Course Teacher: A.

SANDANAKIROUCHENANE
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 bacterial bioinoculants, blue green
algae, azolla and AM fungi. Isolation of yeast and Lactobacillus. Industrial products – wine
and sauerkraut fermentation.
Theory schedule
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
 LECTURE 1
1. Introduction and historical developments in soil microbiology. Contributions of
Beijerinck, Winogradsky, Fleming and Waksman

Introduction and Importance

Soil represents a medium or substrate in which numerous microorganisms live
and bring about a great variety of processes which are responsible for continuation of the
cycle of life in nature. The numerous living forms which spend all or part of their life in
soil ranging from sub microscopic forms to the lower animal forms. With the growing
recognition of the numerous processes carried out by the microorganisms in the soil there
gradually 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 their
functions and activities.
Since soil microbiology concerns with soil microorganisms and their processes it
is closely associated with soil biochemistry. Medical bacteriologists were interested in the
soil as a medium for the growth and survival of disease producing organisms.
Agricultural chemists are also interested in the soil processes that result from the
activities of microorganisms. General bacteriologist, zoologist, botanist were interested in
certain special group of organisms found in soil. Recently, soil microbiology has
expanded to include the study of the role of soil microorganisms in genetic engineering,
in the biological control of pests and diseases, the degradation of pollutants, production
and destruction of radioactive gases and its transfer. Thus microbial participation in
several important processes emphasizes that soil microbiology has become a global
science.
Soil microbiology
· Deals with the microscopic organisms of the soil
· Their population and activities
· Role in various 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 the quantitative and qualitative composition of the microscopic and
ultramicroscopic soil population.
2. Experimental or physiological phase
Study of the physiology and biochemistry of the 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 microbiological activities to soil fertility and crop production.
4. Pedological phase
Importance of microorganisms in soil formation and soil 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.
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.
Important developments in soil microbiology specially biofertilizers:
Soil improving properties of legumes were recognised by man over 2000 years
ago. In the year 1884, French Agricultural chemists Boussingault gave the classical
concept of biological Nitrogen fixation by legumes which was positively established by
Helliriegal and Willfrath in the year 1886. The practical exploitation of biological
nitrogen fixation began to shape by Beijernick. He first isolated nitrogen fixation the
nitrogen fixing bacteria from root nodules of legumes and he named it as Bacillus
radicola in the year 1888. Now it is Rhizobium sp Beijernick also isolated Azotobacter in
the year 1902 and Spirillum lipoferum in the year 1902. He made a commendable
contribution in the field of bacteriology and bio-fertilizers.
Contributions of some important scientist:
1. S.N. Winogradsky (1856-1953)
 He is a great soil microbiologist who worked on nitrifying bacteria and was
credited for reporting known form of chemoautotrophy. He has demonstrated how
a lithotroph fixes carbondioxide to make organic compounds. His major
contributions are listed below:
 Demonstrated the role of bacteria in nitrification process in 1890. Isolated two
groups of nitrifying bacteria
 In 1891 - he has 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.
 He has developed the technique of enrichment culture making use of the principle
of natural selection along with Beijerinck. It is a technique in which
environmental (including nutritional) conditions are controlled to favour the
development of a specific organisms or group of organisms.
2. Beijerinck

- Beijerinck also discovered nitrogen fixation the process by which

diatomic nitrogen gas is converted to ammonium ions and becomes available to
plants. He has discovered that bacteria perform nitrogen fixation by dwelling
inside root nodules of legumes and revealed the typical example of symbiosis
between plants and bacteria.

- Beijerinck discovered the phenomenon of bacterial sulfate reduction, a form

of anaerobic respiration. He learned bacteria could use sulfate as a terminal
electron acceptor, instead of oxygen. This discovery has had an important impact
on our current understanding of biogeochemical cycles. Spirillum desulfuricans,
now known as Desulfovibrio desulfuricans the first known sulfate-reducing
bacterium, was isolated and described by Beijerinck.

- Beijerinck invented the enrichment culture, a fundamental method of

studying microbes from the environment. 
3. 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 the
action of lysozyme.
· He worked with Straphylococcus aureus and observed the inhibition of growth of S.
aureus in the plate due to the growth of Penicillin.
· Florey and chain latter isolated penicillin in pure culture.
4. Selman A. Waksman
· 1922-Isolated Thiobacillus thioxidans
· 1927- he published book on "Principles of soil Microbiology".
· 1939- Identified the soil organism reducing antibiotics
· 1942 , he showed the importance of soil as the source of antagonistic organisms.
· Discovered the antibiotic streptomycin, 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.
Developments and concept of Biofertilizers in India:
In 1920, the legume Rhizobium symbiosis was first studied by N.V. Joshi. In
1939, P.K. Day discovered nitrogen fixation of BGA in rice field. In 1956, the first
commercial production unit of Bio-fertilizers was started in TamilNadu and New Delhi.
The commercial production of Rhizobium was taken up in 1964, when soyabean was
introduced in India. A great demand for Bio-fertilizers and other legumes also emerged.
All India co-ordinated research project on pulse improvement was set up by ICAR in
1968. The use of charcoal, lignite and FYM were recognised as alternate carrier to peat
soils. In 1999, All India Coordinated research Project on Biological Nitrogen fixation was
initiated. In 1983, National project on development and use of Bio-fertilizers was set up
by Ministry of agriculture, Government of India. In the Year 19988 at IARI, New Delhi,
National research Centre for BGA was set up.
 Lecture 2
2. Diversity of soil microorganisms - culturable and unculturable microbial
diversity. Factors influencing the activities of soil microorganisms

Soil contains five major groups of microorganisms. Bacteria, Actinomycetes,

fungi, algae and protozoa. The soil ecosystem includes these microbial groups as well as
the inorganic and organic constituents of a given site. The collections of cells represented
in the community are considered as distinct populations. All the inhabitants of the
particular locality make up the community.
DIVERSITY OF SOIL MICROBES
Bacteria are the most dominant group of M.O in soil and more numerous than the
other four combined. They present in all types of soil but their population decreases as
the depth of soil increases (Horizon A > B > C). The number of cells of bacteria in the
soil is always great, but the individuals are small, (µm in length). Because of the minute
size of the bacteria it probably account for appreciably less than half of the total
microbial cell mass. In transformation process bacteria stand first, due to their rapid
growth and capacity of vigorous decomposition of variety of substrates. Under anaerobic
conditions bacteria dominate the scene and carry on microbiological activities in soil
since fungi and actinomycetes do not grow well in the absence of oxygen.
Soil microbiological population has been divided into two broad groups: a.
autohcthonous and b.zymogenous.
Autochthonous or native microbes; Indigenous, which are characteristic of the
particular soil and found there. The autochthonous population is always uniform and
constant in soil since their nutrition is derived from native soil organic matter eg.
Arthrobacter
· Zymogenous, or fermentative organisms require an external source of nutrition and
their normal population in soil is low (Pseudomonas, Bacillus). When specific
substrates are added to soil, the population is increased. Then gradually declines when
the added substrate is exhausted eg. cellulose decomposers. N utilizing bacteria,
nitrifiers etc
- Transient microbes - comprising organisms that are introduced into the soil by
legume inoculation unintentionally as in the case of agents producing animal and
plant diseases.
Soil bacteria can be classified based on nutrition

Autotrophs synthesize their own food derive energy from light or chemicals.

-depends on preformed food for nutrition, derive energy and C from

Heterotrophs organic compounds.

Photoautotrophs energy from slight, C from CO2

Chemoautotrophs energy from oxidation of inorganic chemicals, C from CO2.

b) Based on the O2 requirement

Aerobic - need O2 for growth
Anaerobic - not require O2 for growth
Facultative anaerobic- live in the presence or absence of O2.
c) Based on structure
Bacilli - Rod shaped

Cocci - Spherical shaped

Spirillum - Spiral shaped

Common bacteria genera found in soil:
Pseudomonas, Arthrobacter, Clostridium, Achromobacter, Bacillus,
Micrococcus, Flavobacterium, Corynebacterium, Sarcina and Mycobacterium.
Another group of bacteria in soil is Myxobacteria belonging to genera Myxococcus,
Chondrococcus, Archangium, Polyangium, Cytophaga and Sporocytophaga.
It is not easy to determine the total population of bacteria in any soil accurately.
Apart from the inherent limitations, of the soil dilution and plate methods, their numbers
vary with the texture, water content and may other parameters especially the availability
of organic substrates in soil.
Importance of bacteria in soil
1. In transformation process bacteria stands first, due to their rapid growth and
capacity of vigorous decomposition of variety of substrate.
2. Involved in nitrogen fixation, P soluilization, S, Fe transformations, Si
 solubilization etc.,

II. Actinomycetes
These are soil organisms which have characteristics common to bacteria and fungi
yet possess some unique features which are making them to be placed in separate group.
They produce slender, branded filaments that develop into a mycelium in all soil.
Taxonomically they are placed under bacteria in the class Schizomycetes but confined to
the order Actinomycetales.
· 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 clumps
and pellets like Fungi.
· Certain actinomycetes resemble mycobacterium and Corynebacterium in all respects
both morphologically and physiologically including susceptibility to virus attack.
Actinomycetes differ from fungi that as they do not have chitin and cellulose
which are commonly found in the cell walls of fungi.
Though the colony characters are not similar to bacteria, some species have
flagella that resemble those of true Bacteria.
Similarities in cell wall composition and sensitivity to antibacterial compounds
was also observed
Common genera in soils
Streptomyces (70%), Nocardia, Micromonaspora, Frankia.
Actinomycetes are sensitive to antibacterial compounds and not antifungal
compounds. They are known to produce Musty odour, an odour reminiscent of freshly
turned soil.
Distribution
Actinomycetes are numerous and widely distributed not only in soil but in a
variety of other habitats including composts, river muds and lake bottoms. Present in
surface soil and also in lower horizons to considerable depth. In abundance they are
second only to the bacteria and the viable counts almost equal to both. Their nutrition is
saprophytic in nature but a few species can cause diseases of plants, domestic animals
and even humans.
The number of actinomycetes increases in the presence of decomposing organic
matter. As a rule, they are intolerant to acidity and their number decline at pH 5.0. The
most conducive range of pH is between 6.5 and 8.0. Waterlogging in soil is unfavourable
for the growth of actinomycetes whereas desertic soils of arid and semi-arid zones sustain
sizeable population of actinomycetes can be isolated in sufficient number even from soil
samples obtained from the C horizon of a soil profile.
Isolation
Population is – 105 – 108 / g in temperate zone, but lower counts in waterlogged
soils, acid peat, arctic, Tundra regions. In alkaline areas, especially when dry, the relative
abundance is high.
Importance
1. Decomposition of resistant components of plant and animal tissue.
2. Carry out transformations at high temperature particularly in the manure and
compost pits. In general temperatures between 25 to 30 0C are conducive for the
growth of actinomycetes although thermophillic cultures growing at 55 and 65 0C are
common in compost heaps where they are numerically extensive and mostly belong
to the genera Thermoactinomyces and Streptomyces.
3. Produce antibiotics
III. Fungi
As the important constituent group of the soil population, they are widely
distributed in most well – cultivated soils. Fungi account for a large part of the total
microbial population. Next to bacteria fungi dominate all soils and possess large diameter
and extensive network of filaments such as hypha and mycelium. Fungi naturally exist in
soil in the form of vegetative mycelium and spores. The hypha itself is rather broad and
has a diameter appreciably greater than that found in the common actinomycetes. In
nature, asexual spores are abundant and widespread, the sexual spores relatively
uncommon. In contrast with bacteria, fungi can be effectively differentiated into Genera
and species on the basis of morphology.
Distribution and Abundance
All the environmental factors which influence the distribution of bacteria and
actinomycetes also influence the fungal flora of soil. The quality and quantity of organic
matter present in soil have a direct bearing on fungal numbers in sol since most fungi are
heterotrophic in nutrition. Fungi are dominant in acid soils because acidic environment is
not conducive for the existence of either bacteria or actinomycetes resulting in the
monopoly of fungi for utilization of native substrates in soil. They are present in neutral
or alkaline soils and some can tolerate pH beyond 9.0. Arable soils contain abundant
fungi since they are strictly aerobic and excess of soil moisture decreases their numbers.
Isolation of fungi from different horizons of soil profiles shows that these organisms
exhibit selective preferences for various depths of soil. Those fungi which are common in
lower depths are rarely encountered on the surface of soils which may be explained on
the basis of the availability of organic matter and the ratio between oxygen and
carbondioxide in the soil atmosphere at varying depths.
Several techniques have been developed for the study of the fungal flora. Each
one has its own advantage. Plate count and burial slide techniques are used for estimation
of fungi in soil.
Estimates of microbial density reveal the presence in soil is ranging a few as
20,000 to as many as 1,000,000 fungal propagules per gram, the propagule being
considered as any spare, or hyphal filament that is capable at giving rise to a colony. The
length of the fungal mycelium has been reported to range from 10 to 100m per g surface
soil, but various up to 500 and sometimes in excess of 1000mt have also been obtained. It
would appear that the weight of fungi ranges from 500 to 5000 kg per ha of surface soil.
Thus the filaments make up a significant part of the soil mass.
Genera of fungi:
Acrostalagmus, Aspergillus, Botrytis, Cephalosporium, Gliocladium, Monilia,
Penicillium, Scopulariopsis, Spicaria, Trichoderma, Trichothecicum, Verticillium,
Alternaria, Cladosporium Pullularia, Cylindrocarpon and Fusarium.
Apart from this many soil yeasts belonging to true Ascomycetes such as
Saccharomyces and those belonging to Fungi Imperfecti such as Candida can also be
isolated. Their numbers in soil are low and their significance in soil is poorly understood.
Soil yeast are – Candida, Debaryomyces, Rhodotorula, Torulopsis.
Economic importance of fungi
1. Involved in the degradation of complex molecules. They can utilize and degrade the
major plant constituents – cellulose, hemicellulose, pectins, starch and lignin.
2. Participate in humus formation from fresh organic residues.
3. Carry out inorganic transformations and influences the formation of stable aggregates
by means of hyphal penetration and the mechanical binding of the particles.
4. Pathogenecity is common character associated with several soil borne F. Fusarium,
 Helminthosporiun.
5. Predator against protozoa. The hyphae penetrate the protozoa with a resulting
decrease in motility of the animal and an eventual total cessation of movement.
6. Nematodes are also trapped by the fungi by hyphal extensions. Eg: Arthropotrys,
Dactylaria, Dactylella, Harposporium
IV. Algae
Soil algae are ubiquitous in nature wherever moisture and sunlight are available.
They are visible to the unaided eye in the form of green scum on the surface of soils.
Numerically they are not as many as fungi, bacteria or actinomycetes. Morphologically,
they may be unicellular or filamentous and belong to the families, Chlorphyceae (green
algae) and Cyanophyceace (Blue-green algae) and few diatoms.
By virtue of the presence of chlorophyll in their cells, algae are photoautotrophic
and use carbondioxide from the atmosphere and give out oxygen. Algae are also known
to occur below the surface of soil and beyond the reach of sunlight. However, they are
not as numerous as the surface algae and the mechanism of their survival is not very
clear. Some the common algae in Indian soil belong to the genera: Chlorella,
Chalmydomonas, Chlorochytrium, Chlorococcum, Protosiphon and Oedogonium.
The blue green algae contain a pigment known as phycocyanin in addition to
chlorophyll which imparts a special blue green colour to these organisms. The dominant
blue green algae in Indian soils belong to the genera: Chlorococcus, Aphanocapsa,
Lyngbya, Oscillatoria, Phormidium, Microcoleus, Cylindrospermum, Anabaena, Nostoc,
Scytonema and Fischerella.
Some of the blue green algae possess specialized cells known as heterocysts
which are implicated in nitrogen fixation. The waterlogged rice soil provides an ideal
environment for the growth of certain blue green algae and the role of such algae in
nitrogen fixation is well established.
Economic importance:
1. Flooded pady field is the environment algae could have a great
agronomic significance. The microbial action may be associated with the release of
oxygen or the excertion of products stimulating plant developments. Under water logged
rice soils, an algal film forms at the liquid surface, make up an appreciable mass.
2. It also colonizes the barren surface and corrode and weather rocks-Contribute
to soil formation. In addition algal layer covers the rocks and on death it favors secondary
colonizers
3. Surface bloom of algae reduces erosion probably by binding together with soil
particles Improve soil structure, texture and add fertility to soil after decay
V. Protozoa
Soil protozoa are unicellular. In general they lack chlorphyll barring few
exceptions. They are characterized by a cyst stage in their life cycle hence tolerate
adverse conditions. Except few genera which reproduce sexually, rest of them
reproduce asexually by fission. The flagellated protozoans belonging to class
Mastigophora are predominant in soil. Important genera are Allation, Bodo, Cerbodo,
Cercomonas and so on.
Protozoa live in soil at the expense of bacteria of the genera Aerobacter,
Agrobacteriu, Bacillus, Escherichia, Micrococcus and Pseudomonas by ingesting
them into their protoplasm. The protozoans prefer certain species of bacteria for their
nutrition. When food base diminishes in soils, the protozoa get encysted for survival.
Protozoa are abundant in the upper layer of the soil and their numbers are directly
dependant on bacterial population. Application of organic manures increases the
number of soil protozoans which is again a reflection on the corresponding increase
in the bacterial flora due to the application of organic matter. Protozoa are abundant
in soil and their main function is to regulate the number of bacteria.
Soil virus
Soil viruses are sub-microscopic and obligately on other soil microbes like
bacteria, actinomycetes, fungi and algae. Some of the plant and animal viruses also
reach soil. Though viruses can be seen only under an electron microscope, the
lysogenic action of specific phages on their hosts can be seen in formof plaques on
agar plates. Bacteriophages – the viruses attacking bacteria, attach themselves to the
host and get released, lysing the host cell. Similarly, actinophages attack
actinomycetes and cyanophages – attack blue green algae have also been studied.
Factors affecting microbial activities in soil
Several environmental conditions affect the density and composition of the
microflora and frequently alter their activities in soil. Primary factors include moisture,
aeration, temperature, pH, organic matter, inorganic fertilizers. Lesser variables are the
secondary factors which include crop rotation season, soil depth, cultivation practices etc.
I. Primary factors
Soil moisture
Soil moisture is one of the important factors influencing the microbial population.
Water is the major component of protoplasm, an adequate supply is needed for vegetative
development. But, when it becomes excessive, proliferation is suppressed due to
limitation in gaseous exchange and lowers the availability of O2 supply creating
anaerobic environment. Most of the organisms prefer a moisture percentage between 20
and 6 per cent. Many bacteria and fungi are able to adjust themselves to different
moisture conditions. Under dry conditions, the bacteria may form spores which can resist
the drought conditions. The fungi may sporulate or form chlamydospores to tide over
adverse conditions. The protozoans also may form cysts and can survive under dry
conditions. Actinomycetes are the chief group of organisms that prefer dry conditions.
At high moisture, it is believed that the concentration of nutrients is diluted and
also the aeration is very much limited and hence only the anaerobic and microaerophilic
organisms can develop better.
Soil air: It is directly linked up with the moisture level of the soil. Most of the
forms are active in aerated soil and under waterlogged soils anaerobic and
microaerophilic forms develop.
Soil temperature: exhibit considerable influence on the microbial population.
Though microorganisms have been found to exist under extreme temperature conditions,
such as –60°C and +60°C the soil temperature usually does not reach such extremes.
Microbial population varies both quantitatively and qualitatively under extreme
conditions. In tropical and subtropical regions, temperatures vary widely in summer and
winter and the population may also be varied. In temperate regions, there is no much
variation in temperatures of summer and winter hence there is no much variation in the
soil population. Soil temperature influences the temperature of air, water and solid phase
of the soil. Thus soil water and temperature exert a combined influence on the microbial
population.
· Soil organic matter: Community size is related to the organic matter content, so that
humus rich localities have the largest biological numbers. Organic matter content
varied with soil types from less than 0.5 per cent in desert soils to 40 per cent in peaty
soils. Organic Matter being the chief source of energy and food for most soil
organisms, it has great influence on the population. Nature of 0.M is responsible for
the differential stimulation of the population. There are several indirect effects of the
 organic matter on soil microflora. It influences the structure and texture of soil
besides enriching with nutrients for plants and microorganisms. Such influences on
the soil also greatly affect the activity of the soil microorganisms.
· Soil pH: It is a key factor influencing the microflora of soil. It influences enzyme
systems and thus plays an important role in microbial activity. In general fungi thrive
better than bacteria and actinomycetes in acid soils. Bacteria flourish well in neutral
and alkaline soils. The saline and alkaline soils have different microflora. Salinity is
due to excess salts, alkalinity is due to high H-ion concentration. Several direct and
indirect effects of H-ion and salt concentration in soil are exerted on microbial
population.
Fertilizer application: Application of fertilizers to the soil improves the microbial
activity because of the availability of more readily obtainable nutrients. Some fertilizers,
may however have inhibitory effect on specific bacterial types. Continuous application of
ammonical fertilizers favours the growth of fungi due to the formation of nitric acid and
which inhibits the growth of bacteria and actinomycetes. Addition of nitrate inhibits the
activity of free living N2 fixing bacteria like Azotobacter. Some of the autotrophs are
encouraged by the addition of fertilizers.
Cropping and vegetation: Two kinds of effects are exhibited by the crop plants. One
is through root exudates, which may have different compounds with reference to the
crops grown vegetation have selective stimulation over population. Continuous
cultivation leads to more microbial activity than the uncultivated land.
HH. Secondary factors
Crop rotation: Crop rotation with different species like legumes, graminaceous
plants, etc. brings about different stimulatory effect on the microflora. Some crops have
deeper roots than others and some are more fibrous. Such variations bring about physical
changes in soil which in turn may have direct and indirect stimulatory effects on the soil
microflora.
Cultural practices: Various cultural practices, such as tillage operations and
irrigation have several physical and chemical changes in soil which are reflected on the
soil microflora. Through subsoil ploughing deeper layers may get better aeration and
there may be quicker multiplication of aerobic organisms. Weeding, irrigation etc. may
influence the microbial populations in the soil.
 Soil depth : Most of the organisms are almost in top layers, largely in upper few
centimeters and decline with greater depth, more active at few cm down and less active at
deeper layers. Low O2 and less sunlight in deeper layers reduce population.
· Season : Cell number are greatest during the spring and autumn and a decline
occur during hot, dry and winter, the cells remain in a state of dormancy for
biochemical inactivity. The influence is due to mainly the alterations in
temperature, moisture as well as availability of organic matter.
III. Specific influences
· Sunlight favours algae and other autotrophs.
· Herbicide application had devasting effect on algae.
· Attach by neighbours – protozoa, nematode, earthworms consume algae.
· Presence of parasites and predators – eg. viruses eat on specific bacteria
· Increase in bacterial population increases protozoans also.
IV. Other factors
· Burning of top soil: Leads to partial sterilization of the top soil and may kill
protozoan population, which may lead to the increase in bacterial population. This
condition may affect biological equilibrium in soil.
· Application of nematicide, fungicide and bactericide may exhibit partial
sterilization. Thus every change in crop production either directly or indirectly,
alters the soil microflora still many more are not clearly understood.

