populations act as feedback mechanisms that limit population
densities. In some cases, negative interactions may result in the elimination of a population that is not
well adapted for continued existence within the community of a given habitat.
Neutralism
Neutralism, actually represents a lack of interaction between two populations. Dormant resting
stages of microorganisms are more likely to exhibit neutralism toward other microbial populations
than are actively growing vegetative cells. Low rates of metabolic activity, which characterize the
resting stages of microorganisms, favour a lack of interaction.
Commensalism
In a commensal relationship between two populations, one-population benefits and the other one is
unaffected. For example, the removal of oxygen from a habitat, as a result of the metabolic activities
of a population of facultative anaerobes, creates an environment that is favourable for the growth of
obligately anaerobic populations. The lowered oxygen tensions favours the anaerobic bacteria, and
assuming that there is lack of competition for the same available substrates; the obligate anaerobes do
not affect the existence of the facultative organisms.
Classification of population Interactions
Effect of Interaction
Name of Interaction Population A Population B
Neutralism 0 0
Commensalism 0 +
Synergism (protocooperation) + +
 Mutualism (Symbiosis) + +
Competition - -
Amensalism 0 or + -
Parasitism + -
Predation + -
0 = no effect + = positive effect - = negative effect

Synergism
Synergism or proto-cooperation between two populations indicates that both populations benefit from
the relationship but that the association is not obligatory. In a specific example of syntrophism,
Streptococcus faecalis and Escherichia coli are able to convert arginine to putrescine together,
although neither organism can carry out the transformation alone. S. faecalis is able to convert
arginine to ornithine, which can then be used by a population of E. coli to produce putrescine; E.coli
growing alone can transform arginine to produce agmatine, but cannot convert arginine to putrescine.
Mutualism
Mutualism or symbiosis is an obligatory interrelationship between two populations that benefits both
of them.
i) Microbe – Microbe Interactions
The relationship between some heterotrophic fungi and their photosynthetic algal or cyanobacterial
partners in the formation of lichens is an excellent example of a mutualistic intermicrobial population
relationship that results in the formation of an essentially new organism.
ii) Plant-Microbe Interactions
Microorganisms establish important relationships with plants such as the nitrogen-fixing symbiosis
between Rhizobium and leguminous plants, Frankia and actinorhizal plants.
The formation of mycorrhizae, which are mutualistic relationships between fungi and plant roots, is
another important symbiotic relationship between microorganisms and plants. The fungus derives
nutritional benefits from the plant roots and contributes to the plant's nutrition. The establishment of
mycorrhizal associations involves the integration of plant roots and fungal mycelia into a unified
morphological unit. Some plants with mycorrhizal fungi are able to occupy habitats that they
otherwise could not inhabit. The importance of this microbe-plant interaction is attested to by the
fact that 95 percent of all plants have mycorrhizae.
iii) Animal-Microbe interactions
a) There are some particularly interesting mutualistic relationships between microorganisms and
animal populations. Some plant-eating insect populations, for example, actually cultivate
microorganisms on plant tissues. The microorganisms degrade cellulosic plant residues, providing a
digestible source of nutrition for the insects, which lack cellulase enzymes and cannot derive any
nutritional benefit from simply eating plant material.
b) The fungal gardens of myrmicine ants, the attini, are an excellent example of an insect population
growing fungi in pure culture. The ants macerate leaf material, mix it with saliva and fecal matter,
and inoculate the prepared substrate with a pure fungal culture. After growth of the fungus, the ants
harvest a portion of the fungal biomass and the byproducts they ingest. Various wood-inhabiting
insects, including ambrosia beetles and some termites, maintain similar mutualistic relationships with
microbial populations. In these cases, the animals rely on the cellulolytic enzymes of microbial
populations to convert plant residues into nutritional sources that they can use. The insect provides
the microorganism with an optimal habitat for growth.
c) Ruminant Ecosystem. Ruminant animals, such as cows, Ilamas, and camels, establish similar
mutualistic relationships with microbial populations. Although plants are the main food sources for
these animals, ruminant animals do not produce cellulase enzymes themselves, instead, they depend
on microbial populations for the degradation of the cellulosic materials they consume. The rumen,
the large first chamber within the stomach of these animals, provides a stable, constant-temperature,
anaerobic environment for the establishment of mutualistic associations with microbial populations.
The plant material ingested by the animal provides a continuous source of nutrients for the
microorganisms within the rumen, very much like what occurs in a continuous fermentor.
d) Bioluminescence. The mutualistic relationship between some luminescent bacteria and marine
invertebrates and fish is particularly interesting. Some fish have specific organs in which they
maintain populations of luminescent bacteria including members of the genera Photobacterium and
Beneckea. The fish supply the bacteria with nutrients and protection from competing
microorganisms. The light emitted by bacteria is used in various ways by different fish. In some
cases, the pattern of light emission is used in sexual mating rituals. In deep-sea and nocturnal fish,
such as the flash light-fish Photoblepharom, the light emitted by the bacteria aids the fish in finding
food sources and warding off predators.
Competition
Competition occurs when two populations are striving for the same resource. Often it focuses on a
nutrient present in limited concentrations, but it may also occur for other resources, including light
and space. As a result of the competition, both populations achieve lower densities than would have
been achieved by the individual populations in the absence of competition. Competitive interactions
tend to bring about ecological separation of closely related populations, a fact known as the
competitive exclusion principle.
Amensalism
Amensalism, or antagonism, occurs when one population produces a substance inhibitory to another
population. The first population gains a competitive edge as a result of its ability to inhibit the
growth of competitive populations. The production of antibiotics, for example, can give the
antibiotic-producing population an advantage over a sensitive strain when competing for the same
nutrient resources. Examples are antibiotic producing bacteria, fungi and actinobacteria. Penicillin
produced by Penicillium sp affects the growth of Staphylococcus aureus and other gram positive
bacteria.
Parasitism
In a relationship of parasitism, the parasite population is benefited and the host population is harmed.
As a rule, parasitic relationships are characterized by a relatively long period of contact, and the
parasite is smaller than the host. The parasite normally derives its nutritional requirements from the
host cell, and in the process the host cell is damaged. Ecto and endophytic interactions of
microorganisms with plants. There are also obligative and facultative parasites.
Predation
Predation involves the consumption of a prey species by a predatory population. The predatory
populations derive nutrition from the prey species, and clearly, the predator population exerts a
negative influence on the consumed prey population. Some microbial species are predators and
others are prey. Many protozoa prey upon bacterial species, and the non-discriminatory consumption
of bacterial populations by protozoan predators is sometimes referred to as grazing. Similarly,
protozoa and invertebrate animal populations graze on algal primary producers. Predation is an
important process in establishing food webs to support the growth of higher organisms. Various
filter-feeding animals are able to remove microorganisms from suspension. This grazing activity is
important in transferring biomass from microorganisms to higher trophic levels in aquatic food webs.
Bacteriophages infecting bacteria is yet another classical example for predation.
An overview of industrially important microorganisms and products

Microorganisms and microbial processes are employed to get industrially important products

The major commercial products of micro organisms can be classified as follows.

1) The microbial cells: eg. Vaccines, brewers yeast, bakers yeast and biofertilizer etc

2) Large molecules like enzymes that are synthesized by the micro organisms

3) Primary metabolic products ie components essential for cell growth eg. Amino acid , Vitamins
etc

4) Secondary metabolites ie Compounds that are not essential for cell growth. Eg. Antibiotics

Major classes of products:

1) Pharmaceutical chemicals: Most common Antibiotics and steroids. Others such as Insulin
Interferens and stomatostain are now being produced by genetically engineered bacteria.

2) Commercially valuable chemicals : solvents, enzymes and intermediary compounds for

synthesis other substances

3) Food supplements : mass production of yeast, bacteria and alga from substrates containing
nitrogen salts and other readily available nutrients provides a good sources

Microbes used in industry

In fermentation industry, various commercial products of important economic value made by

microorganisms are (1) pharmaceuticals, including antibiotics, steroids, human protein, vaccines, and
vitamins; (2) organic acids; (3) amino acids; (4) enzymes; (5) organic solvents; and (6) synthetic
fuels.

Microbes used in industry

Industrial Chemicals Polysaccharides

Saccharomyces cerevisisae Ethanol (from glucose) Leuconostoc Dextran
Kluyveromyces fragilis Ethanol (from lactose) mesenteries
Clostridium acetobutylicum Acetone and Butanol
Xanthomonas Xanthan gum
Asergillus niger Citric acid campestris

Amino Acids and Flavor-enhancing Nucleotides Pharmaceuticals

Corynebacterium L-Lysine Penicillum Penicillins
glutamicum chrysogenum
Corynebacterium 5'- inosinic acid and Cephalosporium Cephalosporins
glutamicum 5' – guanylic acid acremonium
Corynebacterium Streptomyces Amphotericin B,
gluatamicum MSG kanamycins,
Neomycins,
streptomycin,
Tetracyclines
and others
Bacillus brevis Gramicidin 5
Vitamins Bacillus subtilis Bacitracin

Ashbya gossypii Raboflavin Bacillus polymyxa Polymyxin B

Eremothecium ashbyi Riboflavin
Pseudomonas denitrificans Vitamin B12 Rhizopus Steroid
Propionibacterium Vitamin B12 nigricans transformation
shermanii
Steroid
Arthrobacter transformation
simplex
Steroid
Mycobacterium transformation

Insulin, human
Escherichia coli growth hormone,
( a recombinant somatostatin &
DNA technology) interferon
Enzymes Polysaccharides
Aspergillus oryzae Amylases Leuconostoc Dextran
Asergillus niger Glucamylase mesenterioes
Tricholderma reesii Cellulase
 Saccharomyces cerevisiae Invertase Xanthan gum
Kluyveromyces fragilis Lactase
Xanthomonas
Saccharomycopsis lipolytica Lipase
campestris
Asergillus Pectinases & proteases
Bacillus Proteases
Mucor pussilus Microbial rennet
Mucor meihei Microbial rennet
Single cell protein production

Candida lipolytica, Saccharomyces fragilis, Rhodotorula, Aspergillus, Penicillium, Fusarium,

Pseudomonas, Cellulomonas, Scenedesmus acutus, Spirulina maxima & S. platensis
Biofuels

1.Algal lipid FAME-biodiesel

Chlorella, Botryococcus
Chlamydomonas,
2. Ethanol from molasses, Gasohol
lignocellulosic wastes, any
agricultural residues
3. Microbial biomass Bio oil

4. Hydrogen from microbial fuel

process

Lecture No.: 15 Silage production. Bioinoculants – types; biofertilisers - bacterial, fungal

