AGM 301 SOIL AND APPLIED MICROBIOLOGY (1+1)

Aim
 To enlighten the students with the knowledge of microbial diversity in soils
 To high lighten the role of soil microorganisms in soil fertility and plant growth
promotion
 To develop experimental skills in soil microbiology which includes isolation of
beneficial microorganisms from soil and plant and their mass production
 To make students gain expertise in practical aspects of production of industrial
products
Theory
Unit I Introduction to Soil Microbiology
Soil Microbiology- definition and scope.Historical developments in soil microbiology.
Diversity of soil microorganisms - culturable (bacteria, actinobacteria, yeasts, moulds and
algae) and unculturable microorganisms - metagenomic approach - factors influencing the
microbial diversity
Unit II Microbial Processes in soil
Organic matter decomposition and humus formation- C:N ratio.. Carbon cycle.Nitrogen
cycle - biological nitrogen fixation (BNF) – nodulation and biochemistry of BNF.Phosphorus
cycle and sulphur cycle. Microbial transformation of potassium, zinc and silica in soil – role
of soil enzymes
Unit III Soil Microorganisms and plants
Rhizosphere, spermosphere, phyllosphere, epiphytic and endophytic microorganisms and
their significance.and Plant growth promoting rhizobacteria. Soil microorganisms and their
interactions – positive and negative interactions
Unit IV Microbial inoculants
Bioinoculants – types of bioinoculants – nitrogen fixers, P, K, Zn and Si solubilizers and
phosphate mobilizers, sulphur oxidizers and PPFM. BGA and Azolla.Mass production and
quality control of bacterial and fungal bioinoculants.Methods of application of bioinoculants.
Unit V Industrial Microbiology
Industrial utilization of microorganisms - Alcohol fermentation – wine and beer.Antibiotics
and vitamin production.Microbes in food industry – single cell protein, baker’s and brewer’s
yeast and dairy products – cheese and yoghurt.Biofuels- ethanol and biodiesel.

Practical
Enumeration of soil microbial population - quantitative and qualitative methods.Organic
matter decomposition. Isolation of symbiotic nitrogen fixing bacteria, free living, associative
and endophytic nitrogen fixing bacteria. Isolation of phosphobacteria and sulfur oxidizing
bacteria. Isolation of zinc and silicate solubilizing and potassium releasing bacteria.Isolation
of plant growth promoting rhizobacteria (Pseudomonas sp) and phyllosphere (PPFM)
microbes. Examination of AM fungal infection in plants and recovery of AM spores from
soil. Isolation of Blue Green algae. Mass production of bacterialbioinoculants, blue green
algae, azolla and AM fungi. Isolation of yeast and Lactobacillus.Industrial products – wine
and sauerkraut fermentation.
Theory schedule
Theory
1. Introduction and historical developments in soil microbiology. Contributions of
Beijerinck, Winogradsky, Fleming and Waksman
2. Diversity of soil microorganisms - culturable and unculturable microbial diversity.
Factors influencing the activities of soil microorganisms
3. Carbon cycle – C:N ratio. Role of soil microorganisms in the decomposition of
organic matter and humus formation
5. Nitrogen cycle – microbiology and biochemistry of mineralization, ammonification,
nitrification and denitrification
6. Biological nitrogen fixation – free living, associative, endophytic and symbiotic
microorganisms
7. Nodulation in Rhizobium- legume and Frankia – actinorhizal symbioses. Biochemistry
of nitrogen fixation
8. Phosphorus cycle and microbial transformation of phosphorus - phosphate solubilizer
and mycorrhizae
9. Mid Semester Examination
10. Sulphur cycle - sulphur oxidizers; microbial transformation of K, Zn and Si. Role of
soil enzymes in nutrient transformation
11. Importance of soil and plant associated microorganisms – rhizosphere, spermosphere
phyllosphere, epiphytic and endophytes
12. Soil microorganisms and their interactions – positive and negative interactions.
Bioinoculants - types - bacterial, fungal (AMF) and algal bionoculants
13. Mass production of bioinoculants
14. Industrial utilization of microorganisms –alcohol fermentation – alcoholic beverages
 15. Antibiotics production (Penicillin and Streptomycin) and Vitamin production
(Vitamin B2 and Vitamin B12).
16. Microbes in food industry – Single Cell Protein, Baker’s and Brewer’s yeast, Dairy
products – cheese and yoghurt
17. Biofuels – alcohol and biodiesel production
Practical schedule
1. Enumeration of soil microorganisms - quantitative Conn’s direct microscopic method
– qualitative buried slide technique
2. Enumeration of rhizosphere microorganisms and determination of R:S ratio
3. Studying organic matter decomposition by measurement of CO2 evolution
4. Antibiosis in soil – Crowded plate technique
5. Isolation of Rhizobium from root nodules
6. Isolation of Azospirilumand Azotobacter
7. Isolation of Gluconoacetobacter from sugarcane
8. Isolation of phosphobacteria and PPFM
9. Isolation of PGPR (Pseudomonassp)
10. Examination of AM infection in roots and recovery of spores from soil
11. Mass production of bacterial bioinoculants and AM fungi
12. Mass multiplication of blue green algae and Azolla
13. Methods of application of different bioinoculants
14. Isolation of yeast and Lactobacillus
15. Wine fermentation
16. Yoghurt and sauerkraut fermentation
17. Practical Examination
Outcome
 Students will be imparted with the knowledge of microorganisms in soil
 The contribution of soil microorganisms in soil fertility and plant growth promotion
will be made clear
 Students will acquire experimental skills in Soil microbiology which includes
isolation of beneficial microorganisms from soil and plant and their mass production
 Students will gain expertise in practical aspects of production of industrial products.

Text Books
 1. Alexander, M. 1977. Soil Microbiology. John Wiley and Sons. New York
2. Waiter.M.J.,N.L.Morgan,J.S.Rocky and G.Higton.1999. Industrial Microbiology – An
Introduction. Blackwell Scientific
3. e book: Paul , E .A. 2007. Soil Microbiology, Ecology and Biochemistry. 3rd Ed.,
Academic Press, USA
4. e book: Waksman, S. A 1952.Soil Microbiology John Wiley & Sons, Inc.

Reference Books
1. Rangaswamy,G.andBagyaraj, D.J. 1992. Agricultural Microbiology, Asia
Publishing House, New Delhi.
2. Subba Rao,N.S.1999. Soil Microorganisms and plant Growth. Oxford and
IBH, New Delhi
3. Osborn, M., Smith, C.J. 2005. Molecular Microbial Ecology. Taylor and
Francis.
Web Pages
fire.biol.wwu.edu/hooper/416_05Ncycle1.ppt
www.fao.org/docrep/009/a0100e/a0100e05.htm
Lec. No. 1.Introduction and Historical Developments in Soil Microbiology. Contributions of
Beijerinck, Winogradsky, Fleming and Waksman
Introduction and Scope
Soil represents a medium or substrate in which numerous microorganisms liveand bring
about a great variety of processes which are responsible for continuation of thecycle of life in
nature. Numerous living forms either spend all or part of their life insoil ranging from sub
microscopic forms to the lower animal forms. With the growingrecognition of the numerous
processes carried out by the microorganisms in the soil theregradually emerged a branch of
microbiology, which came to known as soil microbiology.It is a branch of soil science
concerned with soil inhabiting microorganisms and theirfunctions and activities.Since soil
microbiology concerns with soil microorganisms and their processes, itis closely associated
with soil biochemistry.
Medical bacteriologists were interested in thesoil as a medium for the growth and survival of
disease producing organisms.Agricultural chemists are also interested in the soil processes
that result from theactivities of microorganisms. General bacteriologist, zoologist, botanist
were interested incertain special group of organisms found in soil. Recently, soil
microbiology hasexpanded 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,
productionand destruction of radioactive gases and its transfer. Thus microbial participation
inseveral important processes emphasizes that soil microbiology has become a globalscience.
Soil Microbiology
 Deals with the microscopic organisms of the soil
 Their population and activities
 Role 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
Study of quantitative and qualitative composition of the microscopic and ultramicroscopic
population in soil
2. Experimental or physiological phase
Study of physiology and biochemistry of organisms, their role in the cycle of life in nature
and their utilization for the formation of valuable metabolic products.

3. Agronomical phase
Application of microorganisms to enhance soil fertility and crop productivity.
4. Pedological phase
Importance of microorganisms in soil formation and governingsoil structure.
History and Development of Soil Microbiology
The fertility of soil depends not only on its chemical composition but also on the qualitative
and quantitative nature of microorganisms inhabiting it. Strictly speaking, the development of
microbiology as a branch of science can be dated back to the time of people who ground less
from glass and saw microorganisms, through them. 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). After
Leeuwenhoek’death, years passed while scientists like LazzaroSpallanzani (1729-1799) and
John Needham (1713-1781) debated whether or not microbes were spontaneously generated.
Although this seems like a waste of time, it was a useful exercise in developing what passed
for the scientific method in the 17th and 18th centuries: state hypothesis, test hypothesis,
revise hypothesis, and savage anyone who disagrees with your hypothesis.
Abiogenesis theory strongly supported by the experimental evidences 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 significant developments are being made in Soil Microbiology.
The next great era in soil microbiology research opened in the mid 19 th and early 20th
centuries with the work of soil microbiologists like Sergei Winogradsky Louis Pasteur and
Selman Waksman.
Sergei Winogradsky (1856-1953) was from MotherRussia, but he’s called "TheFather of Soil
Microbiology"because: he was a man, and he discovered lots of interestingthings before
anyone else did. Winogradsky developed theWinogradsky Column while studying the sulfur
cycle. Heinvestigated microbial growth on CO2 and inorganic ions (chemoautotrophy). He
studied nitrification (a microbial process where NH4+ is ultimately converted into NO3-).
Nitrobacterwinogradskii, one of the nitrifying bacteria, is named after Sergei Winogradsky.
Winogradsky investigated chemoautotrophic oxidation of ferrous iron. Winogradsky also
isolated Clostridium, an anaerobic (growing without air), spore-forming, nitrogen-fixing
(converting N2 to NH3) bacilli (rod-shaped bacterium). Winogradsky’s anaerobic, nitrogen-
fixing bacterium wasn’tnamed Clostridium winogradskii, because Winogradsky didmuch of
his work at an institute named for another greatmicrobiologist of this era--Louis Pasteur and
his friendsnamed asClostridium pasteurianum.
While Pasteur fumed in Paris and Koch plated in Berlin, another great school of
microbiology developed in Leeuwenhoek’s old stomping grounds, Delft. It was led by
MartinusBeijerinck (1851-1931). Beijerinck cultured the first isolates of symbiotic (in
association with another organism) and asymbiotic (free-living) nitrogen-fixing bacteria
(Rhizobium and Azotobacter, respectively).
Meanwhile, in England, everyone knew that Alexander Fleming (1881-1955) was a little
different. They just weren’t quite sure why. Then one day in 1928, Fleming announced that a
fungus, Penicilliumnotatum, contaminating an old plate of Staphylococcus in his lab, was
surrounded by a clear zone of dead and lysed cells. He had discovered the first antibiotic,
Penicillin.
Selman A. Waksman(1888-1973) born in Russia but emigratedto USA and ended up working
at Rutgers University. Waksman is often called "The Father of American Soil Microbiology,"
but you rarely hear about his early work on the microbial ecology of compost. You don’t
hearabout it because around 1944, Waksmanand his researchassociate, Rene Dubos, found a
soil actinomycete,Streptomyces, with antibiotic properties like Fleming hadfound with
Penicillium. It was Waksman who actuallycoined the word "antibiotic" and he won a Nobel
Prize in1952 for discovering streptomycin.Waksman’s laboratory ultimately became devoted
tofinding antimicrobial properties in soil microbes.
History and Development of Soil Microbiology
1838 - J.B. Boussingault Legumes can obtain nitrogen from air when grown in soil
which was not heated
1858- Leishman Nodules are formed on the roots of leguminous plants –
1866- Woronin bacteria are responsible
1879-Frank Nodules on the roots of the plants are formed as a result of
inoculation with microorganisms
1885- Hillriegel and Wilfarth Legumes took nitrogen from the air through the agency of
bacteria existing in the nodules of their roots
1888- Beijerinck Isolated the root nodule bacteria in pure culture
1856- Liebig Nitrification – fertilizer
1890 - S. N. Winogradsky
1902- Omeliansky Found out that anaerobic soil bacteria degrade cellulose