Lecture 3
3. Carbon cycle – C:N ratio. Role of soil microorganisms in
the decomposition of organic matter and humus formation
Soil organic matter (Aerobic decomposition process)
The organic matter subjected to microbial decay in soil comes from several
sources. The chemistry of organic matter is clearly very complex and investigations of
the transformations and the responsible organisms have therefore been extremely
interesting. Soil organic matter comprises residues of plant and animals and these
compounds occur in soil in close combination with inorganic substances. The main
composition of plant residues are polysaccharides, protein sources and lignin. Animals
residues are made up of different composition depends on the sources from which they
are derived.
 The organic constituents of the plants are commonly divided into six categories.
a) Cellulose - Most abundant 15-60% of the dry weight
b) Hemicellulose - 10-30% of the plant dry weight
c) Lignin - 5 – 30 % of the plant dry weight
d) Water soluble fraction - 5-30%, included simple sugar, a. acids,
· ether and alcohol soluble constituents, a fraction containing fat, oils, waxes, resins and
a number of pigments
· proteins. As the plant ages, the content of water soluble constituents, proteins and
minerals decreases and the % of abundance of cellulose, hemicellulose and lignin rises.
Role of soil microbes:
- The soil microorganism play important role in the decomposition of soil organic
matter.
- Bacteria are the dominant group – mostly heterotrophic organisms (use energy
from organic sources such as sugars, starch, cellulose and protein) – are involved.
Autotrophic organism which occupy a small portion of the biomass in soil (and
use inorganic sources such as Fe and S) are not directly involved in organic matter
decomposition.
- Actinomycetes grow on complex substances such as keratin, chitin and other
complex polysaccharides and play active role in humus formation.
- Soil fungi are mostly heterotrophos and use organic residues easily. They form
major part during decomposition process
- Soil algae contribute a small amount of organic matter through their biomass, but
they do not have any active role in organic matter decomposition.
Organic matter decomposition serves two important functions for the microbes
a) Provide energy for growth
b) Supply carbon for the formation of new cell materials
Hence only heterotrophs are actively involved in the process of decomposition.
The relationship between organic matter and plant growth may be direct or
indirect.
· Organic matter is a natural substrate for saprophytic micro organism and provides
nutrition to plants indirectly through the activity of soil microorganisms
· It is essential for the formation of soil aggregates and hence soil structure which
ultimately determines the soil aeration and rooting habit of plants
 · Organic matter helps in the conservation of soil nutrients by preventing erosion
and surface run off of nutrients.
Two different process are happening during organic matter decomposition
Carbon assimilation
The process of converting substrate to protoplasmic carbon is known as
assimilation. Under aerobic conditions 20-40% of the substrate carbon is assimilated, the
remainder is released as CO2. Fungi are more efficient, in their metabolism, since they
convert carbon into cell carbon as filaments and releases less of CO2. 30-40% which is
used to form new mycelium during the decomposition. Compared to fungi, bacteira are
less efficient. Aerobic bacteria are less efficient than anerobic bacteria.
C Mineralization
Conversion of organic C substance to inorganic form of carbon. This is the
positive aspect with respect to plant nutrition.
Immobilization
Assimilation of nutrients and is the mechanism by which micro organism reduce
the quantity of plant available nutrient in soil. Mineralization is considered good than
immobilization.
Process of decomposition
During the decomposition of organic matter three separate simultaneous processes
can be distinguished. The important changes during decomposition are:
1. Plant and animal tissues constituents disappear under the influence of enzymes
2. Synthesis of new microbial cells so that proteins, polysaccharides and nucleic
acids typical of bacteria and fungi appear.
3. Third, certain end products of the breakdown are excreted into surroundings there
to accumulate or to be further metabolized.
Importance of organic matter decomposition
JJJ. Important function is the breakdown of organic matter by which CO2
available for photosynthesis is replenished
KKK. Any compound that is synthesized biologically is subject to destruction by
soil inhabitants, otherwise the compounds would have accumulated in vast
amounts on the earth’s surface
LLL. Since, organic matter degradation is a property of all heterotrophs, it is
commonly used to indicate the level of microbial activity.
Methods to evaluate the decomposition rate
a) Measurement of CO2 evolution or O2 uptake
b) Determination of decrease in organic matter either chemically or by weight loss
c) Observations on disappearance of specific constituents such as cellulose,
hemicellulose or lignin.
Changes during organic matter decomposition
As a result of development of mixed flora on chemically complex natural
products, some components quickly disappear while others are less susceptible to
microbial enzymes and persist. The water soluble fraction contains the least resistant
plant components and is thus the first to be metabolized. Cellulose and hemicellulose on
the other hand disappear not as quickly as water soluble substances, but their persistence
usually is not too great. The lignins are highly resistant and consequently become
relatively 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 occurs.
· Change in (O) (H) oxidation reduction potential (En) – it is shifted to a more
reduced condition (fall in oxidation reduction potential).
The end products of decomposition are
CO2, H2O, NO3, SO4, CH4, NH4 and H2S depending on the availability of air.
Factors influencing the organic matter decomposition
Organic matter level of the soil
Cultivation practices
Temperature
Moisture
pH
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
Importance of C:N ratio:
Nitrogen is a key nutrient substance for microbial growth. If N content of the
substrate is high it is readily utilized and decomposition is faster, if N is poor
decomposition is slower, needs additional N. In general, protein rich substrates are
 readily decomposed. Low N or wide C:N ratio results in slow decay. Optimum level
of C:N ratio for maximum decomposition is 20-25(1.4-1.7% N) Less than this range,
more microbial cells aids in faster mineralization and it likely exceeds
immobilization. Wider the ratio, lesser microbial cells, slower the immobilization and
mineralization increases gradually, resulting in accumulation of Ammonia and
Nitrates. In addition nicrobes scavenge the soil solution to obtain enough N. At
optimum level, there must be an equilibrium between Mineralization and
Immobilization
Anaerobic decay / decomposition
The main products of aerobic carbon mineralization are CO2, water, cells and
humus components. In the absence of O2 organic carbon is incompletely metabolized,
intermediary substances accumulate, and abundant quantities of CH4 and smaller
amounts of H2 are evolved. Energy yield during anaerobic fermentation is low, resulting
in fewer microbial cells per unit of organic carbon degraded. Consequently, organic
matter breakdown is consistently slower under total anaerobiosis than in environments
containing adequate O2. The rate in water logged soils is intermediate between the two
extremes.
When a soil is water logged or flooded there is a shift from aerobic to anaerobic
transformation. Formation and accumulation of organic acids viz., acetic, formic, butyric,
lactic and succinic acids appear too, these are frequently detrimental to root development.
Organic acids accumulate because of the fermentative character of the microflora of wet
soils. The anaerobic carbon transformation is thus characterized by the formation of
organic acids, alcohols, CH4 and CO2 as major end products.
Under anaerobic conditions, decomposition of organic residues takes place by the
activity of both mesophilic, thermophilic microorganism resulting in the production of
CO2, H, ethyl alcohol, and organic acids. Among mesophilis, bacteria are more active
than fungi or actinomycetes in cellulolytic activity. They belong to genus Clostridium
which are numerous in manure pits but rarely encountered in cultivated arable soils. In
compost pits both meso and thermopholic (bacterial and actinomycetes) are important in
the breakdown of cellulose substances.
The primary microbial colonisers initially breakdown the complex CH2O and
proteins into organic acids and alcohols. At a later stage, the methane bacteria which are
strict anaerobes begin to act upon the secondary substrate chiefly lactic and butyric acids
and ferment them into CH4 and CO2.
Degradation processes
(1) Cellulose is a CH2O composed of glucose units bound by -linkage at carbon 1 and 4
of the sugar molecule. The cellulose concentration of higher plants is never fixed and the
concentration. It is a polymer of glucose and is might abundant organic material in nature
changes with age and type of plants. Woody materials have more cellulose and succulent
tissues had poor, but the concentration increases as the plant matures. Cellulose
breakdown in soil is influenced by several environmental factors.
Aerobic organism convert cellulose to 2 major products : CO2 + cell substance,
but certain group releases small amount of organic acids. It is however resistant to
microbial decomposition. When cellulose is associated with pentosans (xylan, mannans)
it undergoes rapid decomposition. When associated with lignin, the decomposition rate is
very low. Degradation is by the enzymes that converts cellulose into glucose.
(Exoenzymes)-
Exoglucanase and Endoglucanase - glucosidase (cellulse complex)

EXO glucanase
Native cellulose Amorphos cellulose + cellobiose

Endoglucanase Endogluconase

-glucosidases

D- Glucose Cellobiose
(cellobiase)

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

Lignin --coniferyl ether -- coniferyl alcohol -- coniferyl aldehyde --

vanillin -- vannillic acid -- protocatechuic acid -- ring cleavage

Table. 1 Genera of microorganisms capable of utilizing different components of

organic mater as reported by several workers: F-Fungi; B-Bacteria;
A-Actinomycetes
Nature of substrate in
Genera of microorganisms
organic matter

Cellulose F Alternaria, Aspergillus, Chaetomium, Coprinus, Fomes,

Fusarium, Myrothecium, Penicillum, Polyporus,
Rhizoctonia, Rhizopus, Trametes, Trichoderma,
Trichothecium, Verticillium, Zygorynchus

B Achrombacter, Angiococcus, Bacillus, Cellfalcicula,

Cellulomonas, Cellvibrio, Clostrium, Cytophaga,
Polyangium, Pseudomonas, Sorangium, Sporocytophaga,
Vibrio

A Micromonospora, Nocardia, Streptomyces,

Streptosporangium

Hemicellulose F Alternaria, Fusarium, Trichothecium, Aspergillus,

 Rhizopous, Zygorynchus, Chateomium, Helminthosporium,
Penicillium, Coriolus, Fomes, Polyporus

B Bacillus, Achromobacter, Pseudomonas, Cytophaga,

Sporocytophaga, Lactobacillus, Vibrio
Clavaria, Clitocybe, Collybia, Flammula, Arthrobotrys,
Lignin F Cephalosporium, Humicola, Mycena, Pholioata
B Pseudomonas, Flavobacterium