(AMF) and algal biofertilisers. Biopesticides – types and mechanism of action

Silage production
Silage is the material produced by controlled fermentation, under anaerobic conditions, of chopped
crop residues or forages with high moisture content. Silage is produced by the activities of naturally-
occurring bacteria that convert some of the plant sugars into organic acids that preserve nutritional
qualities.
Effective silage making is contingent on two major components. Growing a high-yielding crop with
an appropriate quality or specific purpose for dairy production system. Harvesting the crop at the
correct stage and preparing and storing it so that it is preserved effectively and fed with minimum
wastage.
The correct harvesting stage for silage is variable when compared to hay in that silage can potentially
be made at times of year when adequately drying crop for hay can be difficult and the crop may be
far more vegetative and digestible at that time of year. There will generally be an inverse relationship
between yield and quality which needs to be prioritized by the producer with respect to the
total forage needs of the operation. The decline in quality with advancing maturity must be managed
carefully to conserve adequate-quality feed. Maize silage under ideal conditions offers the
opportunity for the highest yield of forage among all the annual forages, but growth and harvest
conditions will significantly impact quality and starch content. Maize and grain sorghum forage are
usually conserved only as silage.
Silage may be chopped and stored in a pit, bunker, or tower, or compressed into round or square
bales and wrapped in plastic to exclude air and allow fermentation to proceed. Silage can also be
deep buried for long-term storage to manage drought.
The plastic wrapping on baled silage can deteriorate, and the silage will not keep as long as bunker or
pit silage.
Major considerations in the preparation of forage for ensiling are as follows:
Fineness of chopping. This influences the capacity to compress silage, exclude air, and stabilize
the anaerobic conditions required for effective silage fermentation. Particle size also affects the rate
of digestion and passage of silage through the rumen when it is fed back to the cows.
Moisture content. When the forage is too wet, the silage will produce excessive amounts
of effluents and be exposed to unfavorable fermentation conditions. This can lead to spoilage, dry
matter losses and feed rejection. Similarly, excessively dry forage is difficult to compress in the stack
to expel air, and fermentation is also restricted or exposed to more aerobic conditions which can
favor development of molds and other spoilage organisms. Water may need to be added to dry forage
and chop length and processing of grains will need to be adjusted to improve compaction and
digestibility of more mature grains and fiber,
The silage should be covered so that air is excluded and potential for aerobic spoilage is prevented.
Aerobic spoilage of silage occurs when it is exposed to air. The breakdown process is similar to
composting.
Brassicas are difficult to ensile, because of their high moisture content. Kale can be conserved as
round bale silage. Brassicas can also be ensiled when sown in mixtures with grasses.
Silage harvest can have some advantages over hay in that it allows for:
Fodder to be harvested earlier in the growth cycle when potentially more vegetative and higher
quality;
This in turn may facilitate more repeat harvests from the same crop;
Target dry matter can be achieved more rapidly allowing paddocks to be re-watered if irrigated and
reducing risk of spoilage with wet weather; or moved into subsequent crop more rapidly;
Losses can be less from shattering and drop of leaves.
However, silage is generally regarded as requiring more capital for storage and is less transportable
and tradable as an alternate commodity.

Bioinoculants – types; biofertilisers - bacterial, fungal (AMF) and algal biofertilisers

Biofertilizers are carrier based inoculants containing cells of efficient strains of specific
microorganisms (mainly bacteria) used by farmers for enhancing the productivity of the soil either
by fixing atmospheric nitrogen, by solubilising soil phosphate or by stimulating plant growth
through synthesis of growth promoting substances. They accelerate certain microbial processes in
the soil which augment the extent of availability of nutrients in a form easily assimilated by plants.
Several microorganisms and their association with crop plants are being exploited in the
production of biofertilizers. N2 fixing organism such as Azospirillum, Rhizobium, Azotobacter,
Gluconacetobacter and PO4 solubilizing bacterial genera Pseudomonas, Bacillus and PO4
mobilizing Arbuscular mycorrhizal fungi are presently used as biofertilizers for commercial
applications. Potassium releasing, zinc solublizing and sulphur oxidizing bacteria. PPFM fro
abiotic stress management.

Biofertilizers may be broadly classified into nitrogenous biofertilizers (NB) phosphate, potassium
and zinc solublizing biofertilizers, phosphate mobilizers and mineralizers.

Biofertilizers are known to make a number of positive contributions in agriculture. For example

1. They supplement fertilizer supplies for meeting the nutrient needs of crops.
2. They can add 20-200 kg N/ha/year (by fixation) under optimum conditions and
solubilize/mobilize, 30-50 kg P2O5/ha
3. They liberate growth promoting substances and vitamins and help to maintain soil fertility.
4. They suppress the incidence of pathogens and control diseases.
5. They are cheaper, pollution free and based on renewable energy sources.
6. They improve soil physical properties, tilth, and soil health in general
7. Improves survival rate of transplanted seedlings
8. Reduces the maintenance period of seedlings at nursery
9. Improves the survival rate of plants in adverse conditions
Lecture No.: 16 Mass production and quality control of bacterial and fungal bioinoculants. BIS
standards– methods of application of bioinoculants

Mass production

I. Mass multiplication of carrier based bacterial biofertilizers

Biofertilizers are carrier-based preparations containing efficient strain of nitrogen fixing or

phosphate solubilizing microorganisms. Biofertilizers are formulated usually as carrier based
inoculants. The organic carrier materials are more effective for the preparation of bacterial
inoculants. The solid inoculants carry more number of bacterial cells and support the survival of
cells for longer periods of time.

Mass culturing of microorganisms in fermentor

Although many bacteria can be used beneficially as a biofertilizer the technique of mass
production is standardized for Rhizobium, Azospirillum, Azotobacter and phosphobacteria and
Gluconacetobacter. The media used for mass culturing are as follows:

Rhizobium : Yeast extract mannitol broth

Azospirillum : Dobereiner's malic acid broth

Azotobacter : Waksmann No.77 broth

Phosphobacteria : Nutrient broth

Gluconacetobacter : LGI broth

Procedure

1. Prepare appropriate broth in 50 ml flasks and inoculate the mother culture in to the flasks.
2. Grow the culture under shaking conditions at 30±2ºC until maximum cell population of 10 10 to
1011 cfu/ml is reached.
3. Under optimum conditions this population level could be attained within 4 to 5 days for
Rhizobium; 5 to 7 days for Azospirillum; 2 to 3 days for phosphobacteria and 6-7 days for
Azotobacter & Gluconacetobacter. The culture obtained in the flask is called starter culture.
4. Use the starter to inoculate the broth in large size flasks of 250 ml, 500 ml, 3 liters and 5 liters
and grow until required level of cell count is reached.
5. For large scale production of inoculant, inoculum from starter culture is transferred to large
flasks/seed tank fermentor.
Processing of carrier material

The use of ideal carrier material is necessary in the production of good quality biofertilizer. Peat
soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can be used as
carrier materials. The neutralized peat soil/lignite is found to be better carrier material for
biofertilizer production.

Characteristics of an ideal carrier

 Cheaper in cost
 Should be locally available
 High organic matter content
 Should not be toxic
 Water holding capacity of more than 50%
 Easy to process, friability and vulnerability.
 Amenab
le for mixing
Schematic
representation
of mass
production of
bacterial
biofertilizers

| 48

Mixing of broth culture with the carrier and packing

1. Pulverize the carrier material and sieve it through 106 µm sieve. If it is peat / lignite, determine
the pH and neutralize it with CaCO3. In case of charcoal, neutralization is not required.
2. Sterilize the carrier in an autoclave and cool it
3. Mix the culture broth with the carrier and adjust to about 40 % moisture content.
4. After mixing allow the impregnated carrier material to undergo curing at 28-30 oC for further
multiplication of bacterial cultures in the carrier.
5. Pack it in polythene bags of medium density (10.089 mm gauge) or low density (0.038-0.051
mm) and heat seal after expelling the air
6. Store the cultures in cool and dry place at a temperature of 15oC but not exceeding 30oC
7. The preparation and maintenance of biofertilizers should be followed as per the BIS (Bureau of
Indian standard) specifications mentioned below:

Storage

 The packet should be stored in a cool place away from the heat or direct sunlight.
 The packets may be stored at room temperature or in cold storage conditions in lots in plastic
crates or polythene / gunny bags.

Product specifications

 There should be more than 108 cells/g of inoculant at the time of preparation and107 cells/ g on
dry weight basis before expiry date.
 It should not have any contaminant at 10-5 dilution

II. Mass production of liquid biofertilizers

All the bacterial biofertilizers except Rhizobium are produced in liquid formulations also. Liquid
biofertilizers are produced through three step process.

1. Preparation of starter culture and seed culture

 Prepare the starter culture from the mother culture in the respective growth medium as given
for carried based inoculants
2. Mass culturing in fermentor

 Do the mass culturing similar to carried based inoculants in the fermentor.

 Harvest the broth once the population reaches the cell load of 1010 cell per ml broth
3. Preparation of liquid formulation

1. Fill the harvested culture in the sterile plastic container of one liter or 500 ml capacity
 2. Add Glycerol @ of one ml per liter broth to arrest the metabolic activities of the cell so as
to avoid bursting of the container under storage
3. Seal the mouth with sterile caps and store under room temperature

Specifications of Biofertilizers

1. Rhizobium
Carrier based*in form of moist /dry powder or
(i) Base
granules, or liquid based

CFU minimum 5x107cell/g of powder, granules

(ii) Viable cell count
or carrier material or1x108 cell/ml of liquid.
(iii) Contamination level No contamination at105dilution
(iv) pH 6.5-7.5
Particles size in case of carrier All material shall pass through 0.15-0.212mm
(v)
based material. IS sieve
Moisture percent by weight,
(vi) 30-40%
maximum in case of carrier based.
Should show effective nodulation on all the
(vii) Efficiency character
species listed on the packet.
*Type o of carrier: The carrier materials such as peat, lignite, peat soil, humus, wood charcoal or
similar material favoring growth of organism.

2. Azotobacter
Carrier based* in form of moist/dry powder or
(i) Base
granules,or liquid based

CFU minimum 5x107cell/g of powder,granules

(ii) Viable cell count
or carrier material or1x108cell/ml ofliquid.
(iii) Contamination level No contaminationat105dilution
(iv) pH 6.5-7.5
Particles size in case of carrier All material shall pass through 0.15-0.212mm IS
(v)
based material. sieve
(vi) Moisture percent by weight, 30-40%
 maximum in case of carrier
based.
The strain should be capable of fixing at least 10
(vii) Efficiency character
mg of nitrogen per g of sucrose consumed.
*Type of carrier:-The carrier material such as peat, lignite, peat soil, humus, wood charcoal or
similar material favouring growth of the organism.

3. Azospirillum
(i) Base Carrier based* in form of moist/dry powder
or granules,or liquid based
(ii) Viable cell count CFU minimum 5x107cell/g of powder,

granules or carrier material or1x108cell/ml

of liquid.
(iii) Contamination No contamination at105dilution
level
(iv) pH 6.5-7.5
(v) Particles size in case of All material shall pass through 0.15-0.212
carrier based material. mm IS sieve
(vi) Moisture percent by 30-40%
weight,maximum in case
of carrier based.
(vii) Efficiency character Formation of white pellicle in semisolid N-
free bromo thymol blue media.

*Type of carrier:-The carrier material such as peat, lignite, peat soil, humus, wood Charcoal or
similar material favouring growth of the organism.

4. Phosphate solubilising Bacteria

(i) Base Carrier based*in form of moist / dry powder or
granules,or liquid based
(ii) Viable cell count CFU minimum 5x107 cell/g of powder, granules or

carrier material or1x108cell/ml of liquid.

(iii) Contamination No contamination at105dilution
level
(iv) pH 6.5-7.5 for moist / dry powder, granulated carrier
 based and 5.0–7.5for liquid based
(v) Particles size in All material shall pass through 0.15-0.212 mm IS
case of carrier based sieve
material.
(vi) Moisture percent by 30-40%
weight, maximum
in case of carrier
based material
(vii) Efficiency The strain should have phosphate solubilizing
character capacity in the range of minimum 30%, when
tested spectrophotometrically. In terms of zone
formation, minimum 5mm solubilization zone in
prescribed media having at least 3mm thickness.