1903- Lipman and Brown Studied ammonification of organic nitrogenous substances by

soil microorganisms and developed the tumbler / beaker
method for studying different types of transformations in soil
 1904- Hiltner
1929- Starkey
1940- Lochhead Rhizosphere Concept
1946- Katznelson
1956- Rovira
1961- Matcura& associates
1909 - Russel and Studied the importance of Protozoa in controlling bacterial
Hutchinson population and activity in soil
1918 - Conn Direct soil examination
1927 – Rossi Contact slide technique
1930 - Cholodny
1921-27- Rayner&Melin Studied about Mycorrhizal Fungi
- Harley
- Gerdemann
- Marx, Trappe
- Hacskaylo
- Browen
1929 - Alexander Fleming Discovered Penicillin
1931- Van Niel Studies on soil bacteria and bacterial photosynthesis
1932- Fred & Associates Worked on nodule bacteria and found cross inoculation
groupings
1936- Garret Ecological classification of soil fungi
1940- Allen and Allen Soil bacteria in general and root nodule bacteria
1944 - Selman A. Waksman Discovered Streptomycin
1945 - Starkey Studies on Iron bacteria
1947- Umbreit Problems of Autotrophy
1956- Ruinen Phyllosphere Concept

Contributions of some important scientists:

1. S.N. Winogradsky (1856-1953)
 Demonstrated the role of bacteria in nitrification process in 1890. Isolated two
groupsof nitrifying bacteria
 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 and Beijerinck developed the technique of enrichment culture
makinguse of the principle of natural selection. It is a technique in which
environmental(including nutritional) conditions are controlled to favour the
development of aspecific organisms or group of organisms.
It involves the successive transfer of microorganisms in desired substrate for theisolation of
sparsely occurring unusual types of microorganism.
2. Beijerinck

 Isolated Azospirillumin 1822

 1858- He was the first to isolate and describe Azotobacterand obtained the pureculture
of Azotobacterchroococcumand A.agilis.
 1888- obtained the pure culture of root nodule bacterium Rhizobium and developed
thetechnique of enrichment culture.
 Obtained the pure culture of Thiobacillusthioparus, T.denitrificansandsulphur
oxidizing bacteria
 1893 - He established the transformation of Nitrogen.

4. Alexander Fleming (England)

 1929 - He discovered the Antibiotic Penicillin which is the important milestone in
 medical microbiology
 He found that natural substances / natural products are having antimicrobial activity
 He reported that Nasal mucous, saliva are having antimicrobial property due to
theaction of lysozyme.
 He worked with Straphylococcusaureusand observed the inhibition of growth of S.
aureusin the plate due to the growth of Penicillin.
 Florey and chain latter isolated penicillin in pure culture.
5. Selman A. Waksman
 1922-Isolated Thiobacillusthioxidans
 1927- Publishedbook on "Principles of Soil Microbiology".
 1939- Identified the soil organismsproducing antibiotics
 1942 - Showed the importance of soil as the source of antagonistic organisms.
 Discovered the antibiotic streptomycin in 1944 for tuberculosis - Mycobacterium
tuberculosis.
 He discovered Streptomycin, Neomycin, Actinomycin antibiotics.
 Studied variety of biochemical reactions carried out by soil microorganisms while
decomposing organic matter.

Lec.No. 2. Diversity of soil microorganisms - culturable and unculturable microbial

diversity. Factors influencing the activities of soil microorganisms
Soil contain five major groups of microorganisms. Bacteria, Actinomycetes,fungi, algae and
protozoa. The soil ecosystem includes these microbial groups as well asthe inorganic and
organic constituents of a given site. The collections of cells representedin the community are
considered as distinct populations.All the inhabitants of the particular locality make up the
community.
I.Bacteriaare the most dominant group of M.O in soil and more numerous thanthe other four
combined. They present in all types of soil but their population decreasesas the depth of soil
increases (Horizon A > B > C). The number of cells of bacteria in thesoil is always great, but
the individuals are small, (μm in length). Because of the minutesize of the bacteria and large
cells or filaments of the other 4 groups, the bacteriaprobably account for appreciably less than
half of the total microbial cell mass. In aeratedsoil, B, will be dominating, alone are
responsible for all the activities in environmentlacking O2 or little O2. In transformation
process Bacteria stand first, due to their rapidgrowth and capacity of vigorous decomposition
of variety of substrates.
Soil microbiological population has been divided into two broad groups. 1. a.autohcthonous
and b.zymogenous.
a. Autochthonous or native microbes; Indigenous, which are characteristic of theparticular
soil and which many be expected always to be found there.eg.Arthrobacter
b. Zymogenous, or fermentative organisms require an external source of and theirnormal
population in soil is low (Pseudomonas, Bacillus). When specific substratesare added to soil,
the population increases. Then gradually declines when theadded substrate is exhausted
eg.cellulose decomposers. N utilizing bacteria,nitrifiersTransient microbes - comprising
organisms that are introduced into the soil bylegume inoculation unintentially as in the case
of agents producing animal and plantdiseases.
Bergey's marginal of determinative bacteriology is universally used for theclassification of
Bacteria.
Ecological classification of soil microorganisms
1. Autochthonous/ native microbes -
2. Allochthonous/ introduced/ fermentative groups -
Nutritional classification
Based on carbon source
1. Autotrophs – Utilize inorganic carbon as a source of carbon
2. Heterotrophs –Derive carbon from organic compounds
Based on source of reducing power
1. Lithotrophs – Inorganic compounds as a source of reducing power
2. Organotrophs - Organic compounds as a source of reducing power

Based on source of energy

1. Chemotrophs- Obtain energy by oxidizing inorganic chemicals
2. Lithotrophs – Obtain energy from radiation/ light
Photoautotrophs - energy from slight, C from CO2
Chemoautotrophs - energy from oxidation of inorganic chemicals, C from CO2.
Several reactions in N transformation of soil depend on the chemoautotrophicorganismsviz.,
Nitrobacterand Nitrosomonasand hence Chemoautotrphy of bacteria insoil is intimately
related to crop production.
b) Based on the O2 requirement
 Aerobic - need O2 for growth(20%) eg. Azotobacter
 Anaerobic - not require O2 for growtheg. Clostridium
 Facultative anaerobic- live in the presence or absence of O2eg. Klepsiella, Bacillus sp
 Microaerophillic-2% O2 requiring microbes eg. Azospirillum
c) Based on structure
 Bacilli - Rod shapedeg. Bacillus, Rhizobium, Azotobacter etc.,
 Cocci - Spherical shapedeg. Streptococcus
 Spirillum - Spiral shapedeg.Azospirillum
Abundance of Bacteria
In Arctic regions -frozen 9-10 m / year bacterial counts of 106 / g areobserved even when
temperature remains below freezing point. Such organism is in astate of dormancy awaiting
the spring to recover. Desert soil is another extreme. Bacteria' exist even in oven dry state.
Sporeforming bacilli are often predominant.
Importance
In transformation process bacteriastands first, due to their rapid growth and capacity
ofvigorous decomposition of variety of substrates - involved in N 2 fixation, P
soluilization,CO2 fixation, S,Fe transformation, Sisolubilizationetc.,

II.Actinomycetes
A transitional group between the simple Bacteria and Fungi are theactinomycetes. These are
microorganisms producing slender, branded filaments thatdevelop into a mycelium in all soil.
They are classified under bacteria, but resemblesfungi in some aspects.
 produces mycelium with extensive branching
 Like Fungi many actinomycetes form aerial mycelium and conidia
 Growth in liquid medium do not resemble (which bacteria is turbid), but form
clumpsand pellets like Fungi.
Certain actinomycetes resemble mycobacterium and Corynebacterium in allrespects both
morphologically and physiologically including susceptibility to virus attack.
Actinomycetes differ from fungi that they do not have chitin and cellulose whichare
commonly found in the cell walls of fungi.
1. The colony characters are not similar to bacteria.
2. Some species have flagella that resemble those of true Bacteria.
3. Similarities in cell wall composition and sensitivity to antibacterial compounds.
Common genera in soils
Streptomyces (70%) ,Nocardia, Micromonaspora, Frankia.
Actinomycetes are sensitive to antibacterial compounds and not antifungalcompounds
Streptomycetes
Musty odour they elaborate; an odour reminiscent of freshly turned soil, themetabolite of
Streptomyces formed geosmin and other volatile products elaborated byStreptomyces are
responsible for the characteristic earth odor/ smell.
Distribution
Actinomycetes are numerous and widely distributed not only in soil but in avariety of other
habitats including composts, river muds and lake bottoms. Present insurface soil and also in
lower horizons to considerable depth. In abundance they aresecond only to the Bacteria and
the viable counts almost equal to both. Saprophyticexistence, but a few species can cause
diseases of plants, domestic animals and evenhumans.
Isolation
Population is around 105to 108/g in temperate zone, but lower counts in waterloggedsoils,
acid peat, arctic, Tundra regions. In alkaline areas, especially when dry, the
relativeabundance is high.
Importance
1. Decomposition of resistant components of plant and animal tissue.
2. Transformations at high temperature particularly in the manure and compost pits
 thermophiles dominant
3. Cause certain soil borne diseases of plants , potato scab of apple, sweet potatopox.
4. cause infection to humans, animals.
5. Importance in microbial antagonism by production of antibiotics and production
of enzymes.
6. Antibiotic Industry
III. Fungi
As the important constituent group of the soil population, they are widelydistributed in most
well – cultivated soils. Fungi account for a large part of the totalmicrobial population.
Though they are not the major inhabitants, they do infact makeupthe significant part of he
biomass because of the large diameter and extensive network ofthe filaments. Fungi exist in
soil in the form of vegetative mycelium and spores.Characteristically the fungi possess a
filamentous mycelium network of individual hyphalstands. The hypha itself is rather
broadand has a diameter appreciably greater than that foundin the common actinomycetes. In
nature consider or asexual spores are abundantand widespread, the sexual spores relatively
uncommon. In contrast with bacteria thehigh can be effectively differentiated into genera and
species on the basis of morphology.
Distribution and Abundance
Several techniques have been developed for the study of the fungal flora. Plate count is burial
slide techniques are used for estimation.Slight audification of pH and addition of
exhibition-.Novobiocin and streptomycininhibit bacteria and actinomycets.Estimates of
microbial density reveal the presence in soil is ranging a few as20,000 to as many as
1,000,000 fungal propagules per gram, the propagule beingconsidered as any spare, or hyphal
filament that is capable at giving rise to a colony. Thelength of the fungal mycelium has been
reported to range from 10 to 100m per g surfacesoil, 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 andtaking the range of 10 to 100 m per gram. It would
appear that the weight of fungi rangesfrom 500 to 5000 kg per ha of surface soil. Thus the
filaments make up a significantpart of the soil mass.Individual species and genera have been
recorded in diverse and highly dissimilarhabits. They are the inhabitants of peats, flooded
soils planted to rice, regions with lowand extremely high salt contents, locations in many
deserts, sites in Antartica, as well as inthe tundra.