Starch serves as a storage product of plants and is present in several specialized parts of
plant too. Starch is more readily broken down than cellulose by soil microbes.
Aerobically the microbes fully utilize starch to produce carbondioxide and low molecular
weight organic acids. Anaerobically fermentation takes place to yield methane, acetic,
lactic and butyric acids. Bacteria, actinomycetes and fungi have the physiological
capacity to utilize starch hydrolyzers, the common organisms found include Aspergillus,
Fomes, Fusarium, Rhizopus, Streptomyces, Nocardia, Micromonospora, Bacillus,
Chromobacterium, Flavobacterium, Micrococcus, Pseudomonas, Clostridium and
Cytophage. The enzymes involved in the hydrolysis are alpha and beta amylases.
Pectin substances are polysaccharides found in the constituents of middle lamella and in
the primary and secondary cell walls of plants. There are three types of pectic substances
viz., pectin, protopectin and pectic acid. Microbes are capable of utilizing pectic
substances as carbon and energy sources are abundant in soil and plant surface. Soil
bacteria such as Bacillus, Clostridium and Pseudomonas and facultative pathogens like
Erwinia more readily produce pectinases. Several fungi, especially the plant pathogenic
ones and Streptomyces are also known to produce pectinases.
Chitin is a polysaccharide whose basic unit is aminosugar. It is the structural component
giving mechanical strength to several plants and animals. This is one of the hardiest
organic molecules for microbial action in soil. Streptomyces and to some extent Nocardia
and Micromonospora readily decompose chitin. Among the fungi, some species of
common soil fungi such as Aspergillus, Penicillium, Mucor, Trichoderma also possess
the capability to break down chitin. Chitinase enzyme is involved in this
The protein content of waste products on hydrolysis by specific enzymes
produced by many varieties of microbes. The protein is split into polypeptides and finally
into simpler aminoacids. Besides protein, other nitrogenous substances which are added
to the soil are first converted into urea which is again decomposed to ammonia and
carbondioxide.
Oils and fats are hydrolysed to glycerol and fatty acids. Glycerol is readily
oxidized to carbondioxide and water and fatty acid produce certain resistant and toxic
substances.
Role of soil microorganisms in the decomposition of organic matter and humus
formation
The plant and animal residues are attacked by soil microbes and are decomposed.
In this process, some substances are volatilized, some are food material for microbes,
others go into soil water yet others are gradually transformed into humus. Humus is a
complex organic residue left in the soil. The nature of this residue depends upon the
composition of organic matter added, the microflora acting on them, and the soil
conditions in which the humus is formed.
To the mass of resistant residues of plant and animal matter in soil, the newly
synthesized microbial cell substances and lysed cell walls are also added. Because of the
constant addition and removal, the humus in soil is in a dynamic equilibrium. Humus is
highly colloidal and occurs in soil as a colloidal complex with clay particles. The
complex carries a negative charge and exhibits base exchange properties. On account of
this property, the humus clay complex is helpful in releasing nutrient elements to the
plants It acts as an acid or base towards soil minerals and mobilizes them for plant use.
Composition:
Humus can be defined as lingo protein complex containing
approximately
45 % lignin compounds
35% amino acids
11% carbohydrates
4% cellulose
7% Hemicellulose
3% fats, wax, resins
6% other miscellaneous substances, including plant growth substances and
inhibitors.
· Age and composition of the humus are dependent on its origin and environment.
· Bacterial and algal protoplasm contribute a good deal to the nutritive value of
 humus
Microbes in humus formation
Soil micro organism take part in humus formation. Some fungi such as Penicillium,
Aspergillus and actinomycetes produce dark humus like substances which serve as
structural units for the synthesis of humic substances.
Properties of humus
· The native organic fraction originates from two sources: the original plant debris
entering the soil and the micro organism with in the soil body. The micro
organism in soil body work upon the former and synthesize microbial protoplasm
and new compounds that become part of the organic fraction.
· Humus exist in a dynamic state
· Chemistry of humus is complex
· It has been pointed out that the organic fraction is derived from
Importance of humus
 The process that converts raw organic matter into humus feeds the soil population
of microorganisms and other creatures, thus maintains high and healthy levels of soil
life
 The rate at which raw organic matter is converted into humus promotes (when
fast) or limits (when slow) the coexistence of plants, animals, and microbes in soil.
 Effective humus and stable humus are further sources of nutrients to microbes, the
former provides a readily available supply, and the latter acts as a longer-term storage
reservoir.
 Decomposition of dead plant material causes complex organic compounds to be
slowly oxidized (lignin-like humus) or to break down into simpler forms
(sugars and amino sugars, aliphatic, and phenolic organic acids), which are further
transformed into microbial biomass (microbial humus) or are reorganized, and
further oxidized, into humic assemblages (fulvic and humic acids), which bind
to clay minerals and metal hydroxides.
 Humus is a colloidal substance, and increases the soil's cation exchange capacity,
hence its ability to store nutrients by chelation. While these nutrient cations are
accessible to plants, they are held in the soil safe from being leached
by rain or irrigation
 Humus can hold the equivalent of 80–90% of its weight in moisture, and therefore
increases the soil's capacity to withstand drought conditions
 The biochemical structure of humus enables it to moderate – or buffer –
excessive acid or alkaline soil conditions
 During the humification process, microbes secrete sticky gum-like mucilages;
these contribute to the crumb structure (tilth) of the soil by holding particles together,
and allowing greater aeration of the soil. Toxic substances such as heavy metals, as
well as excess nutrients, can be chelated (that is, bound to the complex organic
molecules of humus) and so prevented from entering the wider ecosystem
 The dark color of humus (usually black or dark brown) helps to warm up cold
soils in Spring.

Lecture 4
Carbon cycle
The term soil generally refers to the loose material of the earth surface and is the
region that supports the plant life. It consists of five major components such as mineral
matter, water, air, organic matter and living organisms. The proportion of these
components varies with soil type and other soil conditions. To maintain the level of these
components it is essential that they undergo a regular process of recycling. This process
of recycling through various transformations is brought about by different
microorganism.

Carbon cycle

Plant carbon Animal carbon

 C

Soil organic matter

A Microbial cell and decayed residues

D E

Carbondioxide
A- PS
B- Respiration / plant
C- Respiration / Animals
D- Autotrophs
E- Respiration / Microbial mineralization

The most important element in the biological realm and substance that serve as
the cornerstone of the cell structure is carbon. It constituents about 40-50% of all living
organisms. The ultimate source is the CO2 that is present in earth’s atmosphere at the
range of 0.03% and undergoes a cyclic change from an oxidized to reduced state.
Carbon (CO2) is constantly (reduced into organic carbon compounds) being fixed
into organic form by photosynthetic organisms (photosynthesis). Once bound, the carbon
becomes unavailable for use in generation of new plant life. It is thus essential for the
carbonaceous materials to be decomposed and returned to the atmosphere. It is estimated
that 1.3x1014 kg CO2 is fixed annually in the biosphere.
The carbon cycle revolves about CO2 and its fixation and regeneration. The green
plants utilize CO2 as their sole carbon source, and the carbonaceous matter synthesized
serves to supply carbon to other heterotrophic organisms and animals. Upon the death of
plants and animals, microbes assume a dominant role in carbon cycle. The dead tissues
are degraded and transformed into microbial cells and humus or soil organic fraction.
Further decomposition of these materials leads to the production of CO2 and once again
it is recycled.

Lecture 5
5. Nitrogen cycle – microbiology and biochemistry of mineralization,
ammonification, nitrification and denitrification
Biological availability of N, P and K is of considerable economic
importance, since they are the major plant nutrients derived from the soil. Of
the three, N stands out as the most susceptible one to microbial
transformations. This element is the key building block of the protein
molecule upon which all life is based, it is an indispensable component of the
protoplasm of plants, animals and microorganism.
Molecular N2 constitutes about 78% of the earth’s atmosphere but
it is chemically inert and cannot be utilized by more living organism, plant
animals and microorganism therefore depend on a source of combined N such
as ammonia, nitrate or organic N compounds for their growth.
Nitrogen undergoes a number of transformations that involve
organic, inorganic and volatile forms of nitrogen. In addition, a small part of
the large reservoir of N2 in the atmosphere is converted to organic compounds
by certain free living microorganism or by plant microbe association, that
makes the element available to plant growth. The nitrogen present in the
proteins or nucleic acids of plant tissue is used by animals. In the animal body,
the N is converted to other simple and complex compounds. Upon the death,
plants and animals undergo microbial decay and organic N is released as
ammonium, which is then utilized by vegetation or is oxidized to nitrate by
microorganisms. The nitrate from of N is mostly used by the plants or may be
lost by bacteria reduced to gaseous N2, which escapes to atmosphere, there by
completing the cycle. The Nitrogen cycle mainly includes transformations
 such as
· Nitrogen mineralization: In which N containing organic complexes are
decomposed and converted into inorganic compounds for use by plants
· N immobilization: In which N containing inorganic compounds are
assimilated
BIOLOGICAL NITROGEN FIXATION
Atmospheric nitrogen is acted on by certain micro- organism sometimes in
symbiosis with a higher plant, which can use it is as a N source for growth. This
process nitrogen fixation, results in the accumulation of new organic compounds
in the cells of responsible microorganisms. The N2 thus fixed reenters general
circulation when the newly formed cells are inturn mineralized.
By means of these reactions the subterranean microflora regulates the
supply and governs the availability and chemical nature of N in soil.

I. Nitrogen mineralization
The conversion of organic N to the more mobile, inorganic state is known
as nitrogen mineralization. As a consequence of mineralization, ammonium and
nitrate are
generated and organic N disappears. This takes place in distinct microbiological
steps.
1. Proteolysis:
Proteolysis or decomposition of proteins by microbes takes place in many
stages. Proteins consists of aminoacids units linked by peptide bonds. The
 proteins are broken down by microbes with the help of proteolytic enzymes. The
breakdown is done in two stages
Proteinases peptidases
Protein -------------- polypeptides ------------------ aminoacids
It is noted that protein is added to soil fungi first attack the
molecule and reduce it to polypeptides, and then bacteria becomes active and
compete with fungi in further breaking down the polypeptides to aminoacids.
Thus aminoacids formed either taken by plants or assimilated by
microbes or subjected to ammonification process.
Ammonification
The amino compounds are then deaminated to yield ammonia. In aerobic
conditions aminoacids are converted to ammonia by oxidative deamination or
hydrolytic deamination process. Depending on the enzyme system involved in the
hydrolytic process, the end products are obtained. In hydrolytic deamination
organic acids, alcohols, aldehydes, carbondioxide are obtained besides ammonia.
In oxidative deamination process, besides ammonia carbondioxide is liberated.
Ammonification usually occurs under aerobic conditions while under
anerobic conditions protein decomposition leads to conversion of ammonia into
amines and related compounds (eg) clostridium. The anaerobic decomposition of
protein called as putrefaction. These amines are subsequently oxidized in the
presence of O2 to release ammonia.
Ammonification process is brought about by the activity of a multitude of
microbial species. Besides proteins, other containing organic substances are also
attacked by microbes to yield ammonia. Many microbes utilize urea to liberate
ammonia. Members of the genera Bacillus, Proteus, Micrococcus, Sarcina,
Aerobacter quickly convert urea to ammonium carbonate and then to ammonia.
The enzyme involved is urease.

Plants and animals residues contain nucleic acids in small proportions.

When they reach soil they are also attacked by micobes. The hydrolysis of nucleic
acids by aerobic bacteria and fungi is accomplished by nuclease enzyme.
The ammonia produced by the reactions presented may be utilized in
several ways
 1. utilised by some organisms for their growth
2. utilised by higher plants
3. may be volatalised
4. Undergo nitrification process
(2) Nitrification
The biological oxidation of ammonium salts (in soil) to nitrites and the
subsequent oxidation of nitrites to nitrates is called as nitrification. i.e. the
biological convention of N in soil from a reduced to a more oxidized state, called
nitrification.
Nitrification occurs in two steps;
First ammonia is oxidized to nitrite.
2 NH3 + 1½ H2O2 NO2- + 2H+H2O-Nitrosofication
This change is brought about by chemoautotrophic bacteria of the genera
Nitrosomonas, Nitrosolobus, Nitrosococus, Nitrosospira. These bacteria obtain
their energy requirement by the oxidation of NH4+ to NO-2. Among the nitrifiers
Nitrosomonas are most important in soils.
Some heteotrophs involved in denitrification process
Streptomyces, Nocardia
Second step
Nitrite is further oxidized to nitrate
HNO2 + ½ O2 HNO3.
Organisms : Nitrobacter, Aspergillus, Penicillium, Cephalosporium.

Factors influencing the growth of nitrifying bacteria in soil:

Levels of ammonia and nitrite, aeration, moisture, temperature, pH and
organic matter. In acid soils – nitrification is poor. In Waterlogged soils –
deficient in O2 – not congenial for nitrification.
4. Denitrification
The convention of nitrate and nitrite into molecular N2 or nitrous oxide
through microbial processes is known as denitrification. Certain bacteria are
capable of using nitrate as the terminal electron acceptor under anaerobic
conditions. This is called nitrate respiration. As a consequence of nitrate
 respiration, NO3 is reduced to N2 gas or nitrous oxide. Denitirifcation leads to the
loss of N from the soil. It depletes N, and therefore it is not a desirable reaction.

The escape of molecular N into the atmosphere is also known as volatalization.

Denitirfication occur mostly in waterlogged anaerobic soils with a high organic
matter contents. Denitrification of bound nitrogen to gaseous N is mediated by
numerous species of bacteria, which normally use O2 as hydrogen acceptor
(aerobically) and, also use nitrates and nitrites (anerobically).
Anaerobic conversion of nitrate into molecular nitrogen is known as
nitrate respiration.
Bacterial genera which bring about denitirfication Pseudomonas,
Achromobacter, Bacillus, Micrococcus
2NO-3 +10 H N2 + 4H2O+ 2OH- (or)
2NO-2 +6 H N2 +2H2O +2OH- (or)
N2O + 2H N2 + H2O
Since nitrates are used as a source of electron acceptor, there is a net loss
of N from soil. This process is termed also as dissimilatory nitrate reduction.
Many soil bacteria like.
Thiobacillus denitrificans
Oxidize S (chemoautotrophically) and also reduce nitrate to
nitrogen 5S + 6 KNO3 + 2 H2O 3N2 + K2SO4 + 4KHSO4
(or)
5 K2S2O3 + 8 KNO3 + H2O 4N2 + 9 K2SO4 + H2SO4
General pathway of denitrification:
Nitrate is first reduced to nitrite, which is then transformed to nitrous oxide
(NO).
The nitrous oxide is converted to N with N O as an intermediate.
1 2 3 4
2 HNO3 2HNO2 2 NO N2O N2
The enzymes involved
1. Nitrate reductase
2. Nitrite reductase
3. Nitric oxide reductase
 4. Nitrous oxide reductase
- Fallow soils flooded with water are more congenial for denitrification than
well drained and continuously cropped soils.
- Though it is 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 and then ammonia.
HNO3 + 4H2 NH3 + 3H2O
Since organisms are able to obtain cellular N th’h ammonia
assimilation, the process is called as assimilatory nitrate reduction
II. Nitrogen immobilization
The process of microbial assimilation of inorganic nitrogen is referred as
immobilization. In contrast to mineralization microbial immobilization leads to
the biosynthesis of the complex molecules of microbial protoplasm from
ammonium and nitrate. Immobilization results in a marked depression of nitrogen
uptake by the plant.
The mineralization of organic N and the microbial assimilation of
inorganic ions proceeds simultaneously. Both mineralization and immobilization
takes place regardless of the % of N in the organic N in organic matter. On the
death of microorganism, the immobilized N is however released through
mineralization. It is also a loss of nitrogen. NO3 when accumulated in microbial
protoplasm it is referred as assimilatory NO3 reduction.

Lecture – 6
6. Biological nitrogen fixation – free living, associative, endophytic and symbiotic
microorganisms
BIOLOGICAL NITROGEN FIXATION
Fixation of elemental nitrogen in the atmosphere by the microorganism through
areductive process into ammonia is called as BNF. A variety of prokaryotic organism
have the ability to reduce the atmosphere N2, BNF accounts for about 70% of the total N
fixed in the biosphere. The ability to reduce atmosphere N 2 is restricted only to bacteria,
which are belonging to the diverse groups. The root nodule associations were the first to
be recognized for their ability to fix atmosphere N 2. Rhizobia are the first group of
organism realized for its potential of nitrogen fixation.