*Types of Carrier:-The carrier material such as peat, lignite, peat soil, humus, wood Charcoal or
similar material favouring growth of the organism.

Quality Assessment Tests for Biofertilizers

S.No. Test Observation Expected result

Rhizobium

1. Streak on to Yeast Colony character Typical colony of Rhizobium

Extract Mannitol should appear on YEMA & it
 Agar (YEM A) and should not absorb congo red.
Congo Red Yeast
Extract Mannitol
Agar (CRYEMA)

2. Gram stain Reaction Cells must be Gram negative

3. Streak on Glucose Whether growth Growth should not come

Peptone Agar (GPA) is +ve or –ve

4. Measure pH of the - 6.0-7.5

carrier

5. Total plate count No. of colonies 108 cells/g carrier

6. Serological study - Agglutinate with the nominated

antiserum

7. Nodulation study - Nodulation +ve

Azotobacter

1. Streak onto Jensen's Colony character A. chroococcum colonies are

N-free medium gummy, raised with or without
striations, viscous and often
sticky. The pigmentation varies
from very light brown to black.

2. Gram stain Reaction Cells must be gram negative

3. Measure pH of the - 6.5 to 7.5

carrier

4. Measure N-fixation - The minimum amount of fixed

nitrogen should not be less than
10mg/g of sucrose utilized

5. Total plate count - 107/g carrier

Azospirillum
1. Growth in semisolid - 1. Change of the colour of the
medium medium from green to blue
2. Sub surface pellicle formation

2. Growth on solid - 1. Change of the colour of the

medium medium from green to blue

2. Sub-surface pellicle
formation in the semi-solid
broth

Phophobacteria

1. Growth on - 1. Colonies show halo zone

Sperber’sHydroxy around
Apatite medium
the medium

2. The strain should have

phosphateSolubilizing capacity
in the range of minimum 30%,
whentested
spectrophotometrically. In
terms of zone formation,
minimum 5mm solubilization
zone in prescribed media
having at least 3mm thickness.

Potassium Releasers

1. Growth on solid - Colonies show halo zone

medium around the medium

Zinc solubilizers

1. Growth on solid - Colonies show halo zone

medium around the medium

AM Fungal cultures

1. Total viable - Minimum100 /gm of finished

 propagules/ gm of product

Product

2. Infectivity potential - 80 infection points in test

roots/gm of Mycorrhizal
inoculum used

Mass production of AMF

As AM Fungi are obligate symbionts, their mass multiplication in the form of pure culture is
difficult. However, it can be mass multiplied using a host plant like maize, onion, clover, cowpea,
millets, sorghum, cenchrus grass, etc. Several researches in different parts of the world resulted in
different methods of production of AM fungal inoculum as soil based culture as well as carrier based
inoculum. Root organ culture is being used for the production of soil less culture.

As a carrier based inoculum, pot culture is widely adopted method for production. The AM
inoculum was prepared by using sterilized soil and wide array of host crops were used as host. The
sterilization process is a cumbersome one the inert materials such as vermiculite, perlite,
montmorillonite clay etc. are utilized for the production.

Materials required

 Carrier material – Soil and sand / vermiculite / perilite / soilrite

 Seed material of host plant

Production of Root Based Inoculum

Mass multiplication of AM cultures involves a two-step process. In the first step starter culture is
prepared and in the second step, it is mass multiplied.

1. Take soil: sand @ 1:1 in a funnel

2. Place around 20-30 spores of selected AM fungal species in the soil and sand mix and sow maize
seeds.
3. Allow the maize plant to grow
4. Uproot the plant at timely intervals and examine for AM fungal infection in roots and spores in
soil.
5. After 50-60 days of growth, uproot the plant and prepare starter culture by mixing the carrier
material with macerated root pits.
6. Take either soil alone or in combination with carrier materials such as vermiculite / perilite /
soilrite @ 1:10 in mud pots / cement tanks (1m x 1m x 0.3m) and mix thoroughly.
7. Place the starter (1 kg of AM inoculum) culture around 2.5 cm below the soil and sow maize
seeds.
8. Apply 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench at the time of
sowing seeds. Further apply 10 g of urea twice on 30 and 45 days after sowing for each trench
9. Apply 1 g of micronutrient mixture when there will be a symptom for deficiency
10. Allow the plant to grow and check for AM fungal infection periodically
11. After 3-4 months, harvest the plant, mix the root system with soil mix and use as inoculum
consists of a mixture of vermiculite, spores, pieces of hyphae and infected root pieces.
Instead of soil and sand mixture, even vermiculite alone or soilrite + perilite (1:1) mixture can also
be used as a substrate depending up the availability. If an inert material like vermiculite is used
for multiplication, host plants are fertilized in the form of urea, superphosphate and muriate of
potash.

Specification of biofertilizers

Mycorrhizal biofertilizers
Fine Powder / tablets / granules / root biomass
i. Form/base
mixed with growing substrate
Particle size for carrier 90 % should pass through 250micron IS
ii.
based powder formulations sieve(60BSS)
Moisture content percent
iii. 8 -12
maximum
iv. pH 6.0 to7.5
Total viable propagules / 100 gm of finished product with minimum 60
v.
gram of product spores per gram
Inoculum potential:1200IP/g
vi. Infectivity potential (determined by MPN method with10 fold
dilution)
Lecture No.: 17. Biofuel production – methane, hydrogen, alcohol and biodiesel production
Microbiology of methane production

(1) Complex polymers are broken down to soluble products by enzymes produced by
fermentative bacteria which ferment the substrate to short chain fatty acids, hydrogen and
carbon-di-oxide. Fatty acids, longer than acetate are metabolised to acetate by obligate
hydrogen producing acetogenic bacteria.

(2) The major products after digestion of the substrate by these two groups are hydrogen,
carbon dioxide, and acetate. Hydrogen and carbon dioxide can be converted to acetate by
hydrogen oxidising acetogens

(3) Methane by carbon dioxide reducing, hydrogen oxidising methanogens

(4) Acetate is also converted to methane by aceticlastic metanogens

Nearly seventy percent of methane from biogas digesters fed with cattle dung is derived from

acetate.

The first three groups of bacteria include facultative as well as strict anaerobics like
Cellulomonas, Clostridium, Bacillus, Bacteroides, Ruminococcus, Eubacterium etc. While the
methanogenic bacteria include Methanosaraina, Methanothrix, Methano bacterium and
Methanospirillum.

Microbial conversion of organic matter to methane has become attractive as a method of

waste treatment and resource recovery. This process is anaerobic and is carried out by action of
various groups of anaerobic bacteria. Biogas is mixture of methane (50-60%), CO 2 (30-40%),
Hydrogen (5-10%), H2S and nitrogen (traces), produced from the anaerobic digestion of animal,
plant wastes or any cellulos containing waste material. The digester used for biogas production
is called as 'Biogas plant'. A typical biogas plant (Fig.2) using cowdung as raw material consists
of a) digester and b) gas holder. The digesters are either of (i) batch type which are filled once,
sealed and emptied when the raw materials stop producing gas or (ii) continuous type which are
fed with a definite quality of wastes at regular intervals so that gas production is continuous and
regular.

Microbial groups involved in biogas/ methane production

 Complex polymers
(Polysaccharides
Proteins, etc.)

Mono and 1 Propionatre, Butyrate

Oligomers(Sugars, etc.(long chain fatty
amino acids acids)

1
2
1 2

H2 + CO2 3 Acetate

4 5

CH4, C02

Fermentative bacteria

1) Obligately hydrogen producing acetogenic bacteria

2) Hydrogen consuming acetogenic bacteria
3) Carbon-di-oxide reducing methanogens
4) Aceticlastic methanogens
 LECTURE NO. 15.: SILAGE PRODUCTION. BIOINOCULANTS – TYPES;
BIOFERTILISERS - BACTERIAL, FUNGAL (AMF) AND ALGALBIOFERTILISERS.
BIOPESTICIDES – TYPES AND MECHANISM OF ACTION

Silage production
Silage has been used to conserve fodder for more than 3000 years. It is currently the most used
technique for conserving ruminant feed. The process of silage making includes cutting fresh
(green) fodder, compacting it, and storing and fermenting it under controlled conditions in a silo,
where air cannot come in contact with the silage. Any green forage crop can be made into silage.
This fermentation process is divided into 4 phases aerobic phase, the intense fermentation phase,
the stable phase and the aerobic feed-out phase.
Silage undergoes anaerobic fermentation, which starts about 48 hours after the silo is filled, and
converts sugars to acids. Fermentation is essentially complete after about two weeks.
Before anaerobic fermentation starts, there is an aerobic phase in which the trapped oxygen is
consumed. How closely the fodder is packed determines the nature of the resulting silage by
regulating the chemical reactions that occur in the stack. When closely packed, the supply of
oxygen is limited, and the attendant acid fermentation brings about decomposition of
the carbohydrates present into acetic, butyric and lactic acids. This product is named sour silage.
If the fodder is unchaffed and loosely packed, or the silo is built gradually, oxidation proceeds
more rapidly and the temperature rises; if the mass is compressed when the temperature is 60–
70 °C (140–160 °F), the action ceases and sweet silage results. The nitrogenous ingredients of
the fodder also change: in making sour silage, as much as one-third of the albuminoids may be
converted into amino and ammonium compounds; in making sweet silage, a smaller proportion
is changed, but they become less digestible. If the fermentation process is poorly managed, sour
silage acquires an unpleasant odour due to excess production of ammonia or butyric acid
In the past, the fermentation was conducted by indigenous microorganisms, but, now some bulk
silage is inoculated with specific microorganisms to speed up fermentation or improve the
resulting silage. Silage inoculants contain one or more strains of lactic acid bacteria, and the
most common is Lactobacillus plantarum. Other bacteria used include Lactobacillus
buchneri, Enterococcus faecium and Pediococcus species. Ryegrasses have high sugars and
respond to nitrogen fertiliser better than any other grass species. These two qualities have made
ryegrass the most popular grass for silage-making for the last sixty years. 
Bioinoculants
1. Biofertilisers
2. Biopesticides

Biofertilizers
Biofertilizers are defined as preparations containing living cells or latent cells of efficient strains
of microorganisms that help crop plants uptake of nutrients by their interactions in the
rhizosphere when applied through seed or soil. They accelerate certain microbial processes in the
soil which augment the extent of availability of nutrients in a form easily assimilated by plants.
Use of biofertilizers is one of the important components of integrated nutrient management, as
they are cost effective and renewable source of plant nutrients to supplement the chemical
fertilizers for sustainable agriculture. Several microorganisms and their association with crop
plants are being exploited in the production of biofertilizers. They can be grouped in different
ways based on their nature and function.
I. Nitrogen fixers

a. Free living - Aerobic – Azotobacter, Beijerinckia, Anabaena

Anaerobic – Clostridium
Faultative anaerobic – Klebsiella

b. Symbiotic : Rhizobium, Frankia, Anabaena azollae

c. Associative symbiotic Azospirillum

d. Endophytes : Gluconacetobacter, Burkholdria

II. Phosphate solubilizers

Bacteria - Bacillus megaterium var. phosphaticum

B. subtilis, B. circulans, Pseudomonas striata

Fungi : Penicillium digitatum, Aspergillus awamori

III. P mobilizers
 a) AM fungi
b) Ectomycorrhizal fungi
c) Ericoid Mycorrhiza
d) Orchidaceous mycorrhiza
IV. Silicate (potassium releasing) and Zinc solubilizers : Bacillus muscilagenosus,
V. Plant growth promoting Rhizobacteria: Pseudomans spp.,and many more

Importance of biofertilizers
Biofertilizers are known to make a number of positive contributions in agriculture.
 Supplement fertilizer supplies for meeting the nutrient needs of crops.
 Add 20 – 200 kg N/ha (by fixation) under optimum conditions and solubilise/mobilise 30-
50 kg P2O5/ha.
 They liberate growth promoting substances and vitamins and help to maintain soil fertility.
 They suppress the incidence of pathogens and control diseases.
 Increase the crop yield by 10-50%. N2 fixers reduce depletion of soil nutrients and provide
sustainability to the farming system.
 Cheaper, pollution free and based on renewable energy sources.
 They improve soil physical properties, tilth and soil health.
1. Rhizobium
Rhizobia are soil bacteria, live freely in soil and in the root region of both leguminous and non-
leguminous plants. However they enter into symbiosis only with leguminous plants, by infesting
their roots and forming nodules on them. Non legume nodulated by Rhizobia is Trema or
Parasponia sp.
The nodulated legumes contribute a good deal to the amount of N 2 fixed in the biosphere, (50-
200 kg N/ha) varied with crops.