Importance
1. Degrade complex molecules-cellulose,hemicellulose,pectin,starch,lignin
2. Improve soil structure-aggregate formation
3. Pathogenecity in plants,animals and humans
4. Predator against soil protozoa-hyphal penetration reduces mobility,total cessationof
movement
5. Trapping nematodes-Arthrobotrys,Dacylaria,Dactylella,Harposporium
6. Beneficial association with plants-mycorrhizal fungi
IV. Yeast
1. Non filamentous fungi, their presence maybe demonstrated in most soils
2. Soilyeasts are – Candida, Debaryomyces, Rhodotorula, Torulopsis
3. Yeasts have been found incomparable numbers in soils of Antarctica, grasslands,
cultivated fields, and forests.
IV. Algae
They occur in small numbers in soil. They are photosynthetic organisation.Abundant in
habitats in which moisture is adequate and light is accessible. Isolation can bedone in liquid
media and by plating in agar media. Because of their similarities tobacteria, the BGA are
sometimes classified together with bacteria.Photoautotrophic nutrition, in the absence of light
heterotrophy occurs in somespecies of chlorophyta, cyanophyta and diatoms. Algae are
moderately adaptable to environmental changes; persist in unfavourablecircumstances such
as in alkaline and desert soils.Some species colonise the zone under the surface crusts of
limestone and rocks in the desert, liking at sites where the humidity is retained and where
sufficientlight is available.
- Enumeration by MPN 100 to 50,000 / g soil
- biomass – 7 to 300 kg / ha (Chlorophyll ).
- flooded paddy field is the environment algae could have a great agronomic significance.
The microbial action may be associated with the utilization of atmospheric nitrogen, the
release ofO2 or the excretion of products stimulating plant developments. Under water logged
soils, an algal film forms at the liquid surface, make up an appreciable mass.
Common inhabitants :Anabaena, Calothrix, Nostoc, Oscillatoria etc.,
 Involved in CO2 fixation
 Colonizes the barren surface and corrode and weather rocks-Contribute to
soilformation
 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 O2 , provides O2 to the roots and other microorganisms
 N2 fixation and ammonia excretion
 N 2 is released after decomposing of algal biomass
 Production of metabolites
 SCP production
 Β carotene production
V. Protozoa
The subterranean soilcontains protozoa, earthworms, nematodes, and insects. Mostabundant
one is protozoa, the simple from of animal life. Primitive, unicellular organisms; majority of
themare dependent on preformed organic matter - obtaintheir nutritionfrom soluble organic
and inorganic substances, or by a phagotrophicnutrition (directly feedingupon microbial
cells).
 Regulate the bacterial community
 Maintain the biological equilibrium in soil
 Participate in the decomposition of plant remain
Factors affecting microbial activities in soil
Several environmental conditions affect the density and composition of themicroflora and
frequently alter their activities in soil. Primary factors include moisture,aeration, temperature,
pH, organic matter, inorganic fertilizers. Lesser variables are thesecondary 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 forvegetative
development. But, when it becomes excessive, proliferation is suppressed dueto limitation in
gaseous exchange and lowers the availability of O2 supply creatinganaerobic environment.
Most of the organisms prefer a moisture percentage between 20and 6 per cent. Many bacteria
and fungi are able to adjust themselves to differentmoisture conditions. Under dry conditions,
the bacteria may form spores which can resistthe drought conditions. The fungi may sporulate
or form chlamydospores to tide overadverse conditions. The protozoans also may form cysts
and can survive under dryconditions. Actinomycetes are the chief group of organisms that
prefer dry conditions.At moisture, it is believed that the concentration of nutrients is diluted
and alsothe aeration is very much limited and hence only the anaerobic and
microaerophilicorganisms can develop better.
Soil air :It is directly linked up the moisture level of the soil. Most of the formsare active in
aerated soil and under waterlogged soils anaerobic andmicroaerophilic 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 reachsuch extremes. Microbial
population varies both quantitatively and qualitativelyunder extreme conditions. In tropical
and subtropical regions, temperatures varywidely in summer and winter and the population
may also be varied. Intemperate regions, there is no much variation in temperature during
summer and winter; hence soil population will not varymuch in these soils. Soil temperature
influences thetemperature of air, water and solid phase of the soil. Soil water and
temperatureexert a combined influence on the microbial population.
Soil Organic Matter:Community size is related to the organic matter content, sothat humus
rich localities have the largest biological numbers. Organic mattercontent varied with soil
types from less than 0.5 per cent in desert soils to 40 percent in peaty soils. 0. M being the
chief source of energy and food for most soilorganisms, it has great influence on the
population. Nature of 0.M is responsiblefor the differential stimulation of the population.
There are several indirect effectsof the organic matter on soil microflora. It influences the
structure and texture ofsoil, besides enriching with nutrients for plants and microorganisms.
Suchinfluences 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 enzymesystem and
thus plays a important role in microbial activity. In general fungithrice better then bacteria
and actinomycetes in acid soils. Bacteria flourish wellin neutral and alkaline soils. The saline
and alkaline soils have differentmicroflora. Salinity is due to excess salts, alkalinity is due to
high H+ionconcentration. Several direct and indirect effects of H+ion and salt concentrationin
soil are exerted on microbial population.
Fertilizer application :Application of fertilizers, to the soil improves themicrobial 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 tothe formation of nitric acid and which inhibits the
growth of bacteria andactinomycetes. Addition of nitrate inhibits the activity of free living N2
fixingbacteria like Azotobacter. Some of the autotrophs are encouraged by the additionof
fertilizers.
Cropping and vegetation:Two kinds of effects are exhibited by the crop plants.One is
through root exudates, which may have different compounds withreference to the crops
grown vegetation have selective stimulation overpopulation. Continuous cultivation leads to
more microbial activity than theuncultivated land.
II. Secondary factors
Crop rotation :Crop rotation with different species like legumes, graminaceousplants, etc.
brings about different stimulatory effect on the microflora. Some cropshave deeper roots than
others and some are more fibrous. Such variations bringabout physical changes in soil which
in turn may have direct and indirectstimulatory effects on the soil microflora.
Cultural practices :Various cultural practices, such as tillage operations andirrigation have
several physical and chemical changes in soil which are reflectedon the soil microflora.
Through subsoil ploughing deeper layers may get betteraeration 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 fewcentimeters
and decline with greater depth, more active at few cm down and lessactive at deeper layers.
Low O2 and less sunlight in deeper layers reducepopulation.
Season : Cell number are greatest during the spring and autumn and a declineoccur during
hot, dry and winter, the cells remain in a state of dormancy forbiochemical inactivity. The
influence is due to mainly the alterations intemperature, 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 killprotozoan
population, which may lead to the increase in bacterial population. Thiscondition may affect
biological equilibrium in soil.
Application of nematicide, fungicide and bactericide may exhibit partialsterilization.
Thus every change in crop production direct or indirect, alters thesoil microflora, still many
more are not clearly understood.
3.Carbon cycle – C:N ratio. Role of soil microorganisms in the decomposition of organic
matter and humus formation
Carbon cycle
The term soil generally refers to the loose material of the earth surface and is theregion that
supports the plant life. It consists of five major components such as mineralmatter, water, air,
organic matter and living organisms. The proportion of thesecomponents varies with soil type
and other soil conditions. To maintain the level of thesecomponents it is essential that they
undergo a regular process of recycling. This processof recycling through various
transformation is brought about by different microorganism.The most important element in
the biological realm and substance that serve as thecornerstone of the cell structure is carbon.
It constituents about 40-50% of all livingorganisms, yet the ultimate source is the CO2 that
exist in a perennially short supply, only0.03% of the earth’s atmosphere, which undergo a
cyclic change from an oxidized toreduced state.
Aerobic condition Carbon Cycle

Soil organic matter

Photosynthetic organisms Chemoautotrophic bacteria Aerobic respiration

Carbon Dioxide

AnaerobicconditionMethanotrophs
Methane

Photosynthetic bacteria Anaerobic respiration

Acetic acid

Organic acids

Soil organic matter

Carbon (CO2) is constantly (reduced into organic carbon compounds) being fixedinto organic
form by photosynthetic organisms (photosynthesis). Once bound, the carbonbecomes
unavailable for use in generation of new plant life. It is thus essential for thecarbonaceous
materials to be decomposed and returned to the atmosphere. It is estimatedthat 1.3x1014 kg
CO2 is fixed annually in the biosphere. To the lesser extent CO 2 is alsofixed through the
agency of photosynthetic bacteria and other chemolithotrophs with theconversion of so much
of the plant available carbon to organic form each year and thelimited supply in the air, it is
apparent that the major plant nutrient element wouldbecome exhausted in the absence of
microbial transformation.The carbon cycle revolves about CO2 and its fixation and
regeneration. The greenplants utilize CO2 as their sole carbon source, and the carbonaceous
matter synthesizedserves to supply carbon to other heterotrophic organisms and animals.
Upon the death ofplants and animals, microbes assume a dominant role in carbon cycle. The
dead tissuesare degraded and transformed into microbial cells and humus or soil organic
fraction.Further decomposition of these materials leads to the production of CO 2 and once
again itis recycled.
Organic matter decomposition (Aerobic decay)
The organic matter subjected to microbial decay in soil comes from severalsources viz., plant
remains, animal tissues andexcretory products. The chemistry of organic matter is clearly
very complex, andinvestigations of the transformations and the responsible organisms have
therefore beenextremely interesting. The organic constituents of the plants are commonly
divided intosix categories.