Nitrogen for biosynthesis of cell material can be obtained from organic sources
such as amino acids, or inorganic sources. Microbes most commonly use ammonia and
nitrate, but some bacteria can reduce N 2 gas to synthesize organic nitrogen. When
ammonia concentrations are high, the enzyme glutamate dehydrogenase can be used to
incorporate ammonia into organic compounds. However, at low concentrations, the
glutamine synthetase-glutamate synthase system is more efficient. The net reaction
converts α-ketoglutarate and ammonia to glutamate, with the consumption of one ATP.
The activity of glutamine synthetase is controlled in response to ammonia concentration.
At high concentration, the protein is covalently modified by adenylylation; this reduces
its catalytic activity.

2H+ 2H+ 2H+

N=N HN = NH H2N - NH2 2NH3
(Dinitrogen) 2e- (Diimide) 2e- (Hydrazine) 2e- (Ammonia)
Biological Nitrogen Fixation (BNF)
 A number of microorganisms are able to use molecular nitrogen in the atmosphere
as their source of nitrogen for conversion of molecular nitrogen into ammonia through
nitrogenase enzyme produced by microorganisms is known as nitrogen fixation. The
enzymes hydrogenases reversibly catalyse the reduction of H2 ion in to molecular H2
which nitrogenase reduces molecular N2 into NH3 in the presence of molecular H2.
N2 + 6e- +12 ATP + 12 H2O 2 NH4+ + 12 ADP + 12 Pi + 4H+

Mechanism of Nitrogen fixation:

Nitrogen fixing bacteria contain nif gene is responsible for nitrogen fixation
which produce nitrogenase enzyme. The enzyme consists of tow protein Mo-Fe protein
(MW 2, 20,000-2, 70,000) and Fe protein (MW 55000-68,800). The nitrogenase enzyme
complex contains MO-Fe protein (nitrogenase) and Fe protein (Dinitrogen reductase),
ATP molecule, a strong reducing agent ferridoxin or flavodoxin. The Fe protein reduces
dinitrogen (N=N) into two moles of NH3. Active nitrogenase can be reconstituted by the
addition of purified MO-Fe and Fe protein of different microorganisms. Proteins of K.
pnemoniae, B. polymyxa, BGA and photosynthetic bacteria have been confirmed to
reconstitute active nitrogenese capable of reducing acetylene to ethylene.
Nitrogen fixation is a process where some prokaryotic organisms reduce N 2 to
ammonia, which is then incorporated into glutamate by the enzymes described above. A
multimeric enzyme called nitrogenase catalyzes nitrogen fixation. One component of the
complex is the protein dinitrogenase. Together with a cofactor containing iron and
molybdenum atoms, this protein reduces nitrogen gas to ammonia. A second component,
dinitrogenase reductase is necessary to transfer electrons from ferredoxin to
nitrogenase. The reduction of one N2 molecule requires the hydrolysis of 15-20 ATP
molecules. ATP hydrolysis lowers the reduction potential of nitrogenase reductase
sufficiently for the reaction to proceed. Eight electrons are consumed in the reaction,
even though theoretically only six are required. The other two are lost in a molecule of
H2. Nitrogenase reductase is inactivated by O 2. Therefore, in aerobic bacteria, there must
be oxygen-deficient microenvironments in the cell for nitrogen fixation to proceed. The
activity of dinitrogenase is most conveniently assayed by adding acetylene. The enzyme
reduces this triple-bonded molecule to form ethylene, which can be measured by gas
chromatography. The N2 fixing bacteria having nitrogenase enzyme is capable to convert
triple bonded N2 compounds into two moles of ammonia or acetylene convert to ethylene.
2H+
CH = HC H2C = CH2
Acetylene Nitrogenase Ethylene

• Nitrogen reduction starts with the transfer of single electro from flavodoxin to
small Fe protein subunit. At this stage two ATP molecule combine with two Mg 2+
ions to form complex, Mg ATP attaches to Fe protein energize it to transfer
electron from the iron atom in Fe protein to Mo Fe protein. The Mo Fe protein
becomes unstable and to counter balance this electro an H + ion formed by
dissociation of water comes to attch at Mo atom of Mo Fe protein.
 • The second electro in again balanced by another H+ ion when the 3rd electron is to
transferred to Mo Fe protein. The ion H + are displaced by N2 leading to the
evolution of one molecule of H2.
• The 3rd again balance by attachement of one H+ ionto nitrogen is HN=NH. This
process of electron transfer is contained till 8 electron transfer there by reducing
to NH3.
• The net 8e- and 8H+ are involved in reducing N2 to two molecules of NH3. One
electron utilizes two moles of ATP. Hence 16 ATP are used the H 2 evolution is
mediated by transfer of new two electron thereby leading to loss of four ATP
molecules by hydrogen evolution is responsible for the enzyme hydrogenase.
• It is obvious that the process of N2 reduction consumes 12 ATP and it can catalyse
both uptake and evolution of H2. The H2 is transferred by hydrogenase in
Azotobacter at recycled. Bacteria and cyanobacteria show photoevolutio of H2.

Nitrogen fixing bacteria are classified according to their mode of fixation.

1. Non-symbiotic or free-living nitrogen fixer:
Free-living nitrogen fixers are capable of fixing mol. N 2 to cellular nitrogen
independently of other living organism. Ex. Azotobacter, Beijerinckia and Derxia
Azotobacter - Aerobic
Beijerinckia - Aerobic
Clostridium – Anaerobic
Cyanobacteria (Blue green algae)
a. Aerobic bacteria:
Azotobacter, Beijerinckia, Derxia, Achromobacter, Arthrobacter
Species of Azotobacter
A. chroococcum
A. vinelandii
A. beijerinkii
A. paspali
A. agilis
A. insignis
A. macrocytogens
b. Facultative anaerobic bacteria:
 Aerobacter, Kelbsiella and Pseudomonas sp
c. Anaerobic bacteria:
Clostridium, Chlorobium, Chromatium, Rhodopseudomonas, desulfovibrio and
Methanobacterium
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
2. Associative symbiotic nitrogen fixer:
The bacteria reside in or on the roots or plant parts which fix nitrogen.
Azospirillum and Herbaspirillum species are associated symbiotic nitrogen fixer. Ex
Azospirillum sp. A.amazonens is an acid tolerant whereas A. halopraeferans is a salt
tolerant.
Species of Azospirillum
A. lipoferum
A. brasilense
A. amazonense
A. halopareferans andA. irkense
3. Endophytic nitrogen fixer / Endophytes:
Those organisms living inside the leaves, root or stem are called as endophytes.
Ex. Burkholderia sp. Gluconoacetobacterdiazotrophicus
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
B mexicana
A. nilotica
Factors affecting N2 fixation
1. Presence of nitrate or ammonium : More N2, No, N2 fixation
2. Presence of certain inorganic substances
Ca, Co, Mo – influence N2 fixation along with P
3. Availability of energy source – addn. of C source increase N2 fixation
4. pH : Neutral – favours Azotobacter – Acidic- Beijerinkia
5. Soil moisture : Adequate is good for fixation
6. Temperature : Mesophilic – 30°C.
The energy requirement for BNF is very high and it is a major factor determines
the amount of N2 fixed. In, Azotobacter the rate depends on amount of available carbon.
In symbiotic N2 fixers since photosynthesis is the ultimate source of energy the rate of
N2 fixation is influenced by the factors that effect photosynthesis and rate of
translocating photosynthates to the N2 fixing system.
Nitrogenase protection mechansims
1. Leghaemoglobin scavenges O2 to protect nitrogenase in legume rhizobium
symbiosis
2. Confirmatory protection in Azotobacter as well as the higher respiratory rate.
3. Thick walls of Heterocyst protect O2 in BGA, since Nitrogenase are present in the
heterocyst.
4. Microaerophilic nature in Azospirillum
Losses of N by non biological ways
Leaching
20 to 50%of fertilizer N. The most striking loss of N in rice soils where more than
half of the fertilizer N applied get lost through leaching.
Volatalization
Another factor is the volatalizaiotn of ammonia in soil 5-20%.
Fixation of ammonium in soils is the minor contributory factor to overall loss of N2
available for plant growth.
Such losses of N by physical causes and by nitrification and denitirfication
process can be controlled by the application of certain chemicals. Some chemicals have
been designed to control the rate of release of nutrient from nitrogenous fertilizers, while
others retard nitrification in soil by controlling the activity of nitrifying bacteria.

a. Controlled release fertilizers

Urea from isobutyeldene
diurea Crotonilidene Fertilizers, sparingly soluble in water can
diurea S coated urea regulate the release of N from fertilizers
b. Nitrification inhibitors
These are substituted with pyridines, pyrimidines, anilines and isothiocyanates,
Examples
1. 2 chloro 6 (tricholormethyl) – pyridine – (N serve )
2. 2 amino 4 chloro 6 methyl pyridine –( AM.)
N serve inhibits the growth of Nitrosomonas europea and N. agilis.
The seeds of neem conain lipid associates act as nitrification inhibitors and there
by increases the efficiency of urea fertilizers.
Ammonia assimilation
N2 fixation results in NH4 formation which reacts with organic acids and form
amino acids which is mediated by ammonia assimilating enzyme.
GS – Glutamine synthetase
GOGAT – Glutamate synthese
GDH – Glutamate dehydrogenase
Genetics
Nif genes are responsible for N2 fixation.
Nif genes are 22, which are located in 7 or 8 clusters.

Lecture 7
7. Nodulation in Rhizobium- legume and Frankia – actinorhizal symbioses.
Biochemistry of nitrogen fixation
Symbiotic nitrogen fixers:
Species of Rhizobium and Bradyrhizobium make specific associations with
leguminous plants to form root nodules capable of nitrogen fixation. Because the
availability of combined nitrogen in soil often limits the growth of agricultural crops,
these associations are economically important. The association of two organisms
mutually benefited. Rhizobium symbiotic association with legume roots whereas Frankia
symbiotically associated with casuarina. Rhizobium is predominant symbiotic N2 fixing
bacterium. Boussingault showed that leguminous plant could fix atmosphere N 2. Then
Hellriegel and Wilfarth proved that N2 fixed by certain bacteria living in root nodules of
leguminous plants. Later isolates in pure culture by Beijerinck. Winogradsky isolated
Clostridium pasteurianum is an anaerobic N2 fixer. Beijerinck isolated Azotobacter as a
free-living aerobic N2 fixing organism.
Symbiotic nitrogen fixers
Rhizobium (Rhizobium – legume association)
Bradyrhizobium (Bradyrhizobium – soybean association)
Azorhizobium (Azorhizobium- Sesbania rostrata association)
Anabaena azollae (Azolla – Anabaena association)
Frankia (Frankia – Casuarina association)
Cross inoculation groups of rhizobium (CIG)
It (CIG) refers the groups of leguminous plants that will develop effective
nodules. When inoculated with the rhizobia obtained from the nodules from any member
of that legume group.

S. Cross Inoculation
Rhizobium sp. Host it can Nodulate
No. Group
I. Rhizobium Pea Pea, lentils, vicea
Rhizobium
leguminosarum bv.
Viceae
Rhizobium Bean Phaseolus spp.
leguminosarum bv.
Phaseoli
Rhizobium Clover Trifolium spp.
leguminosarum bv. Trifoli
Rhizobium meliloti Alfalfa Alfalfa, clover, fenugreek
Rhizobium loti Lotus Trifoli, lupine
II. Bradyrhizobium Soybean Soybean
B. japonicum, Rhizobium
fredii
Rhizobium sp. Cowpea group Vigna, Arachis, Cajanus,
Dolichus, Sebania, Acacia,
Prosopsi, Green gram and
black gram
Rhizobium sp. Chickpea group Chickpea
III. Stem nodulating Nodulates Sesbania rostrata
IV. Azorhizobium Fast growing
Sinorhizobium soybean nodulator
V. Photorhizobia Nodulants
Aeschynomene sp.
 In the root nodule, it is the bacterium that contains the enzyme nitrogenase. The
cells do not normally produce this enzyme when they are not in root nodules. The O2
level in the nodule is controlled by a plant protein, leghemoglobin. This control is
necessary because the bacteroids carry out aerobic respiration to provide energy for
nitrogen fixation, but nitrogenase is inactivated by oxygen. Plant species are nodulated by
particular species of Rhizobium or Bradyrhizobium. This specificity resides in the early
stages of nodule formation, when the bacterium attaches to the root hair of the plant.
Recognition is mediated by plant lectins, which bind to a bacterial surface
polysaccharide. After specific binding to the root hair tip, nodulation involves (1) entry of
the bacterium into the root hair and formation of an infection thread, (2) travel to the
main root and infection of tetraploid plant cells, and (3) repeated division of the
tetraploid cells and transformation of the bacterial cells into bacteroids capable of
nitrogen fixation.

The plant supplies organic acids produced via photosynthesis to the bacteroid
across the peribacteroid membrane. These are used to synthesize ATP and reducing
power necessary for the reduction of N2 to ammonia. In return, the ammonia generated by
nitrogenase is incorporated by the plant into glutamine and other organic nitrogen
compounds. These are translocated to plant tissues for use in growth. There are other
associations between plants and nitrogen-fixing microbes. In rice paddies, the fern Azolla
contains cyanobacteria that possess specialized cells called heterocysts that fix nitrogen.
Alder trees contain root nodules with the actinomycete Frankia. A looser association
occurs between some tropical grasses and the bacterium Azospirillum lipoferum.
Ammonia assimilation
 • N2 fixation results in NH4 formation which reacts with organic acids and form
amino acids which is mediated by ammonia assimilating enzyme.
• GS – Glutamine synthetase
• GOGAT – Glutamate synthese
• GDH – Glutamate dehydrogenase
Genetics
• Nif genes are responsible for N2 fixation.
• Nif genes are 22, which are located in 7 or 8 clusters.
Nitrogenase protection mechansims
1. Leghaemoglobin scavenges O2 to protect nitrogenase in legume rhizobium symbiosis.
2. Confirmatory protection in Azotobacter as well as the higher respiratory rate.
3. Thick walls of Heterocyst protect O 2 in BGA, since Nitrogenase is present in the
heterocyst.
4. Microaerophilic nature in Azospirillum.

Lecture 8
8. Phosphorus cycle and microbial transformation of phosphorus - phosphate
solubilizer and mycorrhizae
I. Phosphorus cycle
Phosphorus is only second to N2 as an inorganic nutrient required by both plants
and micro organisms. Phosphate constitutes nearly 0.1% of the earth’s crust. They occur
in soil in inorganic and organic forms.
The inorganic forms are derived from parent rocks or through fertilizers
application and manuring with bone meal. They are soluble in water when present as
phosphates of Na, K, Ca, Mg etc.
The organic phosphorus containing compounds are derived from plants and micro
organisms and are composed of nucleic acids, phospholipids, lecittin, phytin and related
compounds.
· Phosphorus in phytin, phospholipids and nucleic acids is found as phosphates
· Phytin is the calcium – magnesium salt of phytic acid
· Phospholipids are compounds in which phosphate is combined with a lipid,
contained 10% of cell phosphorus.
· Inorganic polyphosphates are quite abundant in certain fungi
 · In soil, from15-85% of the total P is organic. Soils rich in organic matter contain
abundant organic P.
· Ratios of organic C to P of 100 to 300:1 N: organic P = 5 to 20: 1
In cultivated soil P present in abundant about 1100 kg/ha but most of them as not
available to plants; only about 1% of the total P is in available form.
Microorganisms bring about a number of transformations of the element.
- Altering the solubility of inorganic compounds of P
- Mineralization of organic compounds with the release of inorganic phosphate
- Converting the inorganic, available anion into cell components, an immobilization
process (analogous to that occurring with N)
- Bringing about an oxidation or reduction of inorganic P compounds
Particularly, important to P cycle are the microbial mineralization and
immobilization reactions.
Solubilization of inorganic phosphorus
Insoluble inorganic compounds of P are largely unavailable to plants, but many
micro organism can bring the PO4 into solution. P solubilizing are 105 to 107 / g soil.
Eg: Pseudomonas striata, Microoccus Bacillus sp., Fusarium, pergillus sp, Solubilises
calcium salts, iron, aluminum, magnesium manganese phosphate.
· P is solubilized by the production of organic acids. The acids convert Ca3 (PO4)2
to di and monobasic phosphates and releases P to plants.
· Solubilization of phosphates by plant roots & micro organism is dependent on soil
pH. In neutrals and alkaline soils having a content of calcium, precipitation of
CaPO4 takes place. Micro organism and plant root readily dissolve such PO4 and
make them available to plants.
· On contrary, acid soils are generally poor in Ca ions and phosphates and
precipitated in the form of ferric or aluminum compounds which are not soluble.
There, it is solubilized by the addition of PO4 solublizing micro organism.