Clover - 130 kg N/ha

Cowpea - 62 – 128 kg N/ha

Beijerinck first isolated and cultivate a microorganism from the roots of legumes in 1888 and he
named this as Bacillus radicola and latter modified as Rhizobium.
Legume plants fix and utilise this N by working symbiotically with Rhizobium in nodules on
their roots. The host plants provide a home for bacteria and energy to fix atmospheric N 2 and in
turn the plant receives fixed N2 (as protein).
Cross inoculation group (CGI)
It refers the group of leguminous plant that will develop nodules when inoculated with the
rhizobia obtained from the nodules from any member of that legume group.

Genera/species Principal and other reported hosts

Rhizobium
R.etli Phaseolus vulgaris, Mimosa affinis
R.galegae Galega orientalis, G.officinalis
R.gallicum Phaseolus vulgaris, Leucaena, Macroptilium,
Onobrychis
R.giardini Phaseolus vulgaris, Leucaena, Macroptilium
R.hainanense Desmodium sinuatum, Stylosanthes, Vigna, Arachis,
Centrosema
R.huautlense Sesbania herbacea
R.indigoferae Indigofera
R.leguminosarum   
                           bv trifolii       Trifolium
                           bv viciae Lathyrus, Lens, Pisum, and Vicia,
                         bv.phaseoli Phaseolus vulgaris
R.mongolense Medicago ruthenica, Phaseolus vulgaris
R.sullae Hedysarum coronarium
R.tropici Phaseolus vulgaris, Dalea, Leucaena, Macroptilium,
Onobrychis
Mesorhizobium
M.amorphae Amorpha fruticosa
M.chacoense Prosopis alba
M.ciceri Cicer arietinum
M.huakuii Astragalus sinicus, Acacia
 M.loti Lotus corniculatus
M. mediterraneum Cicer arietinum
M.plurifarium Acacia senegal, Prosopis juriflora, Leucaena
M.tianshanense Glycyrrhiza pallidflora, Swansonia, Glycine,
Caragana, Sophora
Sinorhizobium
S.abri Abrus precatorius
S.americanus Acacia spp.
S.arboris Acacia senegal, Prosopis chilensis
S.fredi Glycine max
S.indiaense Sesbania rostrata
S.kostiense Acacia senegal, Prosopis chilensis
S.kummerowiae Kummerowia stipulacea
S.meliloti Medicago, Melilotus, Trigonella
S.medicae Medicago truncatula, M. polymorpha, M.orbicularis
S.morelense Leucaena leucocephala
S.sahelense Acacia, Sesbania
S.terangae Acacia, Sesbania
Azorhizobium
A.caulinodans Sesbania rostrata
Allorhizobium
A.undicola Neptunia natans, Acacia, Faidherbia, Lotus
Bradyrhizobium
B.elkanii Glycine max
B.japonicum Glycine max
B.liaoningense Glycine max
B.yuanmingense Lespedeza, Medicago, Melilotus

Description and characteristics

Classification

1. Family : Rhizobiaceae
 2. Genus : Azorhizobium-for stem nodulation
Bradyrhizobium
Rhizobium
Sinorhizobium

Morphology
1. Unicellular, cell size less than 2µ wide, short to medium rod, pleomorphic.
2. Motile with peritrichous flagella
3. Gram negative
4. Accumulate PHB granules.
Physiology
1. Nature : Chemoheterotrophic, symbiotic with legume
2. C source : Supplied by legume through photosynthates,
monosaccharides, disaccharide
3. N source : Fixed atmospheric N2
4. Respiration : Aerobic
5. Growth : Fast (Rhizobium), slow (Bradyrhizobium)
6. Doubling time : Fast growers – 2-4 hrs
Slow growers – 6-12 hrs
7. Growth media : YEMA
Contribution
1. Direct contribution of N symbiotically with legumes.
2. Residual nitrogen benefit for the succeeding crop.
3. Yield increase is by 10-35%.
4. Improve soil structure.
5. Produces exopolysaccharides.
6. Produces plant growth hormone.
Recommended for legumes (Pulses, oilseeds, fodders)
2. Azotobacter
It is a free living N2 fixer, the cells are not prevent on the rhizoplane, but are abundant in the
rhizosphere region. It is classified under the family Azotobacteriaceae. It requires more of
organic matter and depend on the energy derived from the degradation of plant residues.
Beijerinck was the first to isolate and describe Azotobacter.
Species
Cell size, flagellation, pigmentation and production of extracellular slime are considered as
diagnostic features of these bacteria in distinguishing species.
A. chroococcum : Black to brown insoluble pigment.
A. vinelandii, A. paspali, : Green fluorescent and soluble pigments
A. agilis
A. beijerinckii : Yellow to light brown insoluble pigments
A. macrocytogenes : Pink soluble pigments
A. insignis : Yellow brown pigments
Azotobacter cells are polymorphic, gram negative, form cyst and accumulate Poly Beta hydroxy
butyric acid and produces abundant gum.
Morphology
Cell size : Large ovoid cells, size 2.0 – 7.0 x 1.0 – 2.5 µ
Cell character : Polymorphic
Gram character : Negative
Physiology
1. Nature : Chemoheterotrophic, free living
2. C source : Mono, di saccharides, organic acids
3. N source : N2 through fixation, amino acids, NH4+, NO3-
4. Respiration : Aerobic
5. Growth : Ashby / Jensen's medium
6. Doubling time : 3 hours
Benefits
 Ability to fix atmospheric N2 – 20-40 mg BNF/g of C source in laboratory equivalent to
20-40 kg N/ha.
 Production of growth promoting substances like vitamin B, Indole acetic acid, GA.

 Ability to produce thiamine, riboflavin, pyridoxin, cyanogobalanine, nicotinic acid,

pantothenic acid, etc.
 Biological control of plant diseases by suppressing Aspergillus, Fusarium.
Recommended for Rice, wheat, millets, cereals, cotton, vegetables, sunflower, mustard and
flowers.
3. Azospirillum
Azospirillum was isolated by Beijerinck (1922) in Brazil from the roots of Paspalum and named
it as Azotobacter paspali and later named as Spirillum lipoferum. Dobereiner and Day (1976)
reported the nitrogen fixing potential of some forage grasses due to the activity of S. lipoferum in
their roots. Dobereiner coined the term "Associative symbiosis" to denote the occurrence of N 2
fixing spirillum in plants. Taxonomy was re-examined and Tarrand et al. (1978) designated this
organism as Azospirillum.
It is an aerobic or micro aerophilic, motile, gram negative bacterium. Non spore former and
spiral shaped bacterium, inhabiting the plant roots both externally and internally. Being a micro
aerophilic organism, it can be isolated on a semi solid malate medium by enrichment procedures.
Classification
Species : (7) Family – Spirillaceae
1. A. brasilense
2. A. lipoferum
3. A. amazonense
4. A. halopraeferens
5. A. irkense
6. A. dobereinerae
7. A. largimobilis
Morphology

1. Cell size : Curved rods, 1 mm dia, size and shape vary

2. Accumulate : PHB

3. Gram reaction : Negative

4. Development of white pellicles : 2-4 mm below the surface of NFB medium

Physiology
1. Nature : Chemoheterotrophic, associative
2. Sole carbon source : Organic acids, L-arabinose, D-gluconate,
D-fructose, D-glucose, sucrose, Pectin
 3. N source : N2 through fixation, amino acids, N2, NH4+, NO3-
4. Respiration : Aerobic, Microaerophilic
5. Growth media : NFBTB (NFB) medium
6. Doubling time : 1 hr in ammonia containing medium
5.5 – 7.0 hrs in malate containing semisolid medium
1. Mechanism of Action
2. Contribution by BNF
3. Production of PGP substances by bacteria - Increases root hair development, biomass.
4. Production of PGP substances by plant
5. Morphological changes in root cells.
6. Increased activity of IAA oxidase
7. Increase in endogenous IAA
8. Increased mineral and water uptake, root development, vegetative growth and crop yield.
9. Competition in the rhizosphere with other harmful microorganism.
10. Polyamines and amino acids production.
11. Increased extrusion of protons and organic acids in plants.
Benefits
1. Promotes plant growth.
2. Increased mineral and water uptake, root development, vegetative growth and crop
yield.
3. Inoculation reduced the use of chemical fertilizers (20-50%, 20-40 kg N/ha)
4. Increases cost benefit ratio.
5. Reduces pathogen damage.
6. Inhibit germination of parasitic weeds.
7. Restoration of arid zone, margine mangrove ecosystem.
8. Reduces humic acid toxicity in compost.
Recommended for rice, millets, maize, wheat, sorghum, sugarcane and co-inoculant for legumes.
4. Gluconacetobacter diazotrophicus
It is an endophytic N2 fixer and form to occur on large numbers in roots, stem and leaf of
sugarcane and other sugar rich crops. It was first isolated from sugarcane. Cavalcanti and
Dobereiner (1988) reported this new endophytic N2 fixer and recently called as from G.
diazotrophicus. It can tolerate upto 30% sucrose concentration and pH upto 3.0. Optimum
sucrose concentration is 10-15%. Produce surface yellow pellicle on semisolid medium. Does
not grow at pH 7.0. Optimum is 5.5.
Benefits
- Fixes atmospheric N2
- Production of PG hormones (GA, DAA is double than Azospirillum).
- Suitable for sugar rich crops with acidic pH.