 Cellulose - Most abundant 15-60% of the dry weight

 Hemicellulose - 10-30% of the plant dry weight
 Lignin – 5- 30 % of the plant dry weight
 Water soluble fraction - 5-30%, included simple sugar, a. acids,
 Ether and alcohol soluble constituents - a fraction containing fat, oils, waxes, resins
anda number of pigments
 Proteins
As the plant ages, the content of water soluble constituents, proteins andminerals decreases
and the per-cent abundance of cellulose, hemicellulose and lignin rises. Soil organic matter
comprises residues of plant and animals and these compoundsoccur in soil in close
combination with inorganic substances. Animals and plant residuesare 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
organicmatter.
 Bacteria are the dominant group – mostly heterotrophic organisms (use energyfrom
organic sources such as sugars, starch, cellulose and protein) – are involved
 Autotrophic organism which occupy a small portion of the biomass in soil (anduse
inorganic sources such as Fe and S) are not directly involved in organic
matterdecomposition.
 Actinomycetes grow on complex substances such as keratin, chitin and othercomplex
polysaccharides and play active role in humus formation.
 Soil fungi are mostly heterotrophs and use organic residues easily
 Soil algae contribute a small amount of organic matter through their biomass, butthey
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 microorganism and providesnutrition to plants
indirectly through the activity of soil microorganisms. It is essential for the formation of soil
aggregates and hence soil structure whichultimately determines the soil aeration and rooting
habit of plants. Organic matter helps in the conservation of soil nutrients by preventing
erosionand surface run off of nutrients.
Carbon assimilation
The process of converting substrate to protoplasmic carbon is known asassimilation. Under
aerobic conditions 20-40% of the substrate carbon is assimilated, theremainder is released as
CO2. Fungi are more efficient, in their metabolism, since theyconvert carbon into cell carbon
as filaments and release less of CO2. Of which, 30-40% isused to form new mycelium during
the decomposition. Compared to fungi, bacteria areless efficient. Aerobic bacteria are less
efficient than anaerobic bacteria.
Mineralization
It is process of conversion of organic substancesin to inorganic form of carbon.
Immobilization
Assimilation of nutrients and is the mechanism by which microorganisms reducethe quantity
of plant available nutrient in soil. Mineralization is considered better
thanimmobilization.During the decomposition of organic matter, three separate simultaneous
processescan be distinguished. The important changes during decomposition are:
 Plant and animal tissues constituents disappear under the influence of enzymes
 Synthesis of new microbial cells so that proteins, polysaccharides and nucleicacids
typical of bacteria and fungi appear
 Third, certain end products of the breakdown are excreted into surroundings to
accumulate or to be further metabolized.
Importance of organic matter decomposition
 Important function is the breakdown of organic matter by which CO 2 available
forphotosynthesis is replenished
 Any compound that is synthesized biologically is subject to destruction by
soilinhabitants, otherwise the compounds would have accumulated in vast amountson
the earth’s surface
 Since, organic matter degradation is a property of all heterotrophs; it is
commonlyused to indicate the level of microbial activity.
Methods to evaluate the decomposition rate
 Measurement of CO2 evolution or O2 uptake
 Determination of decrease in organic matter either chemically or by weight loss
 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 naturalproducts, some
components quickly disappear while others are less susceptible tomicrobial enzymes and
persist. The water soluble fraction contains the least resistantplant components and is thus the
first to be metabolized. Cellulose and hemicellulose onthe other hand disappear not as
quickly as water soluble substances, but their persistenceusually is not too great. The lignins
are highly resistant and consequently becomerelatively more abundant in the residual,
decaying organic matter. At aerobic conditions when carbonaceous substrates are
incorporated into soil,immediate drop in O2 and an increase in CO2 content of soil air
occur.Change in oxidation - reduction potential (Eh) – it is shifted to a morereduced
condition (fall in oxidation reduction potential).The end products of decomposition areCO2,
H2O, NO3-, SO4-, H2PO4-/ CH4, NH4+ and H2S 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 isfaster
 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 likelyexceeds
immobilization
 Wider the ratio, lesser microbial cells, slower the immobilization andmineralization
increases gradually, resulting in accumulation of Ammoniaand Nitrates
 Microbes scavenge the soil solution to obtain enough N
 At optimum level, there must be an equilibrium between Mineralization
andImmobilization
 Soil N level constrains the maintenance of C:N (organic carbon /soil o.m)
 To make sound soil management
 Arable surface -10:1
 Sub soil -lower
Anaerobic decay / decomposition
The main products of aerobic carbon mineralization are CO 2, water, cells andhumus
components. In the absence of O2 organic carbon is incompletely metabolized,intermediary
substances accumulate, and abundant quantities of CH 4 and smalleramounts of H2 are
evolved. Energy yield during anaerobic fermentation is low, resultingin fewer microbial cells
per unit of organic carbon degraded. Consequently, organicmatter breakdown is consistently
slower under anaerobic condition than in environmentscontaining adequate O2. The rate in
water logged soils is intermediate between the twoextremes.
When a soil is water logged or flooded there is a shift from aerobic to
anaerobictransformation. 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 wetsoils. The anaerobic carbon transformation is thus characterized by the
formation oforganic acids, alcohols, CH4 and CO2 as major end products.
Under anaerobic conditions, decomposition of organic residues takes place by theactivity of
mesophilic and thermophilic microorganisms resulting in the production ofCO 2, H2, ethyl
alcohol and organic acids. Among mesophilis, bacteria are more activethan fungi or
actinomycetes in cellulolytic activity. They belong to genus Clostridiumwhich are numerous
in manure pits but rarely encountered in cultivated arable soils. Incompost pits both
mesophilic and thermopholic (bacterial and actinomycetes) are important inthe breakdown of
cellulose substances.The primary microbial colonizers breakdown initially the complex
(CH2O)n andproteins into organic acids and alcohols. At a later stage, the methane bacteria
which arestrict anaerobes begin to act upon the secondary substrate chiefly lactic and butyric
acidsand ferment them into CH4 and CO2.
Humus
It is a dark coloured and fairly stable soil organic matter with known and unknownphysical
and chemical properties. It is an integral part of the organic matter complex in soil. Humus
can be defined as lingo protein complex containing approximately45 % lignin compounds,
35% amino acids, 11% carbohydrates, 4% cellulose, 7% Hemicellulose, 3% fats, wax, resins,
6% other miscellaneous substances, including plant growth substances andinhibitors.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 ofhumus. Soil microorganisms take
part in humus formation. Some fungi such asPenicillium, Aspergillusand actinomycetes
produce dark humus like substanceswhich 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
otherphysiological effects on plant growth
  Increases the enzyme activities involved in plant metabolism. Since humic acidserves
as hydrogen acceptors.
 Increases the cytochrome oxidase activity in root systems results in growthstimulatory
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
 Aspergillusniger, Penicillium, Bacillus sp., Azotobacterare enhanced
The organic fraction of soil, often termed humus. It is a product of synthetic anddecomposing
activities of the microflora. Since it contains the organic C and N neededfor microbial
development, it is the dominant food reservoir. Because humus is both aproduct of microbial
metabolism and an important food source, the organic fraction is ofspecial interest.
Humus formation
Once the plant or animal remains fall on or are incorporated into the soil, they aresubjected 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 microorganism with in the soil body. The microorganism in soil body works
upon the former and synthesize microbial protoplasmand new compounds that become part of
the organic fraction.Humus exists in a dynamic state. Chemistry of humus is complex.It has
been pointed out that the organic fraction is derived froma) Plant constituents that are
modified by the microflora.b) Constituents of microbial cells and products of microbial
metabolism those are relatively resistant to decay and therefore persist for some time
afterdeath of organism.
In terms of specific elements
The organic fraction contains compounds of C, H, O, N, P, S and small amountsof other
elements. Only a small portion of the total is soluble in water, but much can bebrought 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 ofthe soil that is composed
of a heterogenous group of substances, most having anunknown parentage and an unknown
chemical structure.Lignin and lignin derived molecules have long been considered to be
ofsignificance in the formation of humus.It is possible either that simple aromatics released in
the microbial attack on ligninpolymerize to yield constituents of the soil organic fraction or
that partially altered ligninitself gives rise to humus constituents. The monomeric portions of
humus are similar tothe constituents of lignin.
Degradation processes
Cellulose is a (CH2O)n polysaccharide composed of glucose units bound by β-linkage at
carbon 1 and 4of the sugar molecule. The cellulose concentration of higher plants is never
fixed and theconcentration. It is a polymer of glucose and is might abundant organic material
in naturechanges with age and type of plants. Woody materials have more cellulose
andsucculent tissues had poor, but the concentration increases as the plant matures.Cellulose
breakdown in soil is influenced by several environmental factors.Aerobic organism convert
cellulose to 2 major products : CO 2 + cell substance,but certain group releases small amount
of organic acids. It is however resistant tomicrobial decomposition. When cellulose is
associated with pentosans (xylan, mannans)it undergoes rapid decomposition. When
associated with lignin, the decomposition rate isvery low. Degradation is by the enzymes that
converts celluloseinto glucose(Exoenzymes). They include 1. Exoglucanase,
2.Endoglucanase and 3.Glucosidase (cellulase complex)

Exoglucanase
Native Cellulose Amorphous Cellulose + Cellobiose
Endoglucanase
Endogluconase

Glucosidases
CellobioseD- Glucose
Cellobiase
Most cellulolytic bacteria do not excrete significant amounts of cellulase but fungiare found
to excrete these enzymes. The soluble sugars released by enzymatic hydrolysisare later
utilized by the same or other microorganism for biosynthetic purpose.
Hemicellulose: It is a branched polymer, made up of simple sugars such as either pentoses or
hexosesand their uronic acids. They may beeither homo or hetero polymers. When they are
added to soil, degradation takes place atfaster rate in initial stages. The hemicelluloses such
as mannans are decomposed rapidlywhile galactons (polymer of galactose) are decomposed
slowly. Many soil microorganismsutilize hemicellulose in both aerobic as well as anaerobic
conditions. Themicrobial degradation occurs through the agency of extracellular enzymes
calledhemicellulases.
Lignin :It is the most abundant constituent of plants. It consists of heterocyclic
aromaticorganic molecules containing C, H and O. The degradation is very slow and rate
ofdecomposition depends on the presence of other compounds such as cellulose
andhemicellulose acid. Lignin is highly resistant to microbial degradation. Degradation is
acomplex process.
Lignin coniferyl ether coniferyl alcohol coniferyl aldehyde vanillin
vannillic acid protocatechuic acidring cleavage
Genera of microorganisms capable of utilizing different components oforganic matter
 A- Actinomycetes; B- Bacteria; F- Fungi

Cellulose is a linear homopolymer of d-glucose units linked by 1,4-β-glucosidic bonds. The

length of the chain usually ranges between 4000 and 8000 monomers.

Efficient cellulose hydrolysis to glucose requires the concerted action of endo1,4-β-

gluconase, exo-1,4-β-gluconase, and β-galactosidase. The first enzyme randomly attacks
internal β-glucosidic bonds within the chain. The second enzyme removes cellobiose units
from the non-reducing ends of the chain, and the third enzyme converts cellobiose to glucose.

The presence of active cellulase systems (of all three enzymes) is widespread within the
fungi, aerobic, and anaerobic bacteria. Cellulase enzymes are either produced extracellularly,
mainly in aerobic fungi, or produced as a complex structure called the cellulosome that is
bound to the cell membrane in anaerobic bacteria (e.g. Clostridia), as well as members of the
Neocallimastigales, anaerobic fungi present in the gut of rumens and other herbivores.
Currently, enzymes derived from the aerobic fungal genera Trichoderma and Aspergillus are
most widely used in industrial settings.

Hemicellulose is a heteropolymer of pentoses, hexoses, and sugar acids. Xylans are the most
common form of hemicellulose and are heteropolysaccharide with a backbone consisting of a
relatively short chain (around 200 units) of 1,4-linked β-d-xylopyranose units. In addition,
minor quantities of arabinose, glucuronic acid, and acetic, ferulic, and p-coumaric acids
might be present in xylan. The exact composition of hemicellulose depends on its source.
Enzymes required for the depolymerization of hemicellulose are collectively known as
hemicellulases. The total degradation of xylan requires endo-β-1,4-xylanase, which attacks
the main chain of xylans. Subsequently, β-xylodase degrades the produced
xylooligosaccaharides produced to xylose. In addition, various accessory enzymes are
required for the degradation of various additional components and substitutions within the
xylan polymer.
The presence of the entire suite of enzymes capable of hemicellulose degradation within a
single microorganism is less common than the presence of complete cellulase machinery.
Nevertheless, several microorganisms are known to completely depolymerize hemicellulases
(mainly xylans) to xylose. These include the fungi Penicillumcapsulatum and
Talaromycesemersonii, thethermophilicactinomyceteThermomonosporfusca and the
hyperthermophileCaldicellulosiruptorsaccharolyticus

Lec. No.5 Nitrogen cycle – microbiology and biochemistry of mineralization,

ammonification, nitrification and denitrification
The Nitrogen Cycle
Molecular nitrogen constitutes about 78 per cent of the earth’s atmosphere, but it is
chemically inert and cannot be utilized by most living organisms. Plants, animals and most
organisms, therefore, depend on a source of combined such as ammonia, nitrate or organic
nitrogen compounds for their growth.
Nitrogen undergoes a number of transformations involving organic, inorganic and volatile
forms of nitrogen. Apart of the great reservoir of atmospheric nitrogen is converted into an
organic form by certain free living organisms and by plant-microbe associations which then
make this element available for plant growth.
Upon death, plants and animals undergo microbial decay and organic nitrogen is realised as
ammonia which is then oxidized to nitrate by microorganisms. The nitrate form of nitrogen
is mostly used by plants or may be lost by leaching or reduced to gaseous nitrogen and
subsequently lost to the atmosphere.
The nitrogen cycle mainly includes transformations such as: (i) nitrogen mineralization in
which nitrogen containing complexes are decomposed and converted into inorganic
compounds for use by plants, and (ii) nitrogen immobilization in which nitrogen compounds
are assimilated. Some of the biochemical changes brought about by microorganisms in the
nitrogen cycle are discussed below.