· Phosphorus exists mainly as apatides, with the basic formula M10 (PO4)6 X2.
Commonly the mineral (M) is Ca, less often Al or Fe. The anion (X) is either F -,
Cl-, OH- or CO2-3. Diverse combinations of M and X results in 200 forms of P.
Mineralization of organic phosphorus
Organic form of P is the larger reservoir of P in soil. By the action of bacteria,
fungi and actinomycetes, bound element in remains of the vegetation and in soil organic
matter is made available to succeeding generations of plants.
Among the organic phosphours compounds, lecithin, nucleic acids and phytin
occupy a prominent place. Lecithin contains 9.39 % P2O5, 1.6% N and 65.36% C.
It is a process of convention of organic forms of phosphorus into inorganic
available forms of P a highly significant correlation is observed between the rates of N
and P convention to inorganic forms.
· Mineralization is favoured by warm temperature, with the thermophilic range
being more favourable than mesophilic range.
· Neutral pH increases PO4 release, which favours microbial metabolism
· Quantity of substrate ie presence of organic P. If more P, more of mineralization
· Mineralization is mediated by the enzymes called phosphatases. These enzymes
cleave phosphorus from more frequently encountered organic substrates.
Phytases liberates PO4 from phytic acid or its Ca-Mg, Salt, Phytin. They remove
PO4-s, one at a time, yield penta – tetra, di- and mono PO4 and then finally free
inositol.
Bacillus, Pseudomonas, Aspergillus, Penicillium, Rhizopus can synthesize this
enzyme. Mycorrhizal (fungi) are also able to mineralize the organic forms of P
and increases P uptake by the plants.
4. Immobilization
Process of assimilation of P into microbial nucleic acids, phospholipids or other
protoplasmic substances is called immobilization. It leads to the accumulation of non
utilizable forms of the element.
P accounts for 0.5-1.0% of fungus mycelium and 1.0 to 3.0% of the dry weight of
the bacteria and actinomycetes.
(4) Oxidation reduction reactions
Biological oxidation of reduced phosphorus compounds into oxidized state.
Phosphite (HPO3=) is oxidized to phosphate. A number of hetertrophic (bacteria),
(fungi) & (actinomycetes) utilize phosphite as sole P source. Hypophosphites (HPO2 =)
can also be oxidized to phosphate by heterotrophs.
HPO3= HPO4=
HPO2= HPO4=
Reductive process, reductive pathway has also been functioned. PO4 is reduced
to phosphite and hypophosphite.
H3PO4 H3PO3 H3PO2
Clostridium. butyricum, E. coli form phosphite and hypophosphite from
orthophosphate. It is biochemically analogue to the process of denitirification. Only little
information is available about this process.
P exist in an organic form in the protoplasm on the death of living organism, this
(P) is changed to inorganic phosphoric acid. This is soon converted into insoluble salts of
Ca, Fe, Mg and Al. Phosphorus thus alternates between organic and inorganic, and
soluble and insoluble forms. In soluble P is solubilized by various acids produced by
micro organism.
Microbial activities involved in the cycling of C, N and P are absolutely essential
for maintenance of soil fertility.

MYCORRHIZA
Mycorrhizal fungi have existed since the first plants appeared on dry land more
than 450 million years ago. They form a close symbiotic relationship with plant roots.
 They are called mycorrhizae from the Greek "mukés", meaning fungus, and "rhiza,"
meaning roots. Mycorrhizae form a network of filaments that associate with plant roots
and draw nutrients from the soil that the root system would not be able to access
otherwise. This fungus-plant alliance stimulates plant growth and accelerates root
development.
In a mycorrhizal association, the fungus colonizes the host plant's root tissues,
either intracellularly as in arbuscular mycorrhizal fungi (AMF or AM),
or extracellularly as in ectomycorrhizal fungi. The association is generally mutualistic,
but in particular species or in particular circumstances, mycorrhizae may be
variously pathogenic in the host plants.
Benefits of mycorrhizae
 Produce more vigorous and healthy plants
 Increase plant establishment and survival at seeding or transplanting
 Increase yields and crop quality
 Improve drought tolerance, allowing watering reduction
 Enhance flowering and fruiting
 Optimize fertilizers use, especially phosphorus
 Increase tolerance to soil salinity
 Reduce disease occurrence
 Contribute to maintain soil quality and nutrient cycling
 Contribute to control soil erosion
Mycorrhizas are commonly divided into ectomycorrhizas and endomycorrhizas. The
two types are differentiated by the fact that the hyphae of ectomycorrhizal fungi do not
penetrate individual cells within the root, while the hyphae of endomycorrhizal fungi
penetrate the cell wall and invaginate the cell membrane. Endomycorrhiza
includes arbuscular, ericoid, and orchid mycorrhiza, while arbutoid mycorrhizas can be
classified as ectoendomycorrhizas. Monotropoid mycorrhizas form a special category.
AM fungi
Endomycorrhizas are variable and have been further classified as arbuscular, ericoid,
arbutoid, monotropoid, and orchid mycorrhizas  Arbuscular mycorrhizas, or AM
(formerly known as vesicular-arbuscular mycorrhizas, or VAM), are mycorrhizas whose
hyphae enter into the plant cells, producing structures that are either balloon-like
(vesicles) or dichotomously branching invaginations (arbuscules). The fungal hyphae do
not in fact penetrate the protoplast (i.e. the interior of the cell), but invaginate the cell
membrane. The structure of the arbuscules greatly increases the contact surface area
between the hypha and the cell cytoplasm to facilitate the transfer of nutrients between
them. Arbuscular mycorrhizas are formed only by fungi in the division Glomeromycota.
Arbuscular mycorrhizas are found in 85% of all plant families, and occur in many crop
species.
Ectomycorrhizas, or EcM, are typically formed between the roots of around 10% of
plant families, mostly woody plants including
the birch, dipterocarp, eucalyptus, oak, pine, and rose families, orchids, and fungi
belonging to the Basidiomycota, Ascomycota, and Zygomycota. Thousands of
ectomycorrhizal fungal species exist, hosted in over 200 genera. Ectomycorrhizas consist
of a hyphal sheath, or mantle, covering the root tip and a Hartig net of hyphae
surrounding the plant cells within the root cortex. In some cases the hyphae may also
penetrate the plant cells, in which case the mycorrhiza is called an ectendomycorrhiza.
Outside the root, Ectomycorrhizal extramatrical mycelium forms an extensive network
within the soil and leaf litter.
Ericoid mycorrhiza
Ericoid mycorrhizas are the third of the three more ecologically important types.
They have a simple intraradical (grow in cells) phase, consisting of dense coils of hyphae
in the outermost layer of root cells. There is no periradical phase and the extraradical
phase consists of sparse hyphae that don't extend very far into the surrounding soil. They
might form sporocarps (probably in the form of small cups), but their reproductive
biology is little understood.
Ericoid mycorrhizas have also been shown to have
considerable saprotrophic capabilities, which would enable plants to receive nutrients
from not-yet-decomposed materials via the decomposing actions of their ericoid partners
Arbutoid mycorrhiza
This type of mycorrhiza involves plants of the Ericaceae subfamily Arbutoideae.
It is however different from ericoid mycorrhiza and resembles ectomycorrhiza, both
functionally and in terms of the fungi involved] The difference to ectomycorrhiza is that
some hyphae actually penetrate into the root cells, making this type of mycorrhiza
an ectendomycorrhiza
Monotropoid mycorrhiza
This type of mycorrhiza occurs in the subfamily Monotropoideae of
the Ericaceae, as well as several genera in the Orchidaceae. These plants
are heterotrophic or mixotrophic and derive their carbon from the fungus partner. This is
thus a non-mutualistic, parasitic type of mycorrhizal symbiosis.
Orchid mycorrhiza
All orchids are myco-heterotrophic at some stage during their lifecycle and form orchid
mycorrhizas with a range of basidiomycete fungi.  Their hyphae penetrate into the root
cells and form typical coils.

Lecture 10
10. Sulphur cycle - sulphur oxidizers; microbial transformation of K, Zn and Si.
Role of soil enzymes in nutrient transformation
The inorganic components of soil sulphur in the form SO 4 and constitutes minor
portion of the total sulphur content of the soil. The soil sulphur is in the form of organic
sulphur is metabolized by microorganisms to make it available in an inorganic state for
plant nutrition. Organic sulphur bound in protein of vegetables and animal origin,
protoplasm of microorganisms in the form of sulphur containing amino acids (cysteine
and methionine) and B-vitamins. The conversion of organically bound sulphur to the
inorganic state is termed as mineralization which is mediated through microorganisms.
The sulphur is released either absorbed by plants or escapes to the atmosphere in the form
of oxides. In absence of O2 especially water logged soil, certain microorganism produce
H2S from organic sulphur.
Transformation of sulphur in soil:
• Decomposition of organic sulphur into inorganic sulphur compounds through a
process of mineralization.
• Assimilation of sulphur into the protoplasm of microorganisms a process referred
as immobilization.
• Oxidation of inorganic sulphur into elemental sulphur.
• Reduction of SO4
Bacteria are capable of oxidizing inorganic sulphur compounds either aerobic or
anaerobic. It varies from Thiobacillus to Beggiatoa, Thiothrix and Thioploca and other
sulphur oxidizer (Aspergillus, Penicillium and Microsporeum). Thiobacillus produces
H2SO4 when elemental sulphur added to the soil as a result pH of soil may fall as 2.0
which control potato scab caused by Streptomyces scabies and wort of sweet potatoes
caused by S. ipomea in sulphur amended soils. Similar effects were confirmed that
inoculation of soil with Thiobacilli after addition of sulphur in acid soils.
The application of sulphur coupled with Thiobacilli inoculation rendering alkali
soils fit for cultivation. The formation of H 2SO4 in soil following, addition of elemental
sulphur augments increasing and nutrients mobilization by phosphates, potassium,
calcium, manganese, Al and Mg. Mn deficiency in soil can be corrected by sulphur
application. Thiobacilli can also be used in manufacture of ‘Biosuper’ a form of organic
fertilizer is a mixture of rock PO4 and sulphur is inoculated with Thiobacillus thioxidans.
The H2SO4 dissolves the PO4 enhances phosphorus in plants.
2S + 2H2O + 3O2 2H2 SO4
 Sulphate reducing bacteria like Desulphovibrio desulfuricans is reducing
inorganic sulphate into H2S may diminish availability of sulphur in plants and influence
the agricultural production.
4H2 + Ca SO4 H2S + Ca (OH)2 + 2H2O
H2S resulting from SO4 reduction and amino acid decomposition is oxidized to
elemental sulphur
CO2 + 2H2S (CH2O)x + H2O + 2S
A heterotrophic microorganism oxidizes substrate, depleting the O2 supply and
creates anaerobic condition. Organic acid serves an electron donor for the reduction of
SO4 and sulphides to H2S by Desulfotomaculum. Purple and green sulphur bacteria
(Chromatium and Chlorobium) use H2S as electron donor to reduce CO2. Aerobic sulphur
oxidizing Thiobacillus spp oxidize sulphur into SO4.
Organic matter + O2 Organic acid + CO2
Organic acid + SO42- H2S + CO2
CO2 + H2S (CH2O)x + S
Reduced sulphur compound SO42- + accumulation of S

Lecture 11
11. Importance of soil and plant associated microorganisms – rhizosphere,
spermosphere, phyllosphere, epiphytic and endophytes