5. Azorhizobium
This genus can produce stem nodules. Stem nodulation has been reported in 3 genera of legumes:
Aeschynomene, Neptunia and Sesbania.
Stem nodulating Rhizobium comprises both fast and slow growing types having the generation
time of 3-4 hr and 10 hrs respectively. Those nodulate Aeschynone can cross inoculate with S.
rostrata strains Azorhizobium caulinodans.
 fix N2 in free living conditions without differentiating into bacteroids.
 have O2 protection mechanisms, to fix N2 under free living conditions.
 Mode of entry is through lateral root cracks. No infection thread is formed during
colonization.
 Form both stem and root nodules in S. rostrata.
 Gram negative, motile rods.
 Produces root nodules in rice, wheat.
6. Algal Biofertilizers
The agronomic potential of cyanobacterial N2 fixation in rice fields was recognised in India
during 1939 by De who attributed the natural fertility of tropical rice fields to N 2 fixing blue
green algae. The rice field ecosystem provides an environment favourable for the growth of blue
green algae with respect to their requirements for light, water, high temperature and nutrient
availability.Algal biofertilizers constitutes a perpetual source of nutrients and they do not
contaminate ground water and deplete the resources. In addition to contributing 25-30 kg N ha -1
of biologically fixed N2, they can also add organic matter to the soil, excrete growth promoting
substances, solubilises insoluble phosphates and amend the physical and chemical properties of
the soil.Blue green algae are a group of prokaryotic, photo synthetic microscopic plants,
vigorously named as Myxophyceae, Cyanophyceae and Cyanobacteria. They show striking
morphological and physiological similarities with bacteria and hence are called as cyanobacteria.
Cyanobacteria
They are the photosynthetic bacteria and some of them are able to fix N 2. They can be divided
into two major groups based on growth habit. 1). unicellular forms and 2). filamentous forms.
N2 fixing species are from both groups, found in paddy fields, but the predominant ones are the
heterocystous filamentous forms. Cyanobacteria are not restricted to permanently wet habitats, as
they are resistant to desiccation and hot temperatures, and can be abundant in upland soils.
However wet paddy soils and overlying flood waters provide an ideal environment for them to
grow and fix N2.
Natural distribution
BGA are cosmopolitan in distribution. More widely distributed in tropical zone. Free living
cyanobacteria can grow epiphytically on aquatic and emergent plant as well as in flood water or
on the soil surface. Heterocystous cyanobacteria formed less than 10% of the population of
eukaryotic green algae and the abundance increased with the amount of available phosphorus
and with the pH value over the range 4 – 6.5. In rice soil, population ranges from 10 – 10 7 cfu g-1
soil.
The main taxa of N2 fixing cyanobacteria
Group Genera DNA (mol % GC)
Group-I. Unicelluar: single Gloeothece, 35-71
cells or cell aggregates Gloeobacter,Synechococcus,
Cyanothece, Gloeocapsa,
Synechocystis, Chamaesiphon,
Merismopedia
Group-II. Pleurocapsalean: Dermocarpa, Xenococcus, 40-46
reproduce by formation of Dermocarpella, Pleurocapsa,
small spherical cells called Myxosarcina, Chroococcidiopsis
baeocytes produced through
multiple fission.
Group-III. Oscillatorian: Oscillatoria, Spirulina, 40-67
filamentous cells that divide Arthrospira, Lyngbya,
by binary fission in a single
 plane. Microcoleus, Pseudanabaena.
Group-IV. Nostocalean: Anabaena, Nostoc, Calothrix, 38-46
filamentous cells that Nodularia, Cylinodrosperum,
produce heterocysts Scytonema
Group-V.Branching: cells Fischerella, Stigonema, 42-46
divide to form branches Chlorogloeopsis, Hapalosiphon
The N2 fixing forms generally have a specialized structure known as heterocyst. The BGA have
minimum growth requirement needing only diffused light, simple inorganic nutrients and
moisture. The heterocysts which are modified vegetative cells, because of their thick walls and
absence of photonactin II in photosynthesis, act as ideal sites for N2 fixation under aerobic
conditions. Although the nitrogenase is present in vegetative cells, it remains inactive because of
the presence of oxygenic photosynthesis. They built up natural fertility (C, N) in soil.
N2 fixing BGA: Anabaena, Nostoc, Cylindrospermum, Tolypothrix, Calothrix, Scytonema,
Westiellopsis belonging to orders Nostocales and Stignematales. Many non-heterocystous forms
are also fix N2. eg: But need microaerobic or anaerobic conditions. Gleocapsa fix in aerobic
condition. The species of BGA, known to fix atmospheric N2 are grouped as 3 groups.
(i) Heterocystous – aerobic forms
(ii) Aerobic unicellular forms
(iii) Non-heterocystous, filamentous, micro aerophilic forms.
The blue green algal culture's composite inoculum consisting of Nostoc, Anabaena, Calothrix,
Tolypothrix, Plectonema, Aphanotleca, Gleocapsa, Oscillatoria, Cylindrospermum, Aulosira
and Scytonema.
Contributions of algal biofertilizer
 Important component organic farming.
 Contribute 20 – 25 kg N ha-1.
 Add organic matter to the soil.
 Excrete growth promoting substances.
 Solubilize insoluble phosphates.
 Improve fertilizer use efficiency of crop plants.
 Improve physical and chemical properties of soil.
 Reduce C:N ratio.
 Increase the rice yield by 25-30%.
  Cyanobacteria are more compatible with nitrate N than ammonium N.

It increases FUE of the crop plants through exudation of growth promoting substances and
preventing a part of applied fertilizer N from being lost.
Production of algal biofertilizer
Less cost intensive than bacterial biofertilizer. Because of slow release, the crop plants are able
to utilise more nutrients from soil in presence of algal inoculation.

Production methods
BGA can be mass produced as soil based composite culture (algal flakes) by different methods.
I. Multiplication in trays
 Big metallic trays (6’x 3’x 6”lbh) can be used for small scale production
 Take 10 kg of paddy field soil, dry powder well and spread ,
 Fill water to a height of 3”,Add 250 g of dried algal flakes (soil based) as inoculum
 Add 150 g of super phosphate and 30 g of lime and mix well with the soil
 Sprinkle 25 g carbofuran to control the insects and maintain water level in trays
 After 10 to 15 days, the blooms of BGA will start floating on the water sources
 At this stage stop watering and drain. Let the soil to dry completely
 Collect the dry soil based inoculum as flakes and store in a dry place.
 By this method 5 to 7 kg of soil based inoculum can be obtained.
II. Multiplication under field condition
 Select an area of 40 m2 (20x2m) near a water source which is directly exposed to
sunlight.Make a bund all around the plot to a height of 15 cm and give it a coating with
mud to prevent loss of water due to percolation
 Plot is well prepared and leveled uniformly and water is allowed to a depth of 5-7.5 cm
and left to settle for 12 hrs
 Apply 2kg of super phosphate and 200 g lime to each plot uniformly over the area and
broadcast the well powdered soil based composite starter culture of BGA containing 8-
10 genera at the rate of 5 kg/plot
 Apply Carbofuran at 200g per plot to control soil insects feeding BGAand maintain the
water level at 5 c.m
  The growth of BGA is rapid and in about 10-15 days, a thick mat is formed which floats
on the surface of the water.After 15 days of inoculation, allow the plots to dry up in the
sun, collect the algal flakes and store.
Observations:The floating algal flakes are green or blue green in colour. From each harvest, 30
to 40 kg of dry algal flakes are obtained from 1 cent plot.
Quality of the inoculums : Like bacterial inoculants, the quality of the algal inoculum can be
quantified in terms of cfu. An ideal inoculum should have a cfu value of atleast 10,000 per gram.
Although no appreciable loss in cfu was observed even after 2 years, the safe shelf life can be 1
year.

Field application: The recommended method of application of the algal inoculum is

broadcasting on standing water, about 3-4 days after transplantation. Since size of the inoculum
is too small to ensure uniform distribution over an area of one acre, the inoculum can be mixed
with clean and sieved soil and broadcasted. After the application of algal inoculum, the field
should be kept water logged for about a week's time. Establishment of algal inoculum can be
observed within a week of inoculation in form of floating algal mat.Heterocyst do not develop in
the presence of ammonium but NH4+ ions inhibit the synthesis of nitrogenase in cultures.
- N2 fixing cyanobacteria can degrade both naturally occurring aromatic hydrocarbons and
xenobiotics; since being used for the bioremediation of polluted surface water.
- They are the potential sources of phycobilin proteins.

Acid and salt tolerant cyanobacteria

Acid tolerant cultures - Westiellopsis, Anabaena, Nostoc

Salt tolerant cultures -

They can react to salt stress either through osmotic adjustment or by secretion of excess
polysaccharide. It involves the accumulation of carbohydrates, aminoacids. Salt adaptation
strategy includes two main processes.
(i) Maintenance of low internal contents of inorganic ions by active export mechanism
(Na+/K+ antiporter)
(ii) Synthesis and accumulation of osmoprotective compounds corresponding to the osmotic
potential of the surrounding medium. eg. sucrose, trehalose, glycerol. The physiological
 basis of salt tolerance may be due to extrusion of Na+ and maintenance of low
intracellular Na+, accumulation of K+ or ionic balance between K+ and Na+.
Immobilization of cyanobacterial biofertilizer for rice crop
Free living cyanobacteria, symbiotic BGA (separated from Azolla) can be entrapped in solid
matrix commonly used one is PUF. The immobilized cyanobacteria were intact in the solid
matrix of PUF provided the cyanobiont a niche similar to the cavity of Azolla fronds in a natural
association. Ammonia excretion is found to be more in immobilized cells.