Nitrogen Mineralization
In the process of mineralization proteins, nucleic acids and their components are degraded by
heterotrophic microorganisms with the eventual liberation of ammonia and this is called
ammonification. The microorganisms themselves assimilate a part of the liberated ammonia.
The first step in the process of ammonification 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 anaerobic conditions protein decomposition leads to conversion of
ammonia into amines and related compounds. These amines are subsequently oxidized in the
presence of oxygen to release ammonia. In nature, the breakdown of nitrogenous substance
is brought by the activity of a multitude of microbial species. Almost all bacteria,
actinobacteria and fungi can bring about proteolysis and the amino acids so produced are
utilized for the growth of these organisms.
Nitrification
In the second phase, ammonia is converted into nitrate and this process is called nitrification.
Nitrification occurs in two steps; first, ammonia is oxidized to nitrite:
2NH3 + O2 NO2- + 2H+ + H2O
This change is brought about by chemoautotrophic bacteria of the genera Nitosomonas,
Nitrosolobus, Nitrosococcus and Nitrosospira. These bacteria obtain their energy
requirement by the oxidation of NH4 + to NO2- Of these nitrifying organisms, Nitrosomonas
are the most important in soil.
Besides the chemoautotrophic bacteria, some heterotrophic bacteria such as Streptomyces and
Nocardia have also been known to oxidize ammonia to nitrite.
In the second step, nitrite is oxidized to nitrate:
HNO2 + O2----------------HNO3
This reaction is dependent on the activities of bacteria belonging mainly to be genera
Nitrobacter. Certain fungi belonging to the genera Aspergillus,Penicillium and
Cephalosporium can also carry out nitrification.
Nitrosomonas, first converts ammonia to hydroxylamine, which is then transformed into
some, undefined intermediate, possibly a compound such as nitroxyl (HNO). This
intermediate is then oxidized to nitrite possibly by way of Nitrous oxide as shown below:
NH3---------NH2OH--------(HNO)--------NO--------NO2
Nitrobacter oxidizes nitrite and yields two electrons for each molecule of NO’ 2 transformed.
The last step in the sequence is visualized as involving a hydrated nitrate molecule in which
electrons are removed to yield nitrate. The electrons are then passed on to O 2 to yield H2O as
shown below.
NO2 + H2O-----2H + NO3
 2H + O2 ---H2O
Denitrification
Certain bacteria are capable of using nitrate as the terminal electron acceptor under anaerobic
conditions. As a consequence of such nitrate respiration, nitrate is reduced to nitrogen gas or
nitrous oxide. This process is called as denitrification and leads to the loss of nitrogen from
the soil. Denitrification depletes the soil of an essential nutrient for plant growth and
therefore is not a desirable reaction. Denitrification occurs mostly in waterlogged anaerobic
soils with high organic matter content and the ability to carry outdenitrification is restricted
only to certain bacteria. Fungi and actinomycetes have so far not been implicated. Among the
bacteria important in denitrification are Thiobacillusdenitrificans, Micrococcus denitrificans,
species of Pseudomonas, Bacillus,Paracoccus, Achromobacter and Serratia.
The overall reaction of denitrification is summarized below.
+4H +2H
2HNO3--------------------2HNO2-----------------------2NO
-2H2O -2H2O
+ 2H
- H2O
+2H
N2-----------------------N2O---------------
-H2O
Nitrate is first reduced to nitrite, which is then transformed to nitric oxide (NO). The NO is
converted to N2 with N2O as intermediate.
Although an undesirable reaction, from the point of view of plant nutrition, denitrification is
of major ecological importance since without denitrification the supply of nitrogen on the
earth would have got depleted and NO3 would have accumulated. Also, since high
concentrations of NO3 are toxic, denitrification is a mechanism by which some of the
nitrogen is released back to the atmosphere.
Bacterial genera which bring about denitirficationPseudomonas, Achromobacter,
Bacillus & Micrococcus
2NO3 +10 H+ N2 + 4H2O+ 2OH- (or)
2NO2 +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.
Hence this process is also termed as dissimilatory nitrate reduction.
Thiobacillusdenitrificans
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
2HNO3 2HNO2 2 NO N2O N2
The enzymes involved are
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 an 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. That is the reduction of nitrate to nitrite andthen
ammonia.
HNO3 + 4H2 NH3 + 3H2O
Since organisms are able to obtain cellular N to ammonia assimilation, theprocess is called
as assimilatory nitrate reduction
Nitrogen Immobilization
Sometimes when plant residues or pure carbohydrates are added to the soil, there is a rapid
decrease in the amount of available inorganic nitrogen. This is referred to as “Nitrogen
immobilization”, which results from the microbial assimilation of inorganic nitrogen. The
process immobilization involves the incorporation of ammonia and nitrate into microbial
protein and nucleic acids and is therefore reverse of mineralization. Mineralization and
immobilization, therefore, run counter to each other. On the death of microorganisms, the
immobilized nitrogen is however, released through mineralization.

Lec. No.6 Biological nitrogen fixation – free living, associative, endophytic and symbiotic
microorganisms
Biological Nitrogen Fixation
A variety of prokaryotic organisms are known to have the ability to reduce atmosphere
nitrogen and fixation of the inert atmospheric elemental nitrogen by microorganisms through
a reductive process is called “Biological Nitrogen Fixation”. It is estimated that this
accounts for about 70 per cent of the total nitrogen fixed in the biosphere. Although such
biological nitrogen fixation has been occurring since time immemorial, it was only in 1838
that Boussingault showed that leguminous plants can fix atmospheric nitrogen content of the
soil. This observation led to a better understanding of the practice of crop rotation involving
legume crops. Direct evidence however, to show that pea plants cannot grow in sterile soils
in the absence of an inorganic nitrogen source came from the experiments of Hellriegal and
Wilfarth in 1888. Subsequently, Beijerinck identified the bacteria that fix nitrogen (rhizobia)
has been widely accepted, it is only in the last few years that there have been serious attempts
to understand the process of biological 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. N2 to cellular N independently of other living
organism
2. Associative N fixers
3. Endophytic N fixers
4. Symbiotic N fixers
I. Free living nitrogen fixers
Azotobacter- Aerobic
Beijerinckia – Aerobic and acid prefeering
Clostridium – Anaerobic
Cyanobacteria (Blue green algae) etc.,
II. Associative symbiotic nitrogen fixer
Azospirillum
III. Endophytic nitrogen fixer
Gluconacetobacterdiazotrophicus, Herbaspirillum, Azoarcus etc.,
IV. Symbiotic nitrogen fixers
Rhizobium (Rhizobium – legume association)
Bradyrhizobium(Bradyrhizobium – soybean association)
Azorhizobium(Azorhizobium- Sesbaniarostrataassociation)
Anabaena azollae(Azolla – Anabaena association)
 Frankia(Frankia– Casuarina association)
Species of Azospirillum
A. lipoferum
A. brasilense
A. amazonense
A. halopareferans
A. iarkense
A.oryzae,
A. serapedicae,
A. dobereinerae,
A. formosense,
A.rugosum,
A. picisandA.zeae.

Species of Azotobacter
A. chroococcum - Produces black pigments (melanin)
A. vinelandii - Produces yellow pigments
A. beijerinckii - Produces green yellow fluorescent pigments
A. insignis - Produces yellow – brown pigments
A. macrocytogenes - Produces pink pigments
A. paspali - Produces pink to green fluorescent pigments
A. A. agilis -
B. A.armeniacus -
C. A. nigircans -
D. A.salinestris -
E. A. tropicalis -
F. A.spAR -
G. A.sp DCU26 -
H. A.sp FA8 -

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
A. nilotica
Extent of Biological Nitrogen Fixation
 (N2 fixed X10 metric tons / yr)
Biological 175
Non-biological 80
Total N2 Fixation 255

By the end of 19th century in addition to the rhizobia, many free living aerobic and anaerobic
bacteria were found to have the ability to fix atmospheric nitrogen. It was another 25 years
before the blue green bacteria (blue green algae) were also reported to have the ability to fix
the nitrogen. The various groups of bacteria that are today known to fix atmospheric nitrogen
and the amount that they fix are given in Table. The ability to reduce atmospheric nitrogen is
restricted only to bacteria and the bacteria known to fix atmospheric nitrogen include both
aerobic as well as anaerobic bacteria of diverse types. The relationship varies from
asymbiotic to obligatory symbiotic type with associative symbiosis in between. Among the
free living, the Azotobacter (aerobic) and Clostridium(anaerobic) have been well studied.
The associative symbionts are a new class recognized recently to have the ability to fix
atmospheric nitrogen in association with the roots of grasses and cereal plants. The
symbiotic nitrogen fixers are represented by the genus Rhizobium that occurs in the root
nodules of legumes.
Various Groups of Microorganisms and Nitrogen Fixation
Bacteria Amount of N2 fixed
Group
Free Living Anaerobic - Clostridium 0.25
Aerobic - Azotobacter 0.3
Facultative - Klebsiella
Associative Azospirillumbrasilense
Legumes Rhizobia 50-500
Nonlegumes Azotobacterpaspali 5-25
Rhodospirillum
Blue Green Algae Nostoc, Anabaena 25-30

Azotobacter is a heterotrophic, gram-negative bacteria which many be the principal nitrogen

fixer in neutral soils with high organic matter content. The Bejerinckia are closely related to
Azotobacter and are found in acidic soils. The anaerobic nitrogen fixing bacteria namely the
enterobacters, the bacilli etc. may not be contributing very much to the overall biological
nitrogen fixation process in the biosphere. Blue green bacteria fix nitrogen aerobically
specially in flooded soils and constitute another potential group of organisms for nitrogen
fixation. They are both terrestrial and aquatic and have not only the ability to fix nitrogen,
butare capable of obtaining their carbon requirement through photosynthesis. There are
claims that blue green bacteria are mainly responsible for maintaining the fertility and
productivity of rice field s of the orient. These bacteria grow abundantly in flooded rice
fields and the bacteria mass undergoes decomposition and provides the nitrogen to the rice
crop. In addition to this, these bacteria excrete both ammonia and growth promoting
substances which are utilized by the rice crop. It is believed that nitrogen fixation occurs in
the heterocytes of blue green bacteria although there is some evidence to the country. One
blue green bacterial strain namely Anabenasymbiosis with the water fern Azolla. Although
this blue green bacterium is capable of photosynthesis and nitrogen fixation, it does best only
when it grows in association with Azolla. This symbiotic system is believed to contribute
substantially to the nitrogen economy of rice soils. Other anaerobic nitrogen fixing
photosynthetic bacteria like Chlorobium, Rhodospirillum, etc., might have a role to play
under certain environmental conditions.
Another area of biological nitrogen fixation is the associative symbionts which from a loose
association with the roots of cereal such as wheat, rice and grasses. In this type of associate
symbiosis the bacteria sometimes invade the outer cortical regions of the cereal roots and fix
nitrogen. However, unlike in the Rhizobium leguminous plant association no nodules are
formed. The bacteria grow in the rhizosphere in close contact with the roots and have access
to photosynthates transferred from the shoot to the root. A variety of bacteria have been
identified to have the ability involve in associate symbiosis. Among this bacterium
Azospirillumbrasilenseearlier called Spirillumlipoferumis best studied for its association with
cereal roots. This bacterium is capable of fixing nitrogen both in the free living state as well
as under microaerophilic conditions. Other bacteria that are known to fix nitrogen, in
association with cereal plants are Pseudomonas azotogenesis, Enterobacter, Klepsiella,
Bacillus, etc. The potential of system is appealing because of the association with cereals and
grasses which form the major component of the animal food chain. The magnitude of
nitrogen fixed by this system, however, does not appear to be appreciable at present and
plant response to inoculation with these bacteria has been found only in low fertility soils.
These bacteria may have a role in helping the plants make more efficient use of the limited
nitrogen available in the soil.
The symbiotic bacteria rhizobia, are gram-negative short rods, which are classified based on
their host specificity and their growth rates. Some of these grow relatively faster while others
such as those infecting soybean, grow slowly. These are highly crop specific. The amount of
nitrogen fixed by these bacteria in association with different leguminous crop varies and can
vary from 20-40 kg per hectare in chick pea to 500-600 kg per hectare in some green manure
crops. One major distinguishing and important feature of bacteria is their ability to fix
atmospheric nitrogen in amounts greater than required for their own growth and make this
available to the host plant.
Classification of Rhizobia Based on Host Specificity
Microsymbiont Host
Fast Growing Rhizobia
R. meliloti Alfalfa
R. trifolii Clover
R. leguminosarum Pea
R. phaseoli Beans
R. japonicum Soybeans
R. lupini Lupines
Slow growing Rhizobium
Bradyrhizobium soybean