RHIZOSPHERE AND PHYLLOSPHERE MICROFLORA

The term ‘Rhizosphere’ was introduced by Hiltner (1904) to denote that region of
the soil which is subject to influence of plant roots. The region in the vicinity of roots can
be distinguished into many microhabitats. Rhizosphere means the relationship between
soil microorganism and plant root. Rhizosphere is characterized by greater
microbiological activity than soil away from the plant roots. Rhizoplane is refers that
root surface together with the closely adhering soil particles.
Rhizosphere Effect:
The overall influence of plant roots on soil microorganisms. It can be
demonstrated that rates of metabolic activity of the rhizosphere microorganisms are
different from those of the non-rhizosphere soil. The enzymes of plant and microbial
origin present in the rhizosphere catalyse breakdown of organic materials. These enzymes
include oxidoreductase, hydrolase, lyases, transferase, dehydrogenase and ureases.
R: S ratio:
It is used to find out the degree or extent of influence of the plant roots on soil
microorganisms. The ratio between microbial population in rhizosphere (R) and in soil
(S), the R: S ratio can be calculated by
Number of microorganism in the rhizosphere soil
R: S ratio =
Number of microorganism in the non-rhizosphere soil
It is used to differentiate group of organism in rhizosphere in order to compare the
differential stimulation of microorganisms by the roots. When greater rhizosphere effect
with bacteria ranges from 10 to 20 times more than with actinomycetes or fungi,
rhizosphere effect decline sharply with increasing distance. The number of bacteria
increases in legume rhizosphere than non legume rhizosphere. The rhizosphere organisms
are more when soil moisture is low. Manuring of the soil directly influence of soil
population but the effect on rhizosphere population is more through the plants which get
the benefit of the fertilizer application. Well fertilizer applied plants which stimulates
more organisms in rhizosphere than poorly fertilized plants.
The qualitative differences in the micro flora of the rhizosphere as compared to
the soil, Gram negative, rod shaped, non-sporing bacteria are more in rhizosphere than in
the soil whereas gram positive, cocci and aerobic spore forming bacteria are less in
rhizosphere. In rhizosphere more motile bacteria, chromogenic and proteolytic types,
ammonifiers, fermentors and cellulose decomposers, there is more oxygen consumption
and carbon di oxide productions in the region.
The qualitative and quantitative changes undergone by the soil micro flora in
the root region due to changes in available food supply especially bacteria which
demands amino acids and growth factors such as thiamine and biotin. The corn roots in
acid soil yielded Trichoderma and roots from alkaline soils mainly contained Penicillium.
The growing roots stimulated more nitrate reducers, gelatin liquefiers and starch
hydrolysers. The significant rhizosphere effect in rice plants with application of organic
manures and fertilizers to the paddy soil caused a reduction in the rhizosphere population
due to a general increase in population in soil.
Importance of Rhizosphere in soil:
Biological Nitrogen fixation (BNF) in rhizosphere:
 The Rhizosphere of the plants harbours nitrogen-fixing bacteria of the families
Azotobacteriaceae, Spirilaceae, Enterobacterioceae, Bacillaceae, Psudeomonodaceae and
Achromobacteriaceae. The colonization of Azotobacter limited in the rhizosphere and the
root surface due to acidity caused by root exudates while Azospirillum has been noticed
extensive intrusion within root tissues. Sporangia of Frankia dispersed in the rhizosphere
of casuarina seedlings. Azotobacter chrococcum and A. paspali are known to produce
gibberellins and cytokine like substances and these species of nitrogen fixing bacteria
which colonizers of rhizosphere of grasses. This fixation is a cumulative effective effect
of both free living as well as symbiotic such as BGA, Rhodopseudomonas, Azotobacter,
Beijerinckia, Methylomonas, Clostridium, Desulfovibrio, Klebseilla, Enterobacter,
Flavobacterium, Pseudomonas, Azospirillum and Rhizobium. The bulk of symbiotic
nitrogen fixing in rice rhizosphere of flooded soil comes from root and stem nodules of
leguminous green manure when they are ploughed in to the soil.
Nitrogen fixing bacteria occur in the rhizosphere, stalks and phyllosphere of
sugarcane plants and also reported inside the root cells as an endophytes. The following
BNF have been isolated from the root region of sugarcane Azotobacter vinelandii,
Klebsiella pnemonia, Bacillus polymyxa, Azospirillum brasilense, Derxia gummosa,
Enterobacter cloacae and Erwinia herbicolla. BNF may excrete growth factors or help in
lower oxygen tension are perpuated through continuous practice or vegetative
propagation involving planting of sugarcane sets in the organically rich soil. BNF can be
found in sugarcane rhizosphere even up to the depth of 120 cm.
Alteration of rhizosphere micro flora:
Microbial seed inoculants such as Azotobacter, Beijerinckia, Rhizobium or P-
solubilizing microorganisms may help in the establishment of beneficial microorganism
in the rhizosphere or in the immediate vicinity of growing roots. Azotobacter in wheat
rhizosphere increased upon artificial seed inoculation indicating the efficiency of
bacterization by means of altering and improving the rhizosphere to encourage the plant
growth. Microbial population changes in the rhizosphere micro flora by soil amendments,
foliar application of nutrients and artificial inoculation of seed or soil with preparation
containing live microorganisms especially bacteria which called as Bacterization.
The effects of N, P and K addition on rhizosphere micro flora may results
increase as well as decrease in R: S ratio. Translocation of photosynthates from leaves to
roots is a normal metabolic activity of plants. The recovery of compounds such as 2,3,6-
trichlorobenzoic acids, 2,4,5-T streptomycin, -methoxyphenylacetic acid, 2-methoxy-3-6,
dichlorobenzoic acid, sprayed on leaves from root exudates of plants. Foliar sprays of
urea are known to alter the number and nature of microorganisms. Changes in the
rhizosphere micro flora by foliar sprays of antibiotics, growth regulators, biopesticides
and inorganic nutrients may serves as a new tool in biological control of root diseases.
Association and Antagonistic activities in the Rhizosphere:
The dependence of one microorganism upon another for extra cellular products
chiefly amino acids and growth promoting factors can be regarded as associative effect.
The cellular extract of bacteria, fungi and algae increase the growth of other
microorganism. An increase in amino acid content in plants grown in soil inoculated with
specific microorganism like Azotobacter, Arthrobacter, Pseudomonas and
Agrobacterium are produce B-vitamins, auxins, gibberellins and antibiotics as a result
increased germination. There is an increase in the exudation of organic acids, amino acids
and monosaccharides by plant roots in the presence of microorganisms in rhizosphere. Its
also influence root hair development, mucilage secretion and lateral root development of
several plants. The fungi inhabiting the surface of the root influence the amount of
substance absorbed in to the root system.
The microorganism secretes antibiotics and the resultant biological inhibition of
growth of other susceptible microorganisms in soil. Antagonistic effects may extent to
inhibit Azotobacter and Rhizobium in the root region may leads to decreased nitrogen
fixation and nodulation. Coinoculation of nitrogen fixing Azotobacter and Azospirillum
isolates with Rhizobium appears to have beneficial influence in increasing nodule
number, nitrogen fixation and yield of soybean, pea and clover.
Root Exudates:
The factor responsible for rhizosphere effect is the greater variety of organic
substances available and the root region by the way of exudates from roots are directly or
indirectly influence the quantity and quality of microorganism in the root region. The
substance exuded by plant root includes amino acids, sugar, organic acids, vitamins,
nucleotides etc. The nature and amount of substances exuded are dependent on the
species of plants, age and environmental condition under which they grow the root cap
and areas of active growth are primary region of root exudation and the major sites of
carbon release from seminal wheat roots in to soil to be the zone of root elongation.
 Root exudates containing toxic substances such as glycosides and hydrocyanic
acid may inhibit the growth of pathogen. The changes induced in the rhizosphere due to
virus infection could be restored to normally by spraying the leaves with thiouracil or
gibberellins to overcome foliar abnormalities and stunting symptoms associated with
virus infected plants. Root exudates which neutralizing the soil pH and altering the
microclimate of the rhizosphere through liberation of water and CO 2 may influence
infection of roots by pathogenic fungi.
Fungistasis:
Root exudates influence the proliferation and survival or root infecting pathogens
in soil either through soil fungistasis or inhibition of pathogens in the rhizosphere. The
term soil ‘fungistasis’ is used to explain the inability of non-dormant spores, sclerotia or
propagules to germinate even under most favorable condition of pH, temperature and
moisture in soil. The fungistasis could be released by the rhizosphere effect of plants that
creates a congenial environment for spore germination. The fungistasis of sclerotia of
Sclerotium cepivorum is released by volatile stimulators are associated with roots of
Allium. The alkyl sulphides are produced when alkyl cysteine sulphoxides comes from
roots of Allium, are broken down by soil bacteria.
The macro conidia of Fusarium may germinate in soil and form germ tube in the
form of chlamydospore, the chlamydospore formation indicating that associative effects
of other soil microorganisms. Chlamydospore production of F. solani is accelerated in the
presence of Bacillus licheniformis. Root exudates may provide a food base for the growth
of antagonistic which could suppress the growth of pathogenic microorganisms in soil.
Streptomyces and Trichoderma are effective against Fusarium wilts of plants.
Rhizosphere soil of pigeon pea isolates of Streptomyces from resistant varieties inhibited
the growth Fusarium udum causes wilt. Trichoderma viride in the rhizosphere of
varieties of tomato resistance able to minimize the severity of Verticillium wilt on the
susceptible plants in the presence of the pathogens.
Plant Growth Promoting Rhizobacteria (PGPR):
The term Rhizobacteria implies the ability of certain bacteria to colonize the
rhizosphere very aggressively like Pseudomonas sp. is known as PGPR. Pseudomonas
fluorescens and P. putida when inoculated to potato seed are improved growth of
potatoes (Potato yield 5.33 per cent, sugar beet 4-8 t/ha). Pseudomonas PGPRs increase
potato yields by reducing HCN production by Deleterious Rhizosphere Microorganisms
(DRMO) through siderophore mediated competition for Fe (III) which is required for
HCN production. Because of their versatility in growth and nutrient absorption PGPRs
are competition for substrate and niche exclusion, production of siderophore and
antibiotics leads to beneficial effects of crop plants. Many soil bacteria have shown great
potentiality as biocontrol agents by secreting antibiotics. Agribacterium
radiobacterstrain 84 is a biocontrol agent controlling crown gall disease caused by
Agrobacterium tumifaciens. Bacillus subtilis capable of producing endospore and
tolerating heat can suppress major and minor soil borne disease of carrot, oats and
groundnut. Fluorescent Pseudomonad are believed to improve the growth of plants by
colonizing the root region which produce growth inhibiting cyanide to suppress a root
disease of wheat caused by Gaeumannomyces graminis var tritici.
‘Siderophore’ are low molecular weight, high affinity iron chelators that
transport iron into bacterial cells. Fluorescent Pseudomonad produce yellow green
fluorescent siderophore which specifically recognize and sequester the limited supply of
iron in the rhizosphere and thereby reduce the availability of this trace elements for the
growth of the pathogens. The PGPR function is better in neutral and alkaline soil than
acid soil because the availability of iron in soil decreases with increase in PH. The
phenazin-type antibiotic produced by P. fluorescence controls the diseases of wheat. A
yellow green pigment called pseudobactin (Pseuodomonas B10) inoculated into the soil
was amended with Fe in the form of Fe EDTA (Ethylene DiaminetetraAcetoferate) were
observed effective colonization in rhizosphere which reduced Erwinia carotovora by
scavenging the Fe available in the vicinity and thus reduce disease severity by
minimizing the virulent of pathogens. Similar results were obtained Fe EDTA amended
with pseudobactin B10 effective against disease of wheat caused by Gaeumannomyces
graminis var tritici. QUANTUM 4000 is a growth promoter on groundnut and cotton,
Bacillus subtilis (GBOs), P. fourescens (N12 and Tn5) are the commercial products of
PGPRs.
Transfer of genetic loci encoding the pathways for the synthesis of antifungal
metabolites such as phenazines, phloroglucinols, oomycin (A), pyoluteroin, pyrolinitrin
and HCN. The gene manipulated strains of P. flourescens Hv 37a produce oomycin (A)
under the control of the constitutive lac promoter from E. coli to suppress Pythium
damping off, a disease of cotton. The different strains of Pseudomonas spp recombinant
 through plasmids as results over production of phenazine-1-carboxylase or
phloroglucinol had better ability to suppress the diseases of wheat.
The recombinant strain (R20 (Pn AH7)) was added to soil with salicylate when the
strains of Pseudomonas R20 was genetically engineered with plasmid NAH7 which
encodes the enzymes for salicylate degradation which reduce severity of (DRMOs) in
sugar beet. The combination of biocontrol agent and chemical control (fungicide), dual
application of Pseudomonas and Trichoderma to minimize fungal infections of seedlings
caused by Fusarium, Pythium, Rhizoctonia, Cylindrocarpon and Cylindrocladium. In
USA, the efficacy of a strain of Pseudomonas from the rhizosphere is controlling the
growth of downy brome weed of wheat crop.

Lecture 12
12. Soil microorganisms and their interactions – positive and negative interactions.
Bioinoculants - types - bacterial, fungal (AMF) and algal bionoculants

Interaction among soil microorganism:

Neutralism:
The two different species of microorganisms occupying the same environment
without affecting each other are called neutralism. For example each could utilize
different nutrients without producing metabolic end products that are inhibitory.
Positive association:
Mutualism:
It is defined as the symbiotic relationship in which each organism benefits from
their association are called as mutualism. Rhizobium-legume symbiosis is an example of
mutualism.
Commensalisms:
It is refers to relationship between organisms in which one species of a pair
benefits, the other is not affected, it is termed as commensalisms. Many fungi are able to
dissimilate cellulose to glucose and many bacteria are unable to utilize cellulose but they
can utilize fungal breakdown glucose and organic acids.
Negative association:
Antagonism:
When one species adversely affects the environment for another species is called
as antagonism. Both Staphylococcus aureus and P. aeruginosa are antagonistic towards
Aspergillus terrus. P aeruginosa pigment inhibits germination of Aspergillus spores
whereas S. aureus produce a diffusible anitifungal material that causes distortion and
hyphal swelling in A. terrus.
Competition:
A negative association may result from competition among species for essential
nutrients. The best adopted microbial species will predominate and other species will
eliminate.
Parasitism:
It is defined as a relationship between organisms in which one organism lives in
or another organism. The parasites feed as the cells, tissues or fluids of another organism
(host). All groups of plants, animals and microorganism are susceptible to attack
microbial parasites. Viruses are strict intracellular parasites on bacteria, fungi and algae.
Predation:
Those microorganisms predate another organism for their nutrients are called as
predation. Protozoa engulf bacteria in soil for its nutrition.
Phyllosphere:
The leaf surface can be termed as ‘Phylloplane’ and zone of leaves inhabited by
microorganism as ‘phyllosphere’. In plants, leaves are exposed to atmosphere resulting
in the establishment of typical flora on the surface aided by the cuticle, waxes and
appendages which help in anchorage of microorganisms. These microorganism may die,
survive or proliferate on leaves or leaf sheath depending on the influence of leaf
diffusates or exudates especially amino acids, glucose, fructose and sucrose may provide
niche for nitrogen fixation and secretion of substances capable of promoting the growth
of plants. Ruinen coined the term ‘Phyllosphere’ from forest vegetation where thick
microbial epiphytic association exists on leaves specifically nitrogen fixing bacteria such
as Azotobacter and Beijerinckia and other genera such as Pseudomonas, Phytomonas,
Erwinia, Sarcina, Pseudobacterium etc., nitrogen fixing blue green algae like Anabaena,
Calothrix, Nostoc, Scytonema and Tolypothrix. Some of the fungi and actinomycetes are
Cladosporium, Alternaria, Cercospora, Helminthosporium, Trichoderma,
Colletotrichum, Fusarium, Candida, Penicillium, Cephalosporium, Actinomyces and
Streptomyces.

Spermosphere
The spermosphere is the zone surrounding seeds where interactions between the
soil, microbial communities and germinating seeds take place. The concept of the
spermosphere is usually only applied during germination sensu stricto. Despite the
transient nature of this very small zone of soil around the germinating seed, the microbial
activities which occur there may have long-lasting impacts on plants. The spermosphere
is indirectly characterized by either (i) seed exudates, which could be inhibitors or
stimulators of micro-organism growth or (ii) the composition of the microbiome on and
around the germinating seeds. The microbial communities present in the spermosphere
directly reflect that of the germination medium or are host-dependent and influenced
quantitatively and qualitatively by host exudates. Despite its strong impact on the future
development of plants
Laimosphere
The laimosphere is the microbiologically enriched zone of soil that surrounds
below-ground portions of plant stems; the laimosphere is analogous to
the rhizosphere and spermosphere. The combining form laim- from laimos (Greek:
λαιμός) denotes a connecting organ (neck) while -sphere indicates a zone of influence.
Topographically, the laimosphere includes the soil around any portion of subterranean
plant organs other than roots where exuded nutrients (especially sugars and amino acids)
stimulate microbial activities. Subterranean plant organs with a laimosphere
include hypocotyls, epicotyls, stems, stolons, corms, bulbs, and leaves. Propagules of
soil-borne plant pathogens, whose germination is stimulated by a plant exudates in the
laimosphere, can initiate hypocotyl and stem rots leading to "damping-off". Pathogens
commonly found to cause such diseases are species
of Fusarium, Phoma, Phytopthora, Pythium, Rhizoctonia and Sclerotinia.
Fig. 1. A diagram denoting the location of the laimosphere,
rhizosphere, and spermosphere of the subterranean organs of a plant (Plant and
Soil 37:187-190, 1972).

Fig. 2. Chlamydospores of Fusarium solani f. sp. cucurbitae forming in the laimosphere

of a squash hypocotyl, Magyarosy 1973.

Fig. 3 & 4. Early lesion development in the epidermis of a squash hypocotyl caused
by Fusarium solani stained with tetrazolium salt, Magyarosy 1973.

Fig. 5. Diagram of hypocotyl stem rot leading to "damping-off" caused by Rhizoctonia

solani (Univ.Calif. Agr. Exp. Sta. Service Manual 23, 1957).
 Lecture 13
13. Mass production of bioinoculants
In 1985, Nobbe and Hiltner applied for patent in England and United states for a
legume inoculant that was later marketed as Nitragin. Nitragin was produced on gelatin
and agar nutrient media. However, agar based inoculants were soon replaced by peat
based once because in agar based inoculants mortility was very high during the dry
phase. In India production of biofertilizer on commercial scale started only during late
1960’s when yellow seeded soyabean was introduced for the first time. Recognition of
India peat as suitable carrier for production of biofertilizer further augmented the growth
of biofertilizers industry in India.
Biofertilizers are carrier based preparations containing live or latent cells of
efficient strains of nitrogen fixing, phosphate solubilizing or cellulolytic microorganism
used for application to seed or soil or composting areas with aims of increasing the
number of such microorganism and accelerate certain microbial activity to augment the
extent availability of nutrients in which can easily assimilated by plants. It is ecofriendly
which replace chemical fertilizers that are indispensable for getting maximum yield of
crops.
Biological nitrogen fixers are yielding 175 x 10 6 tonnes/year (67.5 per cent) than
industrially fixed nitrogen yields 40 x 106 tonnes/year (15.3 per cent). For example one
tonne of Rhizobium is equal to 100 tonnes of chemical fertilizer (50 kg of N2 fixed/ha) by
the application of 500 gm of Rhizobium biofertilizer.
Concept of Biofertilizers:
The microorganisms are inducing biochemical transformation in soil,
mineralization, nitrogen fixation, phosphate solubilization and organic matter
decomposition which leads to better availability of nutrients to plants.
Importance of Biofertilizers:
Biofertilizers are cost effective, ecofriendly, improve soil health, compatible with
fungicides and insecticides, genetically modified organisms (GMO) using nif genes
which augment better nitrogen fixation in soil, assimilated nutrients like N, P, zinc, iron,
silica and organic carbon for all crops, it envisages an integration of traditional
techniques with modern technique and safe to both plants and human beings, it is an vital
components of sustainable agriculture.
Types of biofertilizers:
Nitrogen fixers:
i. Non symbiotic or free-living nitrogen fixer:
Azotobacter, Beijerinckia, Clostridium and Derxia are independently living in soil
or freely living in soil and fix nitrogen.
ii. Associative symbiont:
The occurrence of nitrogen fixing Azospirillum inside the roots and aerial parts of
the plant also called ‘Diazotrophic Bioeoeocis’.
iii. Symbiotic nitrogen fixers:
The association of two organisms is mutually benefited. Rhizobium symbiotic
association with legume roots, Frankia symbiotic association with non-legume crop
(casuarina).
Biofertilizer aiding for phosphorus nutrition:
a. Phosphate solubilizing microorganism:
Tropical soils are deficient in phosphorus. Further most of them solubilize
phosphorus and thus make in available for plant growth. It is estimated that in most
tropical soils, 75% super phosphate applied is fixed and 25% is available for plant
growth. These are some fungi such as Aspergillus awamori, penicillium digitatum etc and
bacteria like Bacillus megatherium var. phosphaticum,Bacillus polymyxa, Pseudomonas
striata,Psuedomonas putida etc can solublize unavailable form of P to available form.
Those organisms convert unavailable organic phosphate into soluble inorganic phosphate
are called as Phosphate Solubilizing Bacteria (PSB).
b. Phosphate mobilizer:
Mutualistic symbiosis between some specific root inhibiting fungi and plant roots
which mobilize phosphorus.Vesicular Arbuscular Mycorrihzae (VAM) fungi colonize
roots of several crop plants. They are zygomyceteous fungi belonging to the genera
Glomus, Gigaspora, Acaulospora, Scelercystis etc. These are obligate symbiont and
cannot culture on synthetic media. They help plant growth through improved phosphorus
nutrition and protect the roots against pathogen. Nearly 25-30% of phosphate fertilizer
can be saved through inoculation with effieicient VAM fungi.
Organic matter decomposer:
 Mineralization process, microorganism converts complex organic or element to
inorganic state. Cellulose can be decomposed into glucose enzyme cellulaseproduced by
Cellulomonas.