Mass multiplication of Azolla biomass

Azolla is a floating water fern and it is ubiquitous in distribution. There are six species of Azolla
such as A. caroliniana, A. nilotica, A. filiculoides, A. mexicana, A. microphylla , and A. pinnata
The common species of the water fern occurring is A. pinnata. The plant has a floating branched
stem, deeply bilobed leaves and true roots which grow into the body of water. Each leaf has a
dorsal and a ventral lobe. The dorsal fleshy lobe is exposed to air and contains chlorophyll. It has
an algal symbiont, Anabaena azollae within the central cavity. The ventral lobe is thin, is partly
submerged in water, and lacks chlorophyll. The alga fixes atmospheric nitrogen and is present at
all stages of growth and development of the fern.
Azolla multiplies vegetatively by fragmentation. Sexual reproductive structures - micro sporocarp
(male) and mega sporocarp (female) - form when the conditions are not favourable. When
conditions become favourable the spores germinate into prothalli and produce antheridium and
oogonium respectively. These structures fuse and produce zygote which grows in to young Azolla
plant.
Mass multiplication
Azolla is mass multiplied under field conditions in one cent plots as follows
 Select a wetland field and prepare thoroughly and level uniformly
 Mark the field into one cent plots (20m x2m) by providing suitable bunds and irrigation
channels,Maintain water level to a height of 10 cm
 Sprinkle @ 10 kg of cattle dung mixed in 20 litres of water per plot
  Apply 100 g super phosphate per plot as basal dose,Inoculate fresh Azolla at 8 kg per
plot by spreading over the standing water
 Apply super phosphate 100 g as top dressing on 4 th and 8th day after Azolla inoculation
to each plot and Apply carbofuran (Furadan 3G) granules at the rate of 100 g/plot on 7 th
day after Azolla inoculation to control pests.
 Maintain water level at 10 cm throughout the growth period.( 3 weeks)
Phosphate solubilising Microorganisms
Introduction
Though most soils contain appreciable amounts of inorganic P, most of it being insoluble forms,
cannot be utilized by crops unless they are solubilzied. Soils also contain organic P that could not
be utilized by plants only when it is mineralized. Phosphate solubilizing microorganisms not
only able to solubilize insoluble forms of inorganic P but are also capable to mineralize organic
forms of P, thus improving the availability of native soil P making their P available to plants.
PSM can also solubilize P from rock phosphate (RP), slag or bone meal making their P available
to plants. Thus PSM biofertilizer being economical and environmentally safe offers a viable
alternative to chemical fertilizers.
Microorganisms involved
Many microorganisms can solubilize inorganic phosphates, which are largely unavailable to
plants. Microbial involvement in solubilization of inorganic phosphate was first shown by
Stalstron (1903) and Sacket et al. (1908) gave conclusive evidence for bacterial solubilization of
RP, bonemeal and TCP. Various bacteria and fungi reported to solubilize different types of
insoluble phosphates. Not only solubilizes but also mineralize organic P compounds and release
orthophosphates.
In general PSM constitute 0.5 – 1.0% of soil microbial population with bacteria and out number
the fungi by 2 – 150 folds. But bacteria may loose the P solubilizing ability while sub culturing
and fungi do not lose. Among bacteria, aerobic spore forming bacteria are more effective P
solubilizers.
Mechanism of PO4 solubilization
Different mechanisms were suggested for the solubilization of inorganic phosphates.
Production of organic acids
  Produce monocarboxylic acid (acetic, formic), monocarboxylic hydroxy (lactic, gluconic,
glycolic), monocarboxylic keto, dicarboxyl (oxalic, succinic), dicarboxylic hydroxy
(Malic, maleic) and tricarboxylic hydroxy (citric) acids.
 Solubilization of TCP in liquid medium depends on pH and form of soluble Ca-
complexes. Aliphatic acids are more effective than phenolic acids.
 Organic acids lowers the pH, which helps in the formation of stable complexes with such
cations as Ca++, Mg++, Fe++ and Al++. These complexes are of greater stability than
inorganic PO4 compounds. Such reactions known to prevent the fixation of PO4 ions.
Chelating effect
 Chelation plays an important role in PO4 solubilization PSM produce effective chelating
materials that could solubilize the P.
 The action of organic chelates in solubilizing insoluble PO 4-s and PO4 minerals has been
attributed to the formation of complexes with Ca, Fe or Al, thereby releasing the PO 4 in
water soluble forms.
Production of inorganic acids
 Nitric or sulphuric acids, produced due to oxidation of nitrogenous, compounds or of
inorganic S by nitrifying bacteria and Thiobacillus spp.
 These acids react with insoluble PO4 including rock PO4 and bring about their
solubilization.
Hydrogen sulphide production (H2S)
 H2S may be formed by reduction of S containing amino acids by several heterotrophic
bacteria or by SO4 reducing bacteria. Desulfovibrio, which reduces ferric phosphate to
ferrous sulphide with the release of phosphate.
 H2S produced under anaerobic conditions (water logged) reduces iron in insoluble ferric
phosphate to soluble iron (ferrous sulphide) with the release of P in solution. S
application may also helps to solubilize phosphates.
Effect of carbon dioxide
 CO2 produced by microorganism and plant roots in the rhizosphere encourage
solubilization of insoluble PO4s.
Ca3 (PO4)2 + CO2 + H2O = 2 Ca HPO4 + CaCO3
Proton extrution
Proton release may cause P solubilization at microbial cell surface.
Siderophore production
Siderophores, bind iron tightly to prohibit its reaction with soluble phosphate and rather help
release phosphate fixed as ferric phosphate. It is important in acid soils, where ferric PO 4 is one
of the major forms. The extent of phosphate solubilization depends on the type of organisms
involved. The genus Bacillus showed maximum activity followed by Penicillium and
Aspergillus. Streptomyces was least effective. A. awamori & Penicillium digitatum are
effective fungi and Bacillus polymixa & Pseudomonas striata are effective bacterial
biofertilizers.
Fungal Bioinoculants - Mycorrhizae
Mycorrhiza (fungus root) is the mutualistic association between plant roots and fungal mycelia.
Frank (1885) gave the name "mycorrhiza" to the peculiar association between tree roots and
ectomycorrhizal fungi. 95% of the plant species form mycorrhizae. It can act as a critical linkage
between plant roots and soil. This association is characterized by the movement of plant
produced carbon to fungus and fungal acquired nutrients to plants. Mycorrhizal fungi are the key
components of the rhizosphere are considered to have important roles in natural and managed
ecosystems.
Types of mycorrhiza
Mycorrhizal associations vary widely in structure and function. Two main groups of mycorrhizae
are recognized; the ectomycorrhizae and endomycorrhizae, although the rare group with
intermediate properties, the ectendotrophic mycorrhizae.
Ectomycorrhiza
The fungal hyphae form a mantle both outside the root and within the root in the intercellular
spaces of the epidermis and cortex. No intracellular penetration into epidermal or cortical cells
occurs, but an extensive network called the Hartignet is formed between these cells. Sheath or
Mantle increases the surface area of absorbing roots and offers protection to the roots. Hartignet
can act as storage and transport organ for P.
Ectomycorrhizae are common on trees, including members of the families pinaceae (Pin, Fir,
Spruce, Larch, Semlock), Fagaceae (Willow, Poplar, Chesnut), Betulaceae (Birch, Alder),
Salicaceae (Willow, Poplar) and Myrtaceae. The fungi forming Ectomycorrhizal association are
coming under Basidiomycotina and Ascomycotina. eg: Laccaria laccata, Suillus, Rhizopogan,
Amanita
Endomycorrhizae
Endomycorrhizae consist of three sub groups, but by far the most common are the Arbuscular
Mycorrhizal fungi. Fungi under AM are the members of Endogonaceae and they produce an
internal network of hyphae between cortical cells that extends out into the soil, where the hyphae
absorb mineral salts and water. This fungus do not form an external mantle but lives within the
root. In all forms, hyphae runs between and inside the root cells which includes,
Ericoide mycorrhiza - Associated with some species of Ericaceous plants
Orchid mycorrhiza - associated with orchid plants
Arbuscular mycorrhiza - associated with most of the plant families
Ectendotrophic Mycorrhiza
Ectendotrophic Mycorrhiza are the arbutoid and monotrophoid mycorrhizae. It forms sheath
and produce intracellular penetration.
Arbutoid / Monotrophoid / ectendo – Ectomycorrhizal types
The fungi belong to Basidiomycotina, which covers both Gymnosperms and Angiosperms
plants. The fungal sheath is very thin. Hartignet is present in the intercellular spaces and also
intracellular penetration of hyphae in cortical cells is noticed.
Ericaceous mycorrhiza / Ericoid mycorrhizal: Colonize the plants in the order Ericales. If it
colonises the plants of the family Ericoids, then they are called as Ericoid mycorrhizae. Ericoid
fungus penetrates cortical cells and produce hyphal coils in outer cells of narrow hairy roots of
plants. Fungi belonging to Ascomycetes form this symbiosis. eg. Hysenoschyphus ericae.
Orchid mycorrhiza / orichidaceous mycorrhiza
Associated with plants of orchidaceae. It produces hyphal coils with in roots coming under
Basidiomycetes – Armillaria, Rhizoctonia. Hyphal coils called Pelotons.
Arbuscular Mycorrhizal fungi
AM, an endomorphic mycorrhizae formed by the aseptate phycomycetous fungi are associated
with majority of agricultural crops, growing under broad ecological range.
Class : Zygomycotina
Order : Endogonales
Family : Endogonaceae
Morton and Benny (1990) classification of is given below. 6 genera with 150 species of AMF are
reported by them.
Order Sub order Family Genus
 Endogonales Endogonaceae Endogone
Glomales (i) Glominae (i) Glomaceae Glomus
Sclerocystis
(ii) Acaulosporaceae Enterophosphora
Acaulospora
(ii) Gigasporinae (i) Gigasporaceae Gigaspora
Scutelliospora
.
Colonization Process
Roots do not show visual morphological changes due to AM colonization. AM fungal infection
into a host occurs by germination of spore, hyphal growth through soil to host roots, penetration
of host roots and spread of infection inter and intracellularly in the root cortex. Colonization
occurs under two phases: (1) Extra matrical phase and (2) Intra radical phase.
Extra matrical phase: Events occurring outside the root after the germination of
chlamydospores. Mycelium explores larger soil volume. Fungal growth can be 80-130 times the
length of root. Extra matrical hyphae (EMH) are larger in diameter than inner hyphae. Once the
fungus recognises the plant, appresorium is formed in the host roots and penetration occurs via
the appresorium. EMH ends with resting spores in soil.
Intra radical phase: Events occurring inside the root cortex. After penetrating the cortex, the
fungus may produce intercellular as well as intracellular hyphae in the cortical cells. Forms two
morphological structures namely arbuscules and vesicles inside the cortical cells.
Arbuscules: are the first formed structures after the hyphal entry into the cortical cells.
Arbuscules are the fine dichotomously branched hyphal filaments look like little trees.
Arbuscules start to form approximately 2 days after penetration. They are considered as the
major site of exchange between the fungus and host root. They are short lived (4-13 days) and
degenerate.
Vesicles: Following the formation of arbuscules, some species of fungi also form vesicles in the
roots. Terminal or intercallery hyphal swellings of the hyphae called vesicles. Vesicles contain
lipids and cytoplasm. They act as P storage organ and they ever be present in the root. Size of the
vesicles is about 30-100 µm. In vesicles P can be accumulated as polyphosphates.
EMH, vesicles and Arbuscules play a key role in nutrient transfer particularly in mobilisation of
phosphorus.
Mechanism of action
The beneficial effect on plant growth and yields following inoculation with VAM is attributed to
(i) improved mineral nutrition, especially P (P, Zn, Cu, K, S, NH4)
(ii) Mobilization of nutrients through greater soil exploration.
(iii) Protection of host roots against pathogen infection.
(iv) Improved water relation
(v) Better tolerance to stress like salinity, heavy metal pollution
(vi) Protection against transplantation shock.

Continue d…..

Arbuscules vesicles.
s ta ine d ma s s e s of funga l hypha e ce lls of the orchid a re fille d with coils
(a rrowhe a ds ). of funga l hyphae
Endomycorrhiza Ectomycorrhiza