II. Bradyrhizobium
B. japonicumSoybean - Soybean
B. sppCowpea group - Cowpea group plants
III. Azorhizobium - Stem nodulating – one. NodulatesSesbaniarostrata.
IV. Photorhizobia- NodulantsAeschynomene sp.
V. Sinorhizobium- fast growing soybean nodulator
Lec. No.7. Nodulation in Rhizobium- legume and Frankia – actinorhizal symbioses.
Biochemistry of nitrogen fixation
Steps involved in nodule formation
Biochemistry of Nitrogen fixation
The process of N2 fixation is mediated by the enzyme, called nitrogenase (whichmediates the
reduction of N2 to ammonia) first, this enzyme was extracted from theanaerobic dinitrogen
fixer Clostridumpasteurianum. Latter, this has been isolated formmost other N2 fixing
bacteria.
The mechanism of N2 fixation appears to be quite similar in most N 2 fixingprokaryotes. The
enzyme has been fairly well characterized and the enzymes from thesedifferent systems share
common properties allowing a unified single description ofnitrogenase.
Nitrogenase
Nitrogeanse is a functional enzyme which reduces N2 to ammonia and depends onenergy
source from ATP. The nitrogenase has two components one containing Mo-Fe,designated as
Mo – Fe protein and the other Fe protein . Two components arenecessary for the nitrogenase
activity.
Mo-Fe protein
Consists of 4 subunits and having the molecular not of 22,0000 or 270,000daltons 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 overall reaction is as follows.
General pathway of N2 fixation
This process requires a source of ATP and reductants. 16 molecules of ATP are required to
fix a molecule of N2.
Nitrogenase
N2 + 8H+ + 8e- 2 NH3 + H2
16 Mg ATP 16 Mg ADP +Pi
Lec. No.8 Phosphorus cycle and microbial transformation of phosphorus - phosphate
solubilizer and mycorrhizae
Biological importance of phosphorus
Phosphorus is an element essential to life. It plays both structural and functional roles in
virtually all organisms and is found in cell components such as phospholipids, nucleic acids,
and DNA as described above. Phosphorus, through Phosphorus the phosphate anhydride
bond plays an important role in storing and transferring biochemically useful energy. A free
energy change (∆G 0) of 7.3 kcal mol / 30.6 kJ mol ) of adenosine 5-triphosphate (ATP) is
associated with the hydrolysis of its terminal anhydride bond, yielding adenosine 5-
diphosphate and a phosphate group. Unlike many other anhydrides, phosphate anhydrides
such as ATP are unusually resistant to hydrolysis in the aqueous environment (Westheimer,
1987). At neutral pH and physiological temperature, hydrolysis proceeds at an optimal rate
only in the presence of appropriate enzymes (e.g., ATPase). The relative resistance of
phosphate anhydride bonds to hydrolysis is attributable to the negative charges on the
phosphates at neutral pH and is the probably reason ATP was selected in the evolution of life
as a universal transfer agent of chemical energy in biological systems (Westheimer, 1987).
There is good evidence that P is the dominant element controlling C and N immobilization in
biological systems. In a classic paper, Redfield (1958) hypothesized that P controls the C, N,
and S cycles of marine systems. He noted that the oceanic C:N:P ratio paralleled that of the
plankton and believed the following general relationship occurs: The C:N:P ratios for a
number of terrestrial situations are shown in Table 15.3. The aquatic algae and soil bacteria
have similar C:N ratios of approximately 6:1. The algae have lower C:P ratios than the
bacteria, but fall within the range of C:P found in soil organic matter.
Microorganisms are involved in a range of processes that affect the transformation of soil
phosphorus (P) and are thus an integral component of the soil P cycle. In particular, soil
microorganisms are effective in releasing P from inorganic and organic pools of total soil P
through solubilization and mineralization. The microbial biomass in soil also contains a
significant quantity of immobilized P that is potentially available to plants. Microorganisms
therefore are critical for the transfer of P from poorly available soil pools to plant available
forms and are important for maintaining P in readily available pools. These processes are
likely to be most significant in the rhizosphere of plants.

Phosphorus cycle

Soil Organic matter

Mobilization through Plants Animals
Mycorrhizal Association Mineralization

Microbial Soil Humus Fixed Soil

Phosphorus Phosphorus

Phosphate
Assimilation
Immobilization Mineralization P Solubilisation P Fixation
(Heterotrophs) (Phosphobacteria) (Ca,Mg,Fe,&Al PO4)

Soil Available Phosphorus

Microbial transformations of phosphorus

1. Mineralization
Organically bound P is not directly available to organisms because it cannot be absorbed into
cells in this form. For cellular uptake to occur, P must first be released from the organic
molecule through mineralization. The final stage in the conversion of organically bound P to
inorganic phosphate occurs through the action of phosphatase enzymes. The phosphatase
group of enzymes includes phytase enzymes that catalyze the release of phosphate from
phytin and nuclease enzymes that liberate phosphate from nucleic acids. These enzymes are
produced by up to 70–80% of the microbial population, including bacteria such as Bacillus
megaterium, B. subtilis, Serratia sp., Proteus spp., Arthrobacter spp., and Streptomyces spp.
and fungi such as Aspergillus spp., Penicillium spp., Rhizopus spp., and Cunninghamella spp.
Once P is mineralized, it can be taken up by plants, immobilized by the microbial biomass,
precipitated in inorganic complexes, or sorbed to mineral surfaces.

2. Immobilization
Soil microorganisms can cause fixation or immobilization of P, either by promoting the
formation of inorganic precipitates or by assimilation into organic cell constituents or
intracellular polyphosphate granules. In soils and freshwater sediments, cellular
immobilization is important, though fixation of P by Ca2, Al3 or Fe3 has been observed. In
some marine sediments, where phosphorite minerals occur, the precipitation mechanism is
more important. Microorganisms are indirectly involved in phosphorite precipitation by
making reactive phosphate available, by making reactive calcium available, or by creating or
maintaining the environmental conditions that favor phosphate precipitation.
The extent of immobilization of P is affected by the C:P ratio of the organic materials being
decomposed and the amount of available P in solution. If insufficient P is available in the
substrate for assimilation of the substrate C, inorganic P from the soil solution will be used
and net immobilization occurs. Generally, a C:P- 200 will result in net mineralization, a C:P-
300 results in net immobilization, and C:P ratios between 200 and 300 result in little net
change in soluble P concentrations.
3. Oxidation and Reduction
A number of soil bacteria and fungi have been shown to be capable of oxidizing reduced
phosphorus compounds (e.g., phosphite, hypophosphite) either aerobically (Adams and
Conrad, 1953) or anaerobically (Foster et al., 1978). The biochemical pathway for such a
microbially mediated reaction has been characterized molecularly and genetically, providing
some evidence for a previously underappreciated microbial redox cycle for P. The relatively
high solubility in water of phosphites, hypophosphites, and phosphonates suggests they may
have been important precursors of biochemical P compounds, but the fact that there have
been only trace quantities of phosphite and hypophosphite detected in the current
environment suggests that the existence of microbial pathways of P oxidation might represent
an ancient evolutionary property (Foster et al., 1978; Schink and Friedrich, 2000). There is
increasing appreciation in the literature for the presence of reduced forms of P such as
phosphine (PH 3), phosphites, and organic phosphonates, which provide a small gaseous link
to the P cycle (Glindemann et al., 2005). Microbially mediated reduction of phosphate
remains a controversial topic in the literature (see Morton and Edwards, 2005; Roels and
Verstraete, 2001). The controversy is fuelled by thermodynamic calculations that show that
the reduction of phosphate is energetically unfavourable. However, this does not imply that
reduced P compounds.

4. Phosphate Solublization
1. Organic acids production – Heterotrophic microorganisms
2. Inorganic acid production – Chemoautotrophic microorganisms
3. Microbial respiration – All kinds of microbes
4. H2S Production – Sulphur reducing bacteria
5. Alkaline reaction - All kinds of microbes

5. Phosphate Mobilization –Mycorrhizae

10. Sulphur cycle - sulphur oxidizers; microbial transformation of K, Zn and Si. Role of soil
enzymes in nutrient transformation

11. Importance of soil and plant associated microorganisms – rhizosphere, spermosphere

phyllosphere, epiphytic and endophytes
12. Soil microorganisms and their interactions – positive and negative interactions.
Bioinoculants - types - bacterial, fungal (AMF) and algal bionoculants
Microbial interactions
With in a biological community, various types of interactions can occur between diverse
microbial populations, between microbial and plant populations, 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 faecalisand 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 relationships 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 competitiveexclusion 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.
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.
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.

Lec.No. 13. Mass production of bioinoculants

Fermentoris the culture vessel, where the fermentation process is carried out. The main
function of fermentor is to provide a controlled environment with a particular nutrient media
for the growth of microorganisms to produce the target product that may be either a cell
biomass, a metabolite or bioconversion product
Fermentor consists of two major parts viz., basic and monitoring / controlling parts. Basic
parts are necessary for designing the fermentor unit whereas monitoring or control parts are
necessary for fermentor operation.
a. Basic parts
Fermentation vessel Normally the fermentation vessel is made out of copper/ stainless
steel or mild steel coated with glass or epoxy material with ports for
various outputs, inputs and probes. The vessel should have smooth
inner surface. The vessel should be strong enough to withstand the
interior pressure. It should be resistant to corrosion and free from
any toxic effect for the microbial culture. Based on the capacity of
the vessel it is grouped into three as small, pilot and large sized
fermentor
Top plate Cover (made of steel)
Clamp Top plate compressed onto vessel using clamp
Seal Separates top plate from vessel (glass) to prevent air leakage.
Sealing is an important criteria to maintain airtight condition and
aseptic condition. Sealing ensures air tight condition in spite of
expansion of vessel during fermentation
Drive motor Used to drive mixing shaft
Drive shaft Mixes the medium evenly with its impeller
Marine impeller For plant tissue culture
Baffles Prevent sedimentation on sides and imparts proper mixing
Sparger To provide adequate aeration and agitation to meet the metabolic
requirements of microorganisms. The agitators may be of single
blade, multiple type or impeller type
Exit gas cooler Acts as a condenser; removes as much moisture as possible from
exhaust
Inoculation port To add sterile medium and inoculum
Feed pumps Regulates the flow rates of additives (medium, nutrients) at variable
speed
Peristaltic pumps Fixed speed pumps – used for continuous sampling
Syringe pump Using a syringe – mostly used in batch fermentation
b. Monitoring / Controlling parts

Pt 100 Temperature sensor (platinum resistance electrode)

 Foam probe Kept above the medium level to sense foam formation
pH electrode Senses pH
O2 sensor Monitors dissolved oxygen level
Heater pad Directly heats the medium
Cold finger Used to cool the vessel contents
Variable air flow meter attached to air sparger; indicates rate of air flow
Rota meter
into the vessel
Pressure valve Attached to rota meter for safer operation
Air pump Supply of air
Peristaltic pump To pump in medium, acids, bases, antifoam etc.,
Operation Procedure
1. Fermentor is working under the principle of steam under pressure
2. The medium to be prepared is added directly to the fermentor and sterilized
3. All the sampling / feed inlets and other ports are designed aseptically to ensure zero level
of contamination
4. Necessary foam controls are provided in the fermentor
5. Aerators and agitators are constantly stirred for constant aeration/ agitation
6. After sterilization of the medium, it is allowed to cool to attain room temperature
(28±2ºC)
7. Microbial inoculum is added @ 10% to the fermentor through the inoculation port
8. Then the fermentation is carried out to get the desired product
9. The final products after fermentation are separated out by filtration, foam separation,
centrifugation or by membrane separation techniques
Preparation of mother culture
Following the selection of suitable strain, it will be inoculated into suitable liquid medium.
The incubation is to be made at 28oC+ 2 on a rotary shaker for 2-7 days depending on the type
of bacteria. This is used as a culture for inoculating broth in fermentors.
Mass multiplication and production of carrier based bacterial biofertilizers
1. Pulverize the carrier in a hammer mill and sieve it through 106 micron IS sieve. If it
is peat / lignite, determine the pH and neutralize it with calcium carbonate. In case of
charcoal, neutralization is not required.
2. Sterilize the carrier in an autoclave and cool it.
3. Prepare the starter culture and multiply through shaker culture or in a fermentor
4. Mix the culture broth with the carrier and adjust to about 40 % moisture content.
5. After mixing allow the impregnated carrier material to undergo curing at 28-30oC for
further multiplication of bacterial cultures in the carrier.
6. 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.
7. Store the cultures in cool and dry place at a temperature of 15 oC but not exceeding
30oC.
The preparation and maintenance of biofertilizers should be followed as per the BIS
(Bureau of Indian standard) specifications
Mass production of AM culture
As AM 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.
Materials required
1. Carrier material – Soil and sand / vermiculite / perilite / soilrite
2. Seed material of host plant
Production of root based inoculum
Mass multiplication of AM culture involves two steps. 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 and mix thoroughly.
7. Place the starter culture around 2.5 cm below the soil and sow maize seeds.
8. Allow the plant to grow and check for AM fungal infection periodically
9. After 3-4 months, harvest the plant, mix the root system with soil mix and use as
inoculum.
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, super phosphate and
muriate of potash.