Uses of biofertilizer:
• To increase physiochemical properties of soil like soil structure, soil texture,
water holding capacity, cation exchange and pH by providing nutrients and
organic matter.
• Azotobacter and Azospirillum supply nitrogen and secrete antibiotic which is used
as pesticides.
• Rhizobium which fix 50-150 kg N/ha/year
• Cyanobacteria (BGA) grow pH range of 6.5 to 8.5, used to reclamation of saline
or alkaline soil. It is also secretes plant growth promoting substance (PGP) such
as IAA, IBA, NAA, amino acids, protein and vitamins and add organic matter
into soil
• Algal biofertilizers increase rice yields about 10-45 per cent a fix 40-50 kg N in
soil. Algalization in rice field which yield 25-30 kg N/ha/season which can able to
persist 3 to 4 years
• Azolla supply N2 and organic matter may add soil fertility and tolerance to heavy
metal. VAM increases longevity and surface area of roots which reduce soil
stress, increase resistant in plant
• Application of dried BGA flakes at the rate 10 kg/ha is recommended for 10 days
after transplanting of rice
• PGPR including Pseudomonas fluorescence and P. putida convert non available
organic phosphate into soluble inorganic phosphate
• PGPR also produce siderophore (iron chelating) Pseudobactin) which chelate iron
and make it unavailable to harmful fungi
• Mycorrhiza absorb N, P, K and Ca also convert organic phosphate to protect root
from pathogens and produce growth substance (cytokine)
• Biological nitrogen fixation in the water logged rice fields contributes about 40 to
50 Kg N /ha.
Criteria for strain selection:
The efficient nitrogen fixing strain is evolved or selected in laboratory,
maintained and multiplied on nutritionally rich artificial medium before inoculating on
seed or soil. In soil, the strain has to survive and multiply to compete for infection site on
roots against hostile environment in soil.
Steps for preparing biofertilizers:
The isolated strain is inoculated in small flasks containing suitable medium for
inoculum production. The volume of the starter culture should be a minimum of 1 per
cent to obtain atleast 1 x 109 cells/ml preparations. Carriers carry the nitrogen fixing
organism in the fields. The inoculum is now packed with 109-1010 viable cells/gram. Final
moisture content should be around 40-60 per cent. For large scale production of inoculum
culture fermenters are used.
Mass multiplication biofertilizers:
• Preparation of starter culture
• Preparation of broth culture
• Preparation of carrier
Preparation of starter culture:
Pure culture of efficient strain N2 fixing organism is grown on respective agar
medium. A loopful of inoculum from it is transferred in a 300 ml of liquid medium in the
flask. The flask is then kept on shaker (260 rpm) for 72 to 96 hours. If shaker is not
available incubate it at 28˚C for 5-6 days.
Preparation broth culture:
Each flask containing suitable broth is inoculated with the starter culture in 1:2
proportions aseptically. Incubate the flasks at 28˚C for 2 to 5 days depending upon the
type of organism till the count per ml reaches to 10 9 cells/ml. This broth culture with
population of 109 cells/ml should not be stored for more than 24 hours or stored at 4˚C.

Preparation of carrier inoculants:

Most inculants are the miture of the broth culture and a finely milled, neutralized
carrier material. Carrier is a substance having properties such as non toxicity, good
moisture absorption capacity, free of clumps forming material, easy to sterilize,
inexpensive, easily available and good buffering capacity, so that it can prolonged and
maintained the growth of nitrogen fixing microorganisms which it is carrying. The most
frequently used carrier for inoculant production is peat. A wide range of substitute’s
lignite, coal, charcoal, bagasse, filter mud, vermiculite, polyacrylamide, mineral oil,
vegetable oils etc have been tested as altenative carrier. First of all the carrier like peat is
mined, drained and cleared of stones, roots etc. Then, it is shredded and dried. The peat is
then passed through heavy mills. A material with a particle size of 10-40 µm is collected
for seed coating. Peat with particle size of 500-1500 µm is used for soil inoculant.
Carriers have to be neutralisd by adding precipitated calcium carbonate (pH 6.5-7.0).
After this the carriers are sterilized for use as inoculants.
Isolation and mass production of biofertilizers:
Prepare Jensen’s or Ashby’s mannitol agar media for isolation of Azotobacter
in laboratory sterilize it. Simultaneously, prepare serial dilution, 10 gm of rhizosphere
soil mixed with 90 ml sterile water will give 10 -1 dilution, the 10 fold dilutions of the
above suspension from 10-1 to 10-6 using separate water blanks. Transfer one ml aliquot of
the appropriate dilution to sterile petriplates and pour petriplates with solidifyable
Jensen’s agar incubate at room temperature. The soft, flat, milky and mucoid colonies of
Azotobacter developed after 3 days from incubation. Transfer Azotobacter colonies on
the slant of Ashby’s or Jensen’s agar media.
Preparation inoculums in powder form:
A loopful of Azotobacter inoculum transferred to 300 ml of liquid medium
(Jensen’s broth) in the flask. It is kept on shaker for 72 to 96 hours, the flask containing
Jensen’s broth is inoculated with starter culture is 1:2 proportions aseptically. Incubate
the flask at 28C for 25 days depend upon the type of organism till broth culture
population count 109 cells/ml. Finely powered peat or charcoal powder sieved with 250-
300 mesh (75 micron pore size) and carrier is neutralized with CaCO 3 and sterilized it.
One part of Azotobacter broth is mixed with two parts of carrier powder (1:2
proportions). Then kept for curing at room temperature, break clumps sieve it packed in
polythene bag of 0.5 mm thickness and leaving 1/3rd space for aeration of the bacteria.
Production of Azolla and BGA:
A species of Anabana (A.azollae) is associated with the aquatic fern. Azolla
occurs in a ventral pore in the dorsal lobe of each vegetative leaf. The endophyte fixes
atmosphere nitrogen and resides inside the tissues of the water fern. Azolla being used as
green compost for rice cultivation, multiplities fast and provide higher yield of green
compost (200-300 t/ha/year than conventional green manure plants such as Sesbania,
Crotalaria and Tephrosia which are known to yield 30-50 t/ha/year. The common species
of Azolla occursin India is A.pinnata.
 Azolla nurseries are raised in small plots (50-100 sq.m) or in concrete tank with 5-
10 cm deep water (pH 7-8) containing super phosphate at 4-8 kg P2O5/ha after seeding the
plots with Azolla inoculum at the rate of 0.1 to 0.4 kg per sq.m. At the end of 2 to 3
weeks, when full growth of Azolla takes place, the water is drained and the Azolla growth
is incorporated into the rice fields by ploughing the mass (10-20 t/ha) into the puddled
rice field. Field experiments in India has demonstrated that 10 t/ha of Azolla is equivalent
to 25 to 30 kg N/ha and similarly an application of 20 kg N/ha as Ammonium Sulphate
with Azolla is equivalent to 40 kg N/ha as Ammonium Sulphate.
Trough method of algal production:
Prepare shallow troughs (2m x 1m x 23cm) of galvanized iron sheet or permanent
tanks. Spread 8-10 kg of soil (4 kg/m 2) and mix well with 200g of super phosphate. Add
water (5-15cm) to the troughs and adjust the pH to neutral (pH 7). To prevent insect, add
carbofuron (3% granules, 20g per tray) or Malathion or BHC to the trough. Within 10
days a mat of BGA appears which when dried results in flakes. The trough can be used
again by using the flakes from the previous batch.
Pit method of algal production:
Polythene lined shallow pits can serve as containers instead of trough which
makes it less expensive.
Field method:
Prepare 40 m2 plots ae bunded and flooded with water (2.5cm) with application of
super phosphate at 12 kg/40 m2. Carbofuron, BHC or any other insecticide is used to
avoid insect predator and mosquitoes. A starter culture of BGA is sprinkled over the body
of water. Mats of BGA growth appears when the plot dried up. The flakes of BGA are
collected and used as inoculum at the rate of 10 kg/ha.
Production of VAM:
VAM can be produced on a large scale by pot culture technique. This requires the
host plant mycorrhial fungi and natural soil. The host plant support large scale
productions of inoculum are Sudan grass, strawberry, sorghum, maize, onion, citrus etc.
The starter inoculum of VAM can be isolated from soil by wet sieving and decantation
technique. VAM spores are surface sterilized and brought to the pot culture. Commonly
used pot substrates are sand: soil (1:1 w/w) with a little amount of moisture. There are
two methods of using the inoculum a. using a dried store root soil to plants by placing the
inoculum several centimeters below the seeds or seedling b. using a mixture of soil-roots
and spores in soil pellets and spores are adhered to seed surface with adhesive.
Methods of application of biofertilizers:
Slurry method of inoculation:
Prepare 10 per cent sugar or jaggery solution mixed with 200 gm of inoculants in
one liter of water and make homogenous slurry sprinkled over the seeds. The seeds are
dried in shade and sow within 24 hrs after treatment.
Seedling inoculation:
Crop such as paddy or chilies, the seedling are dipped into slurry inoculants for 1-
2 minutes and transplanted than immediately.

Pasting of sugarcane eye bud:

About 6 to 8 packets of Azotobacter/Azospirillum mixed with 10 kg of soil. The
water is added to the mixture and makes it into a paste. The sets of sugarcane eye bud are
pasted and sets are planted immediately without allowing then drying.
Sugarcane set inoculation:
A slurry of 6 to 8 packets of carrier based inoculum of Azotobacter or
Azospirillum mixed with 50 liter of water. The sets are dipped and planted immediately.
Soil application:
Biofertilizers (bacterial culture) are not used for seed treatment due to there be no
sticky agent present in it. The content of inoculum packets is mixed with 10 to 15 kg of
soil. The mixture in broadcasted in the field.
Quality standards of biofertilizers:
• The inoculant shall be carrier based one
• The inoculant shall contain minimum 109 viable cells of Rhizobia culture of the
carrier on dry weight basis within 15 days of manufacture and 107 with in 15 days
before expiry date marked on pockets
• The inoculant have maximum period of 6 months expiry from the date of its
manufacture
• The inoculant should not be contaminated with other microorganisms
• The pH of the inoculant between ‘6 to 7.5’
• The carrier material should be in the form of powder and it should be neutral
 • The inoculum shall be packed in 50 to 75 micron low density polythene packets.
• Test shall be carried out time to time
• The inoculant shall be stored in cool place preferably 15C, not exceeding 30C. It
should be the duty of manufacturer to instruct retailer and users about precaution
during storage
• The manufacture should maintain records of isolates and identification of cultures
data on effectiveness of pure culture for every season and crop
Each packet shall be marked legibly the following information
i. Name of the product
ii. Leguminous crop intended with inoculant
iii. Name and address of manufacturer
iv. Type of the carrier
v. Batch or code number
vi. Date of manufacture
vii. Date of expiry
viii. Net quantity recommendation for hectare
ix. Storage instruction
‘STORE IN COOL PLACE AWAY FROM DIRECT SUN AND HEAT’

Lecture 14
14. Industrial utilization of microorganisms –alcohol fermentation – alcoholic
beverages

COMMERCIAL PRODUCTION OF BEER

As almost any cereal containing certain sugars can undergo spontaneous

fermentation due to wild yeasts in the air, it is possible that beer-like beverages were
independently developed throughout the world soon after a tribe or culture had
domesticated cereal. Chemical tests of ancient pottery jars reveal that beer was produced
about 5,500 years ago in what is today Iran, and was one of the first-known biological
engineering tasks where the biological process of fermentation is used. Also recent
archaeological findings showing that Chinese villagers were brewing fermented alcoholic
drinks as far back as 7000 BC on small and individual scale, with the production process
and methods similar to that of ancient Egypt and Mesopotamia.

Beer was one of the most common drinks during the middle Ages. It was
consumed daily by all social classes in the northern and eastern parts of Europe where
grape cultivation was difficult or impossible. Though wine of varying qualities was the
most common drink in the south, beer was still popular among the lower classes. Since
the purity of water could seldom be guaranteed, alcoholic drinks were a popular choice,
having been boiled as part of the brewing process. Beer also provided a considerable
amount of the daily calories in the northern regions. In England and the Low Countries,
the per capita consumption was 275-300 liters (60-66 gallons) a year by the Late Middle
Ages, and beer was drunk with every meal. Though probably one of the most popular
drinks in Europe, beer was disdained by science as being unhealthy, mostly because
ancient Greek and more contemporary Arab physicians had little or no experience with
the drink.

Flavoring beer with hops was known at least since the 9th century, but was only
gradually adopted because of difficulties in establishing the right proportions of
ingredients. Before that, gruit, a mix of various herbs, had been used, but did not have the
same conserving properties as hops. Beer flavored without it was often spoiled soon after
preparation and could not be exported. The only other alternative was to increase the
alcohol content, which was rather expensive. Hopped beer was perfected in the towns of
Germany by the 13th century, and the longer lasting beer, combined with standardized
barrel sizes, allowed for large-scale export. The discovery of yeast's role in fermentation
in 1857 by Louis Pasteur gave brewers methods to prevent the souring of beer by
undesirable microorganisms.

Many European nations have unbroken brewing traditions dating back to the
earliest historical records. Beer is an especially important drink in countries such as
Belgium, Germany, Ireland, and the UK, with nations such as France, the Scandinavian
countries, Poland, the Czech Republic, Spain and others having strong and unique
brewing traditions with their own history, characteristic brewing methods, and styles of
beer.
 Unlike in many parts of the world, there is a significant market in Europe (the UK
in particular) for beer containing live yeast. These unfiltered, unpasteurised brews are
awkward to look after compared to the commonly sold dead beers: live beer quality can
suffer with poor care, but many people prefer the taste of a good live beer to a dead one.
While beer is usually matured for relatively short times (a few weeks to a few months)
compared to wine, some of the stronger so-called real ales have been found to develop
character and flavour over the course of as much as several decades.

In some parts of the world, breweries that had begun as a family business by
Germans or other European émigrés grew into large companies, often passing into hands
with more concern for profits than traditions of quality, resulting in a degradation of the
product.

Modern breweries now brew many different types of beer, ranging from ancient
styles such as the spontaneously-fermented lambics of Belgium; the lagers, dark beers,
wheat beers and more of Germany; the UK's stouts, milds, pale ales, bitters, golden ale
and new modern American creations such as Chili Beer, Cream Ale, and Double India
Pale Ales.

Today, the brewing industry is a huge global business, consisting of several

multinational companies, and many thousands of smaller producers ranging from
brewpubs to regional breweries. Advances in refrigeration, international and
transcontinental shipping, marketing and commerce have resulted in an international
marketplace, where the consumer has literally hundreds of choices between various styles
of local, regional, national and foreign beers.

Brewing beer
 All beers are brewed using a process based on a simple formula. Key to the beer
making process is malted grain, depending on the region traditionally barley, wheat or
sometimes rye. Malt is made by allowing a grain to germinate, after which it is then dried
in a kiln and sometimes roasted. The germination process creates a number of enzymes,
notably alfa-amylase and beta-amylase, which will be used to convert the starch in the
grain into sugar. Depending on the amount of roasting, the malt will take on dark colour
and strongly influence the colour and flavor of the beer. Breweries buy malt and this is
not a process that is done in-house.

The malt is crushed in a malt mill to break apart the grain kernels, increase their
surface area, and separate the smaller pieces from the husks. The resulting grist is mixed
with heated water in a vat called a "mash tun" for a process known as "mashing". During
this process, natural enzymes within the malt break down much of the starch into sugars
which play a vital part in the fermentation process. Mashing usually takes 1 to 2 hours,
and during this time various temperature rests (waiting periods) activate different
enzymes depending upon the type of malt being used, its modification level, and the
desires of the brewmaster. The activity of these enzymes convert the starches of the
grains to dextrines and then to fermentable sugars such as maltose.

A mash rest at 104 °F or 40 °C activates β-glucanase, which breaks down gummy

β-glucans in the mash, making the sugars flow out more freely later in the process. A
mash rest from 120 °F to 130 °F (49 °C to 55 °C) activates various proteinases, which
break down proteins that might otherwise cause the beer to be hazy. But care is of the
essence since the head on beer is also composed primarily of proteins, so too aggressive a
protein rest can result in a beer that cannot hold a head. This rest is generally used only
with undermodified (i.e. undermalted) malts which are popular in Germany and the
Czech Republic, or non-malted grains such as corn and rice, which are widely used in
North American beers. Finally, a mash rest temperature of 149 to 160 °F (65 to 71 °C) is
used to convert the starches in the malt to sugar, which is then usable by the yeast later in
the industrial brewing process. Doing the latter rest at the lower end of the range
produces more low-order sugars which are more fermentable by the yeast. This in turn
creates a beer lower in body and higher in alcohol. A rest closer to the higher end of the
range creates more higher-order sugars which are less fermentable by the yeast, so a
fuller-bodied beer with less alcohol is the result.
 Finally the mash temperature may be raised to 165 °F to 170 °F (about 75 °C)
(known as a mashout) to deactivate enzymes. Additional water may be sprinkled on the
grains to extract additional sugars (a process known as sparging).