Reasons for Enhanced P uptake

 Physical exploration of soil.
 Higher affinity towards P
 Lower threshold concentration
 Rhizosphere modification
 Differences in anion and cation absorption due to exudation pattern.
 Siderophore production.
 Selective stimulation of microorganisms in the rhizosphere.
 Increased hyphal area for absorption (EMH).
 Absorb and transport P beyond the depletion zone around the root.
 P absorption by EMH is 1000 times faster than normal hyphae and 3-4 times greater.
Disease resistance
 Resist the parasitic invasion and minimises the loss.
 Mycorrhizal roots harbour more actinomycetes.
  Mycorrhizal roots have elevated levels of phenols, while offers resistance to fungal
hydrolytic enzymes.
 Mycorrhizal infection stimulates biosynthesis of phytoalexins.
Inoculum production
Not cultured on nutrient media. Root based bulk inoculum production technology is used.
Different types of VAM inoculum are produced.
Spore inoculum : Concentrated spore suspension. 100 spores/g inoculum feasible for nursery
application.
Infected root inoculum
Large scale production of infected roots by aeroponic system, contain internal mycelium and
external mycelium. Infected roots may colonise roots after one or 2 days of inoculation.
Soil based inoculum produced using pot culture techniques.
Peat based inoculum (Nutrient Film Technique)
Application Methods (Refer practical manual)
A. Transplanted crops: Inoculum can be applied in nursery
B. Direct sown crops :1. Coating seeds with AM fungal inoculum; 2. Mycorrhizal pellets –
multiseeded pellets. 3. Drilling
Biocontrol agents
Insect control through microbial inoculants
Insect pathogens or entomopathogens include viruses, bacteria, fungi and protozoa. These
pathogens when conserved and augumented, would keep some of the pests under check within
the economic injury levels.
Advantages of using microbial insecticide
 Rapid spread
 Power of search for host
 Persistence, safe
 Aesthetically acceptable control to sub economic levels
 Predictable control
 Virulence
 Easy to produce
 Low cost
 Good storage and
  Easy application are the desirable characteristics of entomopathogens for use as
microbial insecticide.
Different groups of microorganisms showing the potential for IPM programme.
1. Bacterial pathogens
2. Viral pathogens
3. Fungal pathogens
4. Protozoan pathogens
Bacterial pathogens
Bacteria which affect insects are broadly classified as spore formers and non spore formers.
Spore formers include milky disease organisms, Bacillus popilliae, B. lentimorbus, B.
sphaericus, B. thuringiensis. Non–spore formers are Serratia, Pseudomonas, Aerobacter and
Streptococcus. Amongthe genera, Bacillus species are promising organisms for microbial
control.
1. Bacillus thuringiensis
Widely exploited species of Bacillus. It is environmentally safe, active on insects resistant to
chemical insecticide and selective against pest. B. t var. kurstaki was isolated from H. armigera
in 1955. The entomocydal activity of B.t has been attributed mainly to the parasporal protein
crystals, may vary from variety to variety. The crystal proteins are formed during sporulation
stage of the bacterium's life cycle. It is a crystaleceous spore forming organisms. The crystal
proteins are called as endotoxis. There are several varieties of B. t; B. t. var thuringiensis, B.
t .var lentimorpus, B. t .var sotto, and B. t .var canadensis
Toxins of B.t. are of 3 different types
1.  exotoxin : water soluble, heat labile toxin, identified as lecithin
2.  - exotoxin : water soluble, heat stable toxin, highly toxic to several flies. Chemically this is
adenosine nucleotide analog of ATP. Also called as thuringiensis.
Thuringiensis is a specific inhibitor of DNA dependent RNA polymerase and ATP.
3. Delta endotoxin : Most important, called as crystalline toxin.
Symptoms of bacterial infections
1. The body of the insect or larvae becomes soft or dried to a blackscale.
2. Body usually darkened in colour with dark fluids tissues disintegrated with purified
odour.
 3. Body becomes milky white in colour with body fluids white. (eg. Infection in B.
popilliae).
4. Body becomes soft and turns to red colour (Serratia marcesens infection).
5. In Lepidopteran insects, insects may be found hanging by prolegs in plants or twigs.
Formulations
Commercial B.t products Target pest Commercial products
1. B. thuringiensis bv. kurstaki Caterpillars Dipel, Thuricide
2. B. thuringiensis Mosquitoes Acrobe,
bv. israelensis Black flies Vectobac
3. B. thuringiensis Colorado Novodar
bv. tenebrionis Potato beetle Trident
4. B. thuringiensis Wax – moth Florbac
bv. aizawi Diamond back moth
Disadvantages / Limitations
1. Slow in action than chemicals.
2. Method of application and time of application is critical.
3. Environmental stress – Very much influenced by environmental factors like climate,
temperature, pH etc; which decides the efficiency of the pathogen.
4. Geographic strain variation.
A particular strain effective in one place may not be effective in other parts.
5. Host availability
Continuous occurrence of host is must otherwise survival of pathogen is a problem.
6. Presence of antimicrobial or antiviral substances.
Plants have antimicrobial or antiviral substances, which may interfere with microbial
activity
7. Genetic variability
May become ineffective on long run.
8. Problem in mass production
Most of them need live host
Requires technically skilled persons
9. Host specificity
Advantages
1. Host specific :Due to host specificity, they are active against that particular organism,
can be used to control large scale occurrence.
2. Development of resistance is not a problem.
3. Searching capacity is present.
4. Persistent in conducive environment.
5. Non toxic to other organisms.
Fungal pathogens
Entomopathogenic fungi have their ability to decimate insect host populations, particularly
during epizootics. Some fungi have very broad host ranges
eg. Beauveria bassiana
Metarrhizium anisopliae
Verticillium lecanii
Paecilomyces spp.
Metarrhizium : Used in the control of oryctus rhinocerous of coconut. The adult beetle causes
damage to spathe of adult trees and young plants. Multiplied near FYM area. First it was named
as Entomopthora anisopliae and latter renamed as Metarrhizium anisopliae. The other species is
M. flavoviride. M. a was recorded in 200 insect host belonging to different groups orthoptera,
lepidoptera, diptera, hymenoptera, etc.
Commonly called Green muscardine fungus. Mass multiplication is in grubs or in laboratory
media. When young, the colonies 1. mycelium is white in colour and after 4-5 days when conidia
is produced, colour changes to dark green. Toxins are called Destructins A, B, C, D, E and
desmethyl destruxin B. The toxins play a important role in development of symptoms and death
of the organism. LT 50 value for grub is less than adults.
Beauveria : First identified in 1912 more than 10 species are identified. Very few are reported
to be effective in the pest control.B. bassiana is a commercially exploited. Toxins are called
Beauvericins. Toxic to mosquito larvae and also to potato beetle.
Symptoms: Sluggish movement of larvae, loss of appetite, partial paralysis, reduction in
capacity to lay eggs in females, discolouration – are observed initially. Most important stage is
the Sclerotium formation stage.
Microbial control of diseases
Bacterial and fungal agents are widely used for the control of diseases. Important and potential
antagonistic organisms include Pseudomonas and Trichoderma.
Pseudomonas
Bacteria are the most common type soil microorganisms, provide many benefit to plants and
some of them are antagonistic to several pathogenic organisms in the rhizosphere. Among the
potential bacterial antagonist associated with plant roots, the fluorescent Pseudomonas are quite
abundant in the rhizosphere and colonize roots of a wide range of crop plants. Since these
rhizobacteria also promote plant growth, they are called as PGPR. PGPR mediated Induced
Systemic Resistance (IAR) against insects is observed only in few crops. They affect the growth
and development of insects at all stages of growth. They are effective against Helicoverpa, leaf
folder, sheath blight, rice blast and several other fungal diseases.
Method of application: Talc base formulation can be applied through seed, root, foliar and soil.
Mechanism involved in the control
1. Stimulate production of biochemical compounds associated with host defense.
Salicylic acid produced and lipopolysaccharide of P. fluorescens act as local and
systemic signal molecules in inducing resistance in plants.
2. Induce more production of chitinase and peroxidase enzymes, synthesis of phenolics,
lignins, PR proteins.
3. In case of insect control they may affect the feeding behaviour of the larvae resulting in
reduced larval and pupal weight, increased the larval mortality and malformed adult
emergence. Chitinase production is also correlated to the control of insects.
4. The onset of ISR against pest and disease associated with increased accumulation of
defense enzymes viz., PAL, chitinase, PO, PPO, increased amount of phenol and
accumulation of lignin. In addition P. f. promoted the plant growth and attracted the
natural enemies of leaf folder under field conditions.
Trichoderma
It is the widely used biocontrol agent. Among the species of Trichoderma, T. viride, T.
harzianum, T. virens and T. hamatum are used against the management of various diseases of
crop plants.
Advantages as biocontrol agent
 High rhizosphere competence
 High competitive saprophytic ability
 Enhance plant growth
 Induce polysaccharide degrading enzymes
 Ease for mass multiplication
 Broad spectrum of action against various pathogens.
 Excellent and reliable control.
 Environmentally safe.
Factors affecting bioefficacy
Abiotic factors influence the bioefficacy of biocontrol agents in soil. pH, temperature,
moisture, soil type, components of soil atmosphere, inorganic and organic constituents of soil,
quantity of pesticide applied.
Mechanism of disease control
 Competition
 Antibiosis – Trichodermin, dermadin, tricoviridin, sesquiterpene – are the antifungal
metabolites.
 Cellulase production
 Lytic enzymes
Proteases – Proteolytic activity against Sclerotium rolfsii
-1,3 glucanases – degradation of cell wall, results in leakage of protoplasmic contents which
can serve as food material for the antagonist
Chitinases – hydrolysis of chitin.
Indirect mechanism
Induced resistance and plant growth promotion
 LECTURE NO.16 : MASS PRODUCTION AND QUALITY CONTROL OF
BACTERIAL AND FUNGAL BIOINOCULANTS. BIS STANDARDS– METHODS OF
APPLICATION OF BIOINOCULANTS(Refer practical material)

Mass multiplication of carrier based bacterial biofertilizers

Biofertilizers are carrier-based preparations containing efficient strain of nitrogen fixing or
phosphate solubilizing microorganisms. Biofertilizers are formulated usually as carrier based
inoculants. The organic carrier materials are more effective for the preparation of bacterial
inoculants. The solid inoculants carry more number of bacterial cells and support the survival
of cells for longer periods of time.
Mass culturing of microorganisms in fermentor
Although many bacteria can be used beneficially as a biofertilizer the technique of mass
production is standardized for Rhizobium, Azospirillum, Azotobacter and phosphobacteria and
Gluconacetobacter
The media used for mass culturing are as follows:
Rhizobium : Yeast extract mannitol broth.
Azospirillum: Dobereiner's malic acid broth
Azotobacter : Waksmann No.77 broth
Phosphobacteria: Nutrient broth
Gluconacetobacter: LGI broth

Procedure
6. Prepare appropriate broth in 50 ml flasks and inoculate the mother culture in to the flasks.
7. Grow the culture under shaking conditions at 30±2ºC until maximum cell population of
1010 to 1011
cfu/ml is
reached.
8. Under
optimum
conditions this
population
level could be
attained
within 4 to 5
days for
Rhizobium; 5 to
7 days for
| 48
Azospirillum; 2 to 3 days for Phosphobacteria and 6-7 days for Azotobacter &
Gluconacetobacter. The culture obtained in the flask is called starter culture.
9. Use the starter to inoculate the broth in large size flasks of 250 ml, 500 ml, 3 liters and 5
liters and grow until required level of cell count is reached.
10. For large scale production of inoculant, inoculum from starter culture is transferred to
large flasks/seed tank fermentor.

Processing of carrier material

The use of ideal carrier material is necessary in the production of good quality biofertilizer.
Peat soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can be
used as carrier materials. The neutralized peat soil/lignite are found to be better carrier
materials for biofertilizer production.

Schematic representation of mass production of bacterial biofertilizers

Characteristics of an ideal carrier
 Cheaper in cost
 Should be locally available
 High organic matter content
 Should not be toxic
 Water holding capacity of more than 50%
 Easy to process, friability and vulnerability.
 Amenable for mixing
Mixing of broth culture with the carrier and packing
8. Pulverize the carrier material and sieve it through 106 µ sieve. If it is peat / lignite,
determine the pH and neutralize it with CaCO 3. In case of charcoal, neutralization is not
required.
9. Sterilize the carrier in an autoclave and cool it
10. Mix the culture broth with the carrier and adjust to about 40 % moisture content.
11. After mixing allow the impregnated carrier material to undergo curing at 28-30 oC for
further multiplication of bacterial cultures in the carrier.
12. Pack it in polythene bags of medium density (10.089 mm gauge) or low density
(0.038-0.051 mm) and heat seal after expelling the air
13. Store the cultures in cool and dry place at a temperature of 15 oC but not exceeding
30oC
14. The preparation and maintenance of biofertilizers should be followed as per the BIS
(Bureau of Indian standard) specifications mentioned below:
Storage
 The packet should be stored in a cool place away from the heat or direct sunlight.
 The packets may be stored at room temperature or in cold storage conditions in lots
in plastic crates or polythene / gunny bags.
Product specifications
 There should be more than 108 cells / g of inoculant at the time of preparation and10 7
cells/ g on dry weight basis before expiry date.
 It should not have any contaminant at 10-5 dilution
II. Mass production of liquid biofertilizers
 All the bacterial biofertilizers except Rhizobium are produced in liquid formulations also.
Liquid biofertilizers are produced through three step process.
1. Preparation of starter culture and seed culture
 Prepare the starter culture from the mother culture in the respective growth medium as
given for carried based inoculants
2. Mass culturing in fermentor
 Do the mass culturing similar to carried based inoculants in the fermentor.
 Harvest the broth once the population reaches the cell load of 1010 cell per ml broth
3. Preparation of liquid formulation
4. Fill the harvested culture in the sterile plastic container of one liter or 500 ml capacity
5. Add Glycerol @ of one ml per liter broth to arrest the metabolic activities of the cell
so as to avoid bursting of the container under storage
6. Seal the mouth with sterile caps and store under room temperature
Specifications of Biofertilizers
5. Rhizobium
Carrier based*in form of moist /dry powder or
(i) Base
granules,or liquid based

CFU minimum 5x107cell/g of powder, granules

(ii) Viable cell count
or carrier material or1x108 cell/ml of liquid.
(iii) Contamination level No contamination at105dilution
(iv) pH 6.5-7.5
Particles size in case of carrier All material shall pass through 0.15-0.212mm
(v)
based material. IS sieve
Moisture percent by weight,
(vi) 30-40%
maximum in case of carrier based.
Should show effective nodulation on all the
(vii) Efficiency character
species listed on the packet.
*Type o of carrier:The carrier materials such as peat, lignite, peat soil, humus, wood
charcoal or similar material favoring growth of organism.