Mass production of azolla

Methods of application of biofertilizers

1. Seed treatment
14. Industrial utilization of microorganisms –alcohol fermentation – alcoholic beverages
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) Leuconostocm Dextran
Kluyveromycesfragilis Ethanol (from lactose) esenterioes
Clostridium acetobutylicum Acetone and Butanol Xanthomonasc Xanthan gum
Asergillusniger Citric acid ampestris
Amino Acids and Flavor-enhancing Nucleotides Pharmaceuticals
Corynebacteriumglutamicum L-Lysine Penicillumchrys Penicillins
Corynebacteriumglutamicum 5'- inosinic acid and ogenum
Corynebacteriumgluatamicum 5' – guanylic acid Cephalosporiu Cephalosporins
MSG macremonium
Vitamins Streptomyces Amphotericin
Ashbyagossypii Raboflavin Kanamycins,
Eremotheciumashbyi Riboflavin Neomycins,
Pseudomonas denitrificans Vitamin B12 Streptomycin,
Propionibacteriumshermanii Vitamin B12 Tetracyclines and
Enzymes others
Aspergillusoryzae Amylases Bacillus brevis Gramicidin 5
Asergillusniger Glucamylase Bacillus subtilis Bacitracin
 Tricholdermareesii Cellulase Bacillus Polymyxin B
S. cerevisiae Invertase polymyxa
Kluyveromycesfragilis Lactase Rhizopusnigrica Steroid
Saccharomycopsislipolytica Lipase ns transformation
Asergillus Pectinases & proteases
Bacillus Proteases Arthrobacter Steroid
Mucorpussilus Microbial rennet simplex transformation
Mucormeihei Microbial rennet Mycobacterium Steroid
transformation
Escherichia coli Insulin, human
(via rDNA growth hormone,
technology) somatostatin&
interferon

Ethanol from Molasses

On industrial scale, ethanol can be prepared by the fermentation of molasses. Molasses is the
mother liquor left after the crystallization of sugarcane juice. It is a dark coloured viscous
liquid. Molasses contains about 60% fermentable sugar. Ethanol is a volatile, flammable,
clear, colourless liquid. Ethanol is a good solvent. It is also used as a germicide, beverage,
antifreeze, fuel, depressant and chemical intermediate. It can be made by the fermentation
process of material that contains sugar or from the compound which can be converted to
sugar.
The present average alcohol production from molasses in the country is around 2500 million
liters per annum. However, wide variation in ethanol production is observed over the last few
years, due to the fluctuation in sugarcane production. In India alcohol is largely produced in
the form of either i) Rectified Spirit (RS) (95 to96 % v/v ethanol) which is mainly used for
industrial applications in the form of Ordinary Denatured Spirit (ODS) or Special Denatured
Spirit (SDS), ii) Extra Neutral Alcohol (ENA) (96 % v/v ethanol) mainly used for
manufacture of potable liquors and iii) Fuel Ethanol or Absolute Alcohol (AA) (99.8 % v/v)
mainly used for blending with petrol

Alcoholic beverages are a mainstay of human culture and have been produced on a large
scale for centuries. Many different alcoholic beverages are known, with some having world
wide appeal and others more regional appeal. But all alcoholic beverages begin with a
fermentation step in which some fermentable substance, typically a grain, vegetable, or fruit,
is fermented by yeasts or bacteria to yield ethanol and carbon dioxide. The distinctive
character of a given alcoholic beverage is the result of many factors, including natural flavors
present in the fermentable substrate; chemicals other than alcohol produced during
fermentation and of course, the alcohol itself. The undistilled beverages include wine and
beer.
Wine
Fruit juices undergo a natural fermentation by wild yeasts present in them. From these,
particular strains of yeasts have been selected through the years for use in the wine industry.
Wine Varieties
Most wine is made from grapes. Wine can also be made from many other fruits and from
some non fruit sugars, such as honey (Mead). There are a great variety of wines and their
quality and character vary considerably.
1. Dry wines are wines in which the sugars of the juice are almost completely fermented
2. Sweet wines in which some of the sugar is left or additional sugar is added after the
fermentation
3. A fortified wine is one to which brandy or some other alcoholic spirit is added after the
fermentation. Eg. Sherry and port wines.
4. A sparkling wine, such as champagne, is one in which considerable carbon dioxide (CO 2)
is present, arising from a final fermentation by the yeast in the sealed bottle.
Wine Production
First grapes are crushed and the juice (must) is squeezed out. Depending on the grapes used
and on how the must is prepared, either white or red wine may be produced
1. Typical varieties of white wine include Chablis, Rhine wine, sauterne, and chardonnay;
2. Typical red wines include burgundy, Chianti, claret, zinfandel, cabernet, and merlot.
The yeasts that ferment wine are of two types: wild yeasts, which are present on the grapes as
they are taken from the field and are transferred to the juice and strains of the cultivated wine
yeast, Saccharomyces ellipsoideus, which is added to the juice to begin the fermentation.
Wild yeasts are less alcohol-tolerant than commercial wine yeasts and can also produce
undesirable compounds affecting quality of the final product. Thus, the wild yeasts present in
the must are killed by adding sodium metabisulfite (Na 2S2O5)at a level of 50–100 mg / l.
Strains of S. ellipsoideus are resistant to this concentration of sulfite and are added to the
must as a starter culture from a pure culture grown on sterilized grape juice. The wine
fermentation is carried out in fermentors of various sizes, from 200 to 200,000 liters, made of
oak, cement, stone or glass-lined metal. All fermentors must be designed in such a way that
the large amount of CO2 produced during the fermentation can escape but air cannot enter,
and this is accomplished by fitting the vessel with a special one-way valve.
Red and White Wines
1. White wine : A white wine is made either from white grapes or from the juice of red
grapes from which the skins, containing the red coloring matter, have been removed.
2. Red wine : In the making of red wine, the skins, seeds, and pieces of stem, collectively
called pomace, are left in the fermentation. In addition to the color difference, red wine
has a stronger flavor than white because of larger amounts of tannins, chemicals that are
extracted into the juice from the grape skins during the fermentation. In the production of
a red wine, after about five days of fermentation, sufficient tannin and color have been
extracted from the pomace that the wine can be drawn off for further fermentation in a
new tank, usually for 1–2 weeks.
Fermentation: It is carried out in two steps 1. Primary (aerobic) and 2. Secondary
fermentation (anaerobic)
Racking: The next step is called racking; the wine is separated from the sediment, which
contains yeast cells and precipitate and then stored at a lower temperature for aging, flavor
development and clarification.
Clarification: It may be hastened by the addition of fining agents, materials such as casein,
tannin or bentonite clay that absorbs particulates. Alternatively, the wine may be filtered
through diatomaceous earth, asbestos, or membrane filters. The wine is then bottled and
either stored for further aging, or sent to market.
Aging: Red wine is typically aged for months to several years but most white wine is sold
without much aging. During aging, complex chemical changes occur, including the reduction
of bitter components; this improves the flavor and odor, or bouquet, of the wine. The final
alcohol content of wine varies from about 8% to 16% depending on the sugar content of the
grapes, length of the fermentation, and strain of wine yeast used.
Malo lactic Fermentation
Many high-quality dry red wines and a few white wines such as the chardonnays are
subjected to a secondary fermentation following the primary fermentation by yeast. This is
done before bottling and is called the malolactic fermentation. Full-bodied dry red wines are
the typical candidates for malolactic fermentation. In grape varieties used for dry wines a
considerable amount of malic acid can be present in the grapes. The malic acid content of the
grape varies locally due to climatic and soil conditions. Malic acid is a sharp and rather bitter
acid.
During the malolactic fermentation, malic acid is fermented to lactic acid, a softer,
smoother acid, and this makes the wine less acidic and fruity but more complex and
palatable. Many other constituents are produced during the malolactic fermentation, including
diacetyl (2,3-butanedione), a major flavoring ingredient in butter; this also helps to impart a
soft, smooth character to the wine. The malolactic fermentation is catalyzed by species of
lactic acid bacteria including Lactobacillus, Pediococcus, and Oenococcus. These organisms
are extremely acid tolerant and can carry out the malolactic fermentation even if the initial
pH of the wine is below pH 3.5.
Commercial wineries typically use starter cultures of selected malolactic fermentation
organisms and then store the wine in barrels especially for this purpose. Inocula for future
rounds of malolactic fermentation come from lactobacilli that become attached to the insides
of the barrel. The malolactic fermentation can take several weeks. The final product is often
much smoother and far superior to the more sharp-tasting and bitter starting material.
Beer Fermentation: Beers and ales are alcoholic beverages produced from the fermentation
of grains and other sources of starch. The amount of alcohol in brewed products is much
lower than wine, and levels of CO2 are much higher. Thus the two products, beer and wine
are quite different fermented beverages.
Brewing is the production of alcoholic beverages from malted grains. Typical malt beverages
include beer, ale, porter, and stout. Brewing includes 1. Preparation of wort and
2.Fermentation of wort.
1. Preparation of Wort
i. Malting: Malt is prepared from germinated barley seeds and it contains natural enzymes
that digest the starch of grains and convert it to glucose. Because brewing yeasts are unable to
digest starch, the malting process is essential for the generation of fermentable substrates.
ii. Mashing:The fermentable liquid for brewing is prepared by a process called mashing. The
grain of the mash may consist only of malt or other grains such as corn, rice, or wheat. The
mixture of ingredients in the mash is cooked and allowed to steep in a large mash tub at warm
temperatures. During the heating period, enzymes from the malt cause digestion of the
starches and liberate glucose, which will be fermented by the yeast. Proteins and amino acids
are also liberated into the liquid, as are other nutrient ingredients necessary for the growth of
yeast.
iii. Preparation of Wort: After mashing, the aqueous mixture, called wort, is separated by
filtration. Hops, an herb derived from the female flowers of the hops plant, are added to the
wort at this stage. Hops add flavors to the wort but also have antimicrobial properties, which
help to prevent bacterial contamination in the subsequent fermentation. The wort is boiled for
several hours, so that desired ingredients are extracted from the hops, undesirable proteins
present in the wort are coagulated and removed, and the wort is sterilized. The wort is filtered
again, cooled and transferred to the fermentation vessel.
2. The Fermentation Process
Brewery yeast strains are of two major types: top fermenting and bottom fermenting.
The main distinction between the two is that top-fermenting yeasts(Saccharomyces
cerevisiae) remain uniformly distributed in the fermenting wort and are carried to the top by
the CO2 gas generated during the fermentation, whereas bottom-fermenting yeasts
(Saccharomycescarlsbergensis), settle to the bottom. Top yeasts ferment at higher
temperatures (14–23°C) than bottom yeasts (6–12°C) and thus complete the fermentation in a
shorter time (5–7 days for top fermentation versus 8–14 days for bottom fermentation).
After bottom yeasts complete lager beer fermentation, the beer is pumped off into large
tanks where it is stored in the cold (about -1°C) for several weeks. Then, the beer is filtered
and placed in storage tanks from which it is packaged and sent to market.
Top-fermented ale is stored for only short periods at a higher temperature (4–8°C),
which assists in development of the characteristic ale flavor.
Different types of Beer
Type Alcohol
(% w/v)
Weiss beer 2.73
Lager beer 3.93
Export beer 4.40
Bock beer 4.69
Ale beer 4.75
Distilled Alcoholic Beverages
Distilled alcoholic beveragesare obtained by heating fermented liquids to volatilize
alcohol and other constituents. The distillate is condensed and collected, a process called
distilling. A product much higher in alcohol content is obtained by distilling than is possible
by fermentation alone. Alcohol concentrations in distilled products vary from as little as 20%
to as high as 95%. The “proof” rating, used primarily for labeling distilled spirits in the
United States, is defined as twice the alcohol concentration. Thus a whiskey that is 80 proof
contains 40% ethanol by volume. Examples for different distilled products are given below
1. Malt brews - Whiskey
2. Wine - Brandy
3. Fermented molasses - Rum
4. Fermented grain or potatoes - Vodka and
5. Fermented grain and juniper berries - Gin
Aging :The distillate contains not only alcohol but also other volatile products from either the
yeast fermentation or released from the ingredients. Some of these products add desirable
flavor, whereas others are undesirable. To eliminate the undesirable products, the distilled
product is aged, usually in oak barrels. The aging removes undesirable products and allows
desirable new flavors and aromatic ingredients to develop. The fresh distillate is typically
colorless, whereas the aged product is often brown or yellow. The character of the final
product is partly determined by the manner and length of aging; aging times of 5–10 years are
common, but some very expensive distilled spirits are aged for 20 years or more.