6. Azotobacter
(i) Base Carrier based* in form of moist/dry powder or
 granules,or liquid based

CFU minimum 5x107cell/g of powder,granules

(ii) Viable cell count
or carrier material or1x108cell/ml ofliquid.
(iii) Contamination level No contaminationat105dilution
(iv) pH 6.5-7.5
Particles size in case of carrier All material shall pass through 0.15-0.212mm IS
(v)
based material. sieve
Moisture percent by weight,
(vi) maximum in case of carrier 30-40%
based.
The strain should be capable of fixing at least 10
(vii) Efficiency character
mg of nitrogen per g of sucrose consumed.
*Type of carrier:-The carrier material such as peat, lignite, peat soil, humus, wood charcoal
or similar material favouring growth of the organism.

7. Azospirillum
(i) Base Carrier based* in form of moist/dry powder
or granules,or liquid based
(ii) Viable cell count CFU minimum 5x107cell/g of powder,

granules or carrier material or1x108cell/ml

of liquid.
(iii) Contamination No contamination at105dilution
level
(iv) pH 6.5-7.5
(v) Particles size in case of All material shall pass through 0.15-0.212
carrier based material. mm IS sieve
(vi) Moisture percent by 30-40%
weight,maximum in case
of carrier based.
(vii) Efficiency character Formation of white pellicle in semisolid N-
free bromo thymol blue media.
*Type of carrier:-The carrier material such as peat, lignite, peat soil, humus, wood
Charcoal or similar material favouring growth of the organism.

8. Phosphate solubilising Bacteria

(i) Base Carrier based*in form of moist / dry powder or granules,or
liquid based
(ii) Viable cell CFU minimum 5x107 cell/g of powder, granules or carrier
count
material or1x108cell/ml of liquid.
(iii) Contamination No contamination at105dilution
level
(iv) pH 6.5-7.5 for moist / dry powder, granulated carrier based and
5.0–7.5for liquid based
(v) Particles size All material shall pass through 0.15-0.212 mm IS sieve
in case of
carrier based
material.
(vi) Moisture 30-40%
percent by
weight,
maximum in
case of
carrier based
material.
(vii) Efficiency The strain should have phosphate solubilizing capacity
character in the range of minimum 30%, when tested
spectrophotometrically. In terms of zone formation,
minimum 5mm solubilization zone in prescribed media
having at least 3mm thickness.
*Types of Carrier:-The carrier material such as peat, lignite, peat soil, humus, wood
Charcoal or similar material favouring growth of the organism.

Quality Assessment Tests for Biofertilizers

S.No. Test Observation Expected result

Rhizobium
1. Streak on to Yeast Colony character Typical colony of Rhizobium
Extract Mannitol should appear on YEMA & it
Agar (YEM A) and should not absorb congo red.
Congo Red Yeast
Extract Mannitol
Agar (CRYEMA)
2. Gram stain Reaction Cells must be Gram negative
3. Streak on Glucose Whether growth Growth should not come
Peptone Agar is +ve or –ve
(GPA)
4. Measure pH of the - 6.0-7.5
carrier
5. Total plate count No. of colonies 108 cells/g carrier
6. Serological study - Agglutinate with the nominated
antiserum
7. Nodulation study - Nodulation +ve
Azotobacter
1. Streak onto Jensen's Colony character A. chroococcum colonies are
N-free medium gummy, raised with or without
striations, viscous and often
sticky. The pigmentation varies
from very light brown to black.
2. Gram stain Reaction Cells must be gram negative
3. Measure pH of the - 6.5 to 7.5
carrier
4. Measure N-fixation - The minimum amount of fixed
 nitrogen should not be less than
10mg/g of sucrose utilized
5. Total plate count - 107/g carrier
Azospirillum
1. Growth in semisolid - 3. Change of the colour of the
medium medium from green to blue
4. Sub surface pellicle formation
2. Growth on solid - 1. Change of the colour of the
medium medium from green to blue
2. Sub-surface pellicle
formation in the semi-solid
broth
Phophobacteria
1. Growth on - 1. Colonies show halo zone
Sperber’sHydroxy around
Apatite medium the medium
2. The strain should have
phosphateSolubilizing capacity
in the range of minimum 30%,
whentested
spectrophotometrically. In
terms of zone formation,
minimum 5mm solubilization
zone in prescribed media
having at least 3mm thickness.
Potassium Releasers
1. Growth on solid - Colonies show halo zone
medium around the medium
Zinc solubilizers
1. Growth on solid - Colonies show halo zone
medium around the medium
AM Fungal cultures
 1. Total viable - Minimum100 /gm of finished
propagules/ gm of product
Product
2. Infectivity potential - 80 infection points in test
roots/gm of Mycorrhizal
inoculum used

II. Mass production of AMF

As AM Fungi are obligate symbionts, their mass multiplication in the form of pure culture is
difficult. However, it can be mass multiplied using a host plant like maize, onion, clover,
cowpea, millets, sorghum, cenchrus grass, etc. Several researches in different parts of the world
resulted in different methods of production of AM fungal inoculum as soil based culture as well
as carrier based inoculum. Root organ culture is being used for the production of soil less
culture.

As a carrier based inoculum, pot culture is widely adopted method for production. The AM
inoculum was prepared by using sterilized soil and wide array of host crops were used as host.
The sterilization process is a cumbersome one the inert materials such as vermiculite, perlite,
montmorillonite clay etc. are utilized for the production.
Materials required
 Carrier material – Soil and sand / vermiculite / perilite / soilrite
 Seed material of host plant
Production of Root Based Inoculum
Mass multiplication of AM cultures involves a two-step process. In the first step starter
culture is prepared and in the second step, it is mass multiplied.

12. Take soil: sand @ 1:1 in a funnel

13. Place around 20-30 spores of selected AM fungal species in the soil and sand mix and
sow maize seeds.
14. Allow the maize plant to grow
15. Uproot the plant at timely intervals and examine for AM fungal infection in roots and
spores in soil.
 16. After 50-60 days of growth, uproot the plant and prepare starter culture by mixing the
carrier material with macerated root pits.
17. Take either soil alone or in combination with carrier materials such as vermiculite /
perilite / soilrite @ 1:10 in mud pots / cement tanks (1m x 1m x 0.3m) and mix
thoroughly.
18. Place the starter (1 kg of AM inoculum) culture around 2.5 cm below the soil and sow
maize seeds.
19. Apply 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench at the
time of sowing seeds. Further apply 10 g of urea twice on 30 and 45 days after sowing
for each trench
20. Apply 1 g of micronutrient mixture when there will be a symptom for deficiency
21. Allow the plant to grow and check for AM fungal infection periodically
22. After 3-4 months, harvest the plant, mix the root system with soil mix and use as
inoculum consists of a mixture of vermiculite, spores, pieces of hyphae and infected
root pieces.
Instead of soil and sand mixture, even vermiculite alone or soilrite + perilite (1:1) mixture
can also be used as a substrate depending up the availability. If an inert material like
vermiculite is used for multiplication, host plants are fertilized in the form of urea,
superphosphate and muriate of potash.

Specification of Biofertilizers

MycorrhizalBiofertilizers
Fine Powder / tablets / granules / root biomass
i. Form/base
mixed with growing substrate
Particle size for carrier 90 % should pass through 250micron IS
ii.
based powder formulations sieve(60BSS)
Moisture content percent
iii. 8 -12
maximum
iv. pH 6.0 to7.5
Total viable propagules / 100 gm of finished product with minimum 60
v.
gram of product spores per gram
vi. Infectivity potential Inoculum potential:1200IP/g
 (determined by MPN method with10 fold
dilution)
Observations
 Take periodical sampling of carrier and liquid biofertilizers under storage and record
the population
 Take periodical sampling of the AM inocula and look for root infection percentage
and colonization.

LECTURE No.17: BIOFUEL PRODUCTION – METHANE, HYDROGEN, ALCOHOL

AND BIODIESEL PRODUCTION

Biofuels include ethanol, biodiesel, hydrogen, butanol, methane etc.,

1. Alcohol production:
Production of ethyl alcohol from sugary material is one of the oldest known microbiological
processes. Alcohol is an important solvent and raw material used in a variety of Chemical
Industries. Although industrial alcohol is produced synthetically from ethylene, production of
alcohol by fermentation of cheap sugary materials such as molasses by yeast is an important
industry. Most fruit juices undergo a natural fermentation caused by wild yeasts that are present
on the fruit. Selected strains of Saccharomyces cerevisiae are employed for production. The
alcohol tolerance and sugar tolerance are important criteria used in the selection of yeast strains.
The raw material generally used is either crude cane molasses or beet molassess which contain
absolute 50 percent fermentable sugar.
The production process involves the dilution of molasses to a suitable sugar concentration (15-
16%), addition of small quantity of nitrogen source (Urea, ammonium sulphate or ammonium
phosphate), adjustment of PH to about 5.0 addition of an activity growing yeast culture. The
fermentation is carried out in big deep tanks of steel or stainless steel and it is allowed to
continued for 24-36 hrs at about 25-30oC. Then the cells are allowed to settle. The fermented
wash is then distilled and passed through rectifying columns to recover ethyl alcohol. CO 2 is
produced in large amount during fermentation. The yield is 50% of the fermentable sugar
concentration. Further purification is done by fractional distillation.
In some distillers, yeast is recovered and used as animal feed. Recently cell recycle technique is
followed. Basically this involves the reuse of cell mass that is produced during the fermentation.
By this 5-10% of substrate used for cell growth has been saved, saving in cost of inoculum,
reduction of fermentation time by 24-36 hrs in batch fermentation.
Biodeisel
Any oil can be converted into biodiesel (fatty acid methyl or ethyl ester) through
transesterification process. Lipids from fungi and algae are used to produce biodiesel. Algae like
Botryococcus,Chlorella, Scendesmus Chlamydomonas etc are exploited for biodiesel production.
Butanol
Butanol is produced using Clostridium acetobutylicum
Hydrogen
Hydrogen producing bacteria and algae are used
Methane
Biogas or methane from any cellulosic waste or biomass