15. Antibiotics production (Penicillin and Streptomycin) and Vitamin production (Vitamin B2
and Vitamin B12).
Antibiotics
Antibiotic pr

oduction
The major antibiotics used in medicine and the microorganisms used for producing these
antibiotics are shown in Table.
Some Antibiotics Produced by Microorganisms
Antibiotic Microorganism
Amphotericin B Streptomyces nodosus
Bacitracin Bacillus licheniformis
Carbomycin Streptomyces halstedii
Chlorotetracycline Streptomyces aureofaciens
Chloramphenicol Streptomyces venezuelae of total chemical
synthesis
Erythromycin Streptomyces erythreus
Fumagillin Aspergillusfumigatus
Griseofulvin Penicilliumgriseofulvin, P.nigricans
Penicilliumurticae
Kanamycin Streptomyces kanamyceticus
Neomycin Streptomyesfradiae
Novobiocin Streptomyces niveus, S. spheroides
Nystatin Streptomyces noursei
Oleandomycin Streptomyces antibioticus
Oxytetracycline Streptomyces rimosus
Penicillin Penicilliumchrysogenum
Polymyxin B Bacillus polymyxa
Streptomycin Streptomyces griseus
Tetracycline Dechlorination and hydrogenation of
Chlorotetracycline; direct fermentation in
dechlorinated medium
Vira A (adenine Streptomyces antibioticus
arabinoside)
Production of Penicillin
In a typical process for manufacturing penicillin, an inoculum of Penicilliumchrysognumis
produced by inoculating a dense suspension of spores of the fungus onto a wheat bran-
nutrient solution. The cultures are allowed to incubate for approximately 1 week at 24 0C and
are then transferred to an inoculum tank.
During the first day of the fermentation, there is a large increase in the biomass of penicillum
mycelia. The carbohydrate substrate is rapidly used during this early phase, providing the
necessary carbon and energy for the production of fungal mycelia. At a later stage, reduction
of the carbohydrate concentration provides the necessary nutritional starvation conditions that
favor penicillin production. The nitrogen required to support fungal growth comes from the
corn steep liquor. The production of penicillin, a secondary metabolite (idiolite) not required
for the growth of the fungus, lags behind the accumulation of fungal biomass (trophophase).
The accumulation of penicillin occurs in the (idiophase), which begins on the second day and
reaches its maximal concentration a few days later.
When the fermentation is completed, the concentration of penicillin having reached maximal
achievable levels, the liquid medium containing the penicillin is separated from the fungal
cells, using a rotating vacuum filter. Penicillin is recovered from the filtrate, using various
extraction procedures.
The resulting penicillin (called penicillin G) can be chemically and enzymatically modified to
make a variety of penicillins with slightly different properties. These semi‐synthetic
penicillins include penicillin V, penicillin O, ampicillin and amoxycillin.
Production of Streptomycin
Streptomycin and various other antibiotics are produced using strains of Streptomyces
griseus. As in penicillin fermentation, spores of S. griseus are inoculated into a medium to
establish a culture with a high mycelial biomass for introduction into an inoculum tank, with
subsequent use of the mycelial inoculum to initiate the fermentation process in a production
tank. The basic medium for the production of streptomycin contains soybean meal as the
nitrogen sources, glucose as the carbon source and sodium chloride.
Vitamins
Vitamins are used as supplements for human food and animal feeds. Vitamin B12 and
riboflavin are the most important vitamins produced by microbial fermentation process. For
industrial production of vitamin B12, Propionibacterium and Pseudomonas are employed
especially Propionibacteriumfreudenreichii is used
Riboflavin is synthesized by many microorganisms, including bacteria, yeasts, and fungi. The
fungus Ashbyagossypii produces several grams per liter of riboflavin and is therefore the
main organism used in microbial production.
Production of some vitamins using microorganisms
Vitamin Culture Medium Fermentatio Yield
n Conditions
Riboflavin Ashybya gossypii Glucose 6 days at 360C 4.25 g/L
collagen, soya aerobic
oil, glycine
L- Sorbose (in Gluconobacter D-Sorbitol, 30% 45 hours at 70% based on
vitamin C oxidans subsp corn steep liquor 300C, aerobic substrate used
synthesis) suboxidans
5-keto luconic Gluconobacter Glucose, CaCO3 33 hours at 100% based on
aid (in vitamin oxidans subsp. corn steep liquor 300C substrate used
C synthesis) suboxidans
Vitamin B12 Propionibacteriu Glucose corn 3 days at 300C 23 mg/L
m shermanii steep liquor, anaerobic +4
ammonia cobalt, days, aerobic
pH 7.0

Commercial Production of Riboflavin

Commercial production of riboflavin by direct fermentation often uses the fungal species
Eremotheciumashbyii or Ashbyagossypii. Riboflavin production using such fungi employs a
medium containing glucose and/or corn oil/ corn oil may be added even when glucose is used
as the primary growth substrate to increase yields of riboflavin. The fermentation using
Ashbyagossypii to produce riboflavin is normally carried out at 28 - 30 0C, pH 6-7.5 for
approximately 4-5 days. After growth of the yeast, the cells are recovered and used as a feed
supplement for animals to supply needed riboflavin.
16. Microbes in food industry – Single Cell Protein, Baker’s and Brewer’s yeast, Dairy
products – cheese and yoghurt
Single protein
Single cell protein refers to the cells of microscopic organisms (bacteria, yeasts, moulds and
microalgae) grown in large scale cultures for use primarily as a source of protein in human or
animal diets.
Microorganisms involved: Candida lipolytica, Saccharomyces fragilis, Rhodotorula,
Aspergillus, Penicillium, Fusarium, Pseudomonas, Cellulomonas, Scenedesmusacutus,
Spirulina maxima and S. platensis.
Substrates used for production of single cell protein:
1) High energy substrates: Petroleum, methanol and ethanol.
i) Candida lipolytica and Pseudomonas sp. which utilize waxy residues of crude
petroleum are used as animal feeds.
 ii) One commercial single cell protein product is made by growing the organism
Methylophilus methylotrophicus on methanol. The product "Pruteen" (Imperial
chemical industries, UK) is marketed as an animal feed.
iii) Another organism Torula yeast is produced by using ethanol and used for human
consumption.
2) Waste products:
i) Molasses is used for growing yeasts (Bakers yeast) but is also used in Taiwan and
Japan for growing Chlorella heterotrophically.
ii) Sulphite waste liquor from paper mills can be used as a substrate for yeast Torula sp.
iii) Whey from cheese making contains 4% lactose is used for culturing Kluyveromyces
marxianus in America.
iv) Starch – rich wastes from potato or rice processing are converted to single cell protein
by two step process.
In the first step, Starch is hydrolysed to glucose and maltose by an organism
Saccharomyces fibuligera. Another step, Candidautilis utilizes simple sugar to form the
bulk of the single cell protein. This process reduces the biological oxygen demand of the
wastes by 90% and thus reduces difficulties in disposal (Swedish Sugar Corporation).
v) Sewage effluents: Sewage effluents are used for the culture of Chlorella,
Scenedesmus, Spirulina and Photosysthetic bacteria.
3) Renewable Sources: eg. Wood and other plant residues containing lignocellulosic
materials need pre-treatment to separate the lignin, hemicellulose and cellulose
components. Once free, cellulose can be hydrolysed to fermentable sugars by acid
treatment or by cellulases (Trichoderma spp.). Yeast single cell protein (Candida
spp.) has been grown on wood hydrolysates in the USSR.
Production of single cell protein
1. Bacteria, yeasts and moulds are generally cultured in well-aerated liquid media in a
fermentor by the continuous culture method.
2. Algae are grown in batch or semi continuous cultures in ponds with large surface area
3. Yeasts and bacteria are harvested by centrifugation.
4. Algae and moulds are collected by filtration.
5. The concentrated biomass is washed, dried and used either directly (e.g. in animal
feeds) or (for human consumption) its protein content may be extracted and used as a
nutrient supplement or as a functional ingredient.
Advantages of single cell protein over plant and animal proteins
 1. It is rich in protein. The crude protein ranges from 40 – 85%.
2. The yield co efficient of SCP (g cell mass produced/g carbon utilised), is much higher
than that of either plants or animals.
3. SCP can be produced rapidly and unlike agricultural crops, production can be
continuous throughout the year.
4. A wide range of raw materials, including wastes from other industries, can be used as
substrates. In some cases, this has the additional advantage of reducing the BOD of
certain trade effluents.
Disadvantages of single cell protein
1. Microbial protein tend to be deficient in sulphur containing amino acids, particularly
L-methionine. This can be overcome by supplementing SCP products with
methionine or its hydroxy analogues.
2. Some microbial components, example cell walls cannot be digested by non –
ruminant animals or man.
3. SCP is rich in nucleic acids, mainly RNA. Digestion of purine nucleosides yields uric
acids, which deposit in joints and kidneys, leading to gout and kidney stone. Nucleic
acids may be removed by enzymatic treatment.
4. Some SCP can affect man adversely in ways not always indicated by animal feeding
experiments.
5. Cost wise SCP is significantly more expensive than either soybean or fish meal.

Dairy products – cheese and yoghurt

Cheeses
Cheese may be the most popular fermented milk product, using more than one-third of all
milk produced in the United States each year for its production. Both soft and hard cheeses
are produced by culturing milk for an extended period of time. Certain types of cheeses can
be made simply by straining the moisture out of sour cream or yogurt. Some other types of
cheese, however, require additional steps in the culturing and fermentation process. Over
2,000 varieties of cheeses exist, with some of the most notable being cheddar, feta, cream,
goat and blue.
Yogurt
A staple of the Middle Eastern diet for thousands of years, yogurt is a fermented food that
holds the same level of protein and fat as the milk from which it is produced. It is also a
source of calcium and vitamins B2, B6 and B12. Yogurt, like other fermented milk products,
is primarily cultured from cows milk, but can be made from goat's milk. Microorganisms can
also be used to ferment non-dairy milks, including coconut milk, almond milk and soy milk,
into yogurt.
Baker’s and Brewer’s yeast

17. Biofuels – alcohol and biodiesel production

Ethanol production
Molassess/ cellulose/starch Glucose Ethanol+ CO2 +H2O
Raw materials
Microroganisms used
1. Saccharomyces cerevisiae
2. Kluveromycesmarxians
3. Xymomonasmobilis

Biodiesel production - Transesterification

Animal/plant/microalgal oil + Methanol Fatty acid methyl ester (FAME) + Glycerol
Catalyst (Biodiesel)
Biodiesel is defined as non-petroleum-based diesel fuel consisting of alkyl esters (mainly
methyl, but also ethyl, and propyl) of long chain fatty acids. Biodiesel could be produced
from various animal and plant sources by esterification of triglycerides with methanol. In
addition, biodiesel could be produced from various species of microalgae.

An overview of microalgalbiomass and biofuel production

Lab scale model

Industrial scale
 1. Circular pond

2. Raceway pond

3. Tubular bioreactor

4. Flat plate bioreactor