After the mashing, the mash is pumped to a lauter tun where the resulting liquid is
strained from the grains in a process known as lautering. The lauter tun generally
contains a slotted "false bottom" or other form of manifold which acts as a strainer
allowing for the separation of the liquid from the grain.

At this point the liquid is known as wort. The wort is moved into a large tank
known as a "cooking tun" or kettle where it is boiled with hops and sometimes other
ingredients such as herbs or sugars. The boiling process serves to terminate enzymatic
processes, precipitate proteins, isomerize hop resins, concentrate and sterilize the wort.
Hops add flavor, aroma and bitterness to the beer. At the end of the boil, the hopped wort
settles to clarify using hop filters.

The wort is then moved into a temperature controlled cylindrical-conical

"fermenter" where yeast is added or "pitched" with it. The yeast converts the sugars from
the malt into alcohol, carbon dioxide and other components through a process called
fermentation or glycolysis. After a week to three weeks, the fresh (or "green") beer is
cooled close to freezing temperature, yeast is purged and the beer is allowed to "lager" or
rest. After this conditioning for a week to several months, the beer is often filtered to
remove remaining yeast and particulates. The "bright beer" is then ready for serving or
packaging.

Converting Sugar to Alcohol and Carbon Dioxide

The overall chemical reaction:

C6H12O6 --------------> 2C2H5OH + 2CO2 + E

There are four main families of beer styles determined by the variety of yeast used
in their brewing.

Ale (top-fermenting yeasts)

Ale yeasts ferment at warmer temperatures between 15°C and 20°C (60°F to
68°F), and occasionally as high as 24°C (75°F). Pure ale yeasts form a foam on the
surface of the fermenting beer, because of this they are often referred to as "top-
fermenting" yeast - though there are some ale yeast strains that settle at the bottom. Ales
are generally ready to drink within three weeks after the beginning of fermentation,
however, some styles benefit from additional aging for several months or years. Ales
range in color from very pale to black opaque.

Lager (bottom-fermenting yeasts)

While the nature of yeast was not fully understood until Emil Hansen of the
Carlsberg brewery in Denmark isolated a single yeast cell in the 1800s, brewers in
Bavaria had for centuries been selecting these cold-fermenting Lager yeasts by storing or
"Lagern" their beers in cold alpine caves. The process of natural selection meant that the
wild yeasts that were most cold tolerant would be the ones that would remain actively
fermenting in the beer that was stored in the caves. Some of these Bavarian yeasts were
stolen and brought back to the Carlsberg brewery around the time that Hansen did his
famous work.

Lager yeast tends to collect at the bottom of the fermenter and is often referred to
as "bottom-fermenting" yeast. Lager is fermented at much lower temperatures, around
10°C (50°F), compared to typical ale fermentation temperatures of 18°C (65°F). It is then
stored for 30 days or longer close to the freezing point. During the storing or "lagering"
process, the beer mellows and flavors become smoother. Sulfur components developed
during fermentation dissipate. The popularity of lager was a major factor that led to the
rapid introduction of refrigeration in the early 1900s.
 Today, lagers represent the vast majority of beers produced, the most famous
being a light lager called Pilsner which originated in Pilsen, Czech Republic (Plzen in
Czech language). It is a common misconception that all lagers are light in color: lagers
can range from very light to deep black, just like ales.

Biochemistry of brewing process

The Brewing Process

Work in the brewery is typically divided into 7 steps: Mashing, Lautering,
Boiling, Fermenting, Conditioning, Filtering, and Filling.

Mashing
Mashing is the process of mixing milled grain (typically malted grain) with water,
and heating this mixture up with rests at certain temperatures to allow enzymes in the
malt to break down the starch in the grain into sugars, typically maltose.

Lautering
 Lautering is the separation of the extracts won during mashing from the spent
grain. It is achieved in either a lauter tun, a wide vessel with a false bottom, or a mash
filter, a plate-and-frame filter designed for this kind of separation. Lautering has two
stages: first wort run-off, during which the extract is separated in an undiluted state from
the spent grains, and sparging, in which extract which remains with the grains is rinsed
off with hot water.
Lauter tun
A lauter tun is the traditional vessel used for separation of the extracted wort.
While the basic principle of its operation has remained the same since its first use,
technological advances have led to better designed lauter tuns capable of quicker and
more complete extraction of the sugars from the grain.

The false bottom in a lauter tun has thin slits to hold back the solids and allow
liquids to pass through. The solids, not the false bottom, form a filtration medium and
hold back small solids, allowing the otherwise cloudy mash to run out of the lauter tun as
a clear liquid. The false bottom of a lauter tun is today made of wedge wire, which can
provide a free-flow surface in the bottom of the tun.

In the past the run-off tubes flowed through swan-neck valves into a wort
collection grant. While visually stunning, this system led to a lot of oxygen uptake. Such
a system has mostly been replaced either by a central wort-collection vessel or the
arrangement of outlet ports into concentric zones, with each zone having a ring-shaped
collection pipe. Brewhouses in plain public view, particularly those in brewpubs, often
maintain the swan-neck valves and grant for their visual effect.
A quality lauter tun has rotating rake arms with a central drive unit. Depending on
the size of the lauter tun, there can be between two and six rake arms. Cutting blades
hang from these arms. The blade is usually wavy and has a plough-like foot. Each blade
has its own path around the tun and often the whole rake assembly can be raised and
lowered. Attached to each of these arms is a flap which can be raised and lowered for
pushing the spend grains out of the tun. The brewer, or better yet an automated system,
can raise and lower the rake arms depending on the turbidity (cloudiness) of the run-off,
and the tightness of the grain bed, as measured by the pressure difference between the top
and bottom of the grain bed.
 A system will introducing sparge water into the lauter tun. Most systems have a
ring of spray heads that insure an even and gentle introduction of the sparge water. The
watering system should not beat down on the grain bed and form a channel.

Large breweries have self-closing inlets on the bottom of the tun through which
the mash is transferred to the lauter tun, and one outlet, also on the bottom of the tun, into
which the spent grains fall after lautering is complete.
Some small breweries use a combination mash/lauter tun, in which the rake
system cannot be implemented because the mixing mechanism for mashing is of higher
importance. The stirring blades can be used as an ersatz rake, but typically they cannot be
moved up and down, and would disturb the bed too much were they used deep in the
grain bed.

Mash Filter
A mash filter is a plate-and-frame filter. The empty frames contain the mash,
including the spent grains, and have a capacity of around one hectoliter. The plates
contain a support structure for the filter cloth the plates, frames, and filter cloths are
arranged in a carrier frame like so: frame, cloth, plate, cloth, with plates at each end of
the structure. Newer mash filters have bladders that can press the liquid out of the grains
between spargings. The grain does not act like a filtration medium in a mash filter.

Boiling
Boiling the won extracts, called wort, ensures its sterility, and thus prevents a lot
of infections. During the boil hops are added, which contribute bitterness, flavor, and
aroma compounds to the beer, and, along with the heat of the boil, causes proteins in the
wort to coagulate and the pH of the wort to fall. Finally, the vapors produced during the
boil volatilize off flavors, including dimethyl sulfide precursors.
The boil must be conducted so that is it even and intense. The boil lasts between
50 and 120 minutes, depending on its intensity, the hop addition schedule, and volume of
wort the brewer expects to evaporate.

Whirlpool
At the end of the boil, the wort is set into a whirlpool. The so-called teacup effect
forces the more dense solids (coagulated proteins, vegetable matter from hops) into a
cone in the center of the whirlpool tank.
 In most large breweries, there is a separate tank for whirlpooling. These tanks
have a large diameter to encourage settling, a flat bottom, a tangential inlet near the
bottom of the whirlpool, and an outlet on the bottom near the outer edge of the whirlpool.
A whirlpool should have no internal protrusions that might slow down the rotation of the
liquid. The bottom of the whirlpool is often slightly sloped toward the outlet.

A better alternative to a whirlpool are hop filters. Hops are removed from the
bitter wort using stainless steel filters. The main advantages of his system are better hop
filtrations, lower equipment cost and less floor surface.

Wort cooling
After the hop filtration, the wort must be brought down to fermentation
temperatures before yeast is added. In modern breweries this is achieved through a plate
heat exchanger. A plate heat exchanger has many ridged plates, which form two separate
paths. The wort is pumped into the heat exchanger, and goes through every other gap
between the plates. The cooling medium, usually water, goes through the other gaps. The
ridges in the plates ensure turbulent flow. A good heat exchanger can drop 95 °C wort to
20 °C while warming the cooling medium from about 10 °C to 80 °C. The last few plates
often use a cooling medium which can be cooled to below the freezing point, which
allows a finer control over the wort-out temperature, and also enables cooling to around
10 °C. After cooling, oxygen is often dissolved into the wort to revitalize the yeast and
aid its reproduction.

Fermenting
Fermentation, as a step in the brewing process, starts as soon as yeast is added to
the cooled wort. This is also the point at which the product is first called beer. It is during
this stage that sugars won from the malt are metabolized into alcohol and carbon dioxide.
Fermentation tanks come in all sorts of forms, from enormous tanks which can look like
storage silos, to five gallon glass carboys in a home brewer's closet.

Most breweries today use cylindroconical tanks, or CCTs, have a conical bottom
and a cylindrical top. The cone's aperture is typically 60°, an angle that will allow the
yeast to flow toward the cones apex, but is not so steep as to take up too much vertical
space. CCTs can handle both fermenting and conditioning in the same tank. At the end of
fermentation, the yeast and other solids which have fallen to the cones apex can be
simply flushed out a port at the apex.

Fermentation tanks are typically made of stainless steel. If they are simple
cylindrical tanks with beveled ends, they are arranged vertically, as opposed to
conditioning tanks which are usually laid out horizontally.

A very few breweries still use wooden vats for fermentation as wood is difficult to
keep clean and infection-free and must be repitched more or less yearly.
After high kraeusen a bung device (German: Spundapparat) is often put on the
tanks to allow the CO2 produced by the yeast to naturally carbonate the beer. This bung
device can be set to a given pressure to match the type of beer being produced. The more
pressure the bung holds back, the more carbonated the beer becomes.

Conditioning
When the sugars in the fermenting beer have been almost completely digested, the
fermentation slows down and the yeast starts to settle to the bottom of the tank. At this
stage, the beer is cooled to around freezing, which encourages settling of the yeast, and
causes proteins to coagulate and settle out with the yeast. Unpleasant flavors such as
phenolic compounds become insoluble in the cold beer, and the beer's flavor becomes
smoother. During this time pressure is maintained on the tanks to prevent the beer from
going flat.

If the fermentation tanks have cooling jackets on them, as opposed to the whole
fermentation cellar being cooled, conditioning can take place in the same tank as
fermentation. Otherwise separate tanks (in a separate cellar) must be employed.

Filtering
Filtering the beer stabilizes the flavor, and gives beer its polished shine and
brilliance. Not all beer is filtered.

Filters are many types. Many use pre-made filtration media such as sheets or
candles, while others use a fine powder made of, for example, diatomaceous earth, also
called kieselguhr, which is introduced into the beer and recirculated past screens to form
a filtration bed.
 Filters range from rough filters that remove much of the yeast and any solids (e.g.
hops, grain particles) left in the beer, to filters tight enough to strain color and body from
the beer. Normally used filtration ratings are divided into rough, fine and sterile. Rough
filtration leaves some cloudiness in the beer, but it is noticeably clearer than unfiltered
beer. Fine filtration gives a glass of beer that you could read a newspaper through, with
no noticeable cloudiness. Finally, as its name implies, sterile filtration is fine enough that
almost all microorganisms in the beer are removed during the filtration process.

Packaging
Packaging is putting the beer into the containers in which it will leave the
brewery. Typically this means in bottles and kegs, but it might include cans or bulk tanks
for high-volume customers.

Secondary fermentation
Secondary fermentation is an additional fermentation after the first or primary
fermentation. Some beers may have three fermentations.

Bottle fermentation
Some beers undergo a fermentation in the bottle, giving natural carbonation. This
may be a second or third fermentation. They are bottled with a viable yeast population in
suspension. If there is no residual fermentable sugar left, sugar may be added. The
resulting fermentation generates CO2 which is trapped in the bottle, remaining in solution
and providing natural carbonation.

Cask conditioning
Beer in casks are managed carefully to allow some of the carbonation to escape.

In shorter forms
Brewing is the term used in making beverages, both alcoholic and non-alcoholic,
through fermentation. Brewing, on this page, refers to the beer-making process.

The following figure schematically illustrates the entire brewing process.

The brewing process involves many biochemical reactions. It is very complex.
The products from the brewing are not only carbon dioxide and alcohol, but they also
include the following by-products:
  acetaldehyde (green apple aroma)
 phenolic (flavour and aroma of medicine, plastic, Band-Aids, smoke, or cloves)
 solvent (reminiscent of acetone or lacquer thinner)
 hydrogen sulfide (reminiscent of rotten eggs or burnt matches)

This process consists of the following steps:

 Milling
 Mashing
 Brewing
 Cooling
 Fermenting
 Conditioning
 Racking

Malting
Malting is the process of preparing barley for brewing. It is comprised of three
steps, with each step unlocking the starch within the barley. The steps are:

1) Steeping
 The barley is sprayed with water and soaked for about 40 hours.

2) Germination
The soaked barley grows rootlets in the germination room. A large number of
enzymes, such as α- and β- amylase, are used to convert the grain into sugar. The goal of
germination is to break the starches into shorter length molecules.

3) Kilning
After the germination, the barley is dried up in a kiln to preserve the enzymes in
the grain. It is important to note that the flavour of beer is related to the temperature at
which it is kilned. A higher temperature results in a heavier flavour.

Milling
Milling is the cracking of the grain from malting. This is done so that the starch
has a greater surface area, allowing it to contact enzymes in the mashing processing.

Mashing
This step serves to convert the starch, from the malting process, into sugar. At the
end of the mashing, the sugar-rich water that is strained through the bottom of the
mashing tun is called wort. There are two types of mashing.
 Infusion Mashing: the starch is mixed with hot water and delivered to a cooking
vessel called a mash tun. The mash is directly heated by water to achieve
temperature changes. Grist, the grain separated from the shell, is coarsely ground,
making a high proportion of well modified malt.
 Decoction Mashing: is carried out with more finely ground grist. A portion of the
grains is passed to a series of vessels and returned to decoction mashing vessel,
where the grains are heated to boiling.

Brewing
The wort is sent to a brew kettle called COPPER, where it is boiled. During the
wort boiling process, hops, which are the female cones of hop plants, are added to create
the bitterness, flavour and aroma of beer. A number of changes occur in the wort, such as
coagulation of protein, evaporation of the wort, and flavour and colour changes. In this
process, it consumes about half of the energy in the brewing process.
Cooling
After the brewing, the wort is filtered and quickly cooled to a point that yeast can
be safely added. This is the preparation of fermentation. The yeast does not grow in a
high temperature condition.

Fermenting
During this step, the yeast metabolizes substances that can be dissolved in the
wort. The major products of carbohydrate metabolism are ethanol, carbon dioxide and
heat. The yeast multiplies around 3 to 5 times during this process.

Conditioning
At this stage, the beer is referred to as “green beer,” or immature beer, because the
flavour of the beer is not of acceptable quality. The immature beer has to be held for a
period of time to refine the flavour of beer. Sometimes, sugar or a small amount of wort
is added to boost the yeast metabolism. In this process, the acidic gas hydrogen sulfide is
released from beer.

Racking
During this step, beer becomes mature. The beer will be filtered and packed after
this stage.
Production process of Wine

Wine is the product made by the “normal alcoholic fermentation of the ripe grape
juice.

Classification and Definitions:

Wine may be classified in many ways.

Classification of wines
Wine is classified in three major categories. Table wines, also called still or
natural wines, are consumed primarily as complements to food. Sparkling wines, for
example, champagne, distinguishable by their effervescence, are drunk for the most part
on festive occasions. Fortified wines, such as sherry or vermouth, are drunk for the most
commonly drunk before or after meals and are also frequently used in cooking. These
wines are termed fortified because their alcoholic and sugar content are increased and
their fermentation arrested by the controlled addition of a more potent liquor, usually a
grape brandy, during the wine-making process; this results in an alcoholic content of 15
to 22 per cent by volume, as against 9 to 14 per cent for most table wines.

Table wine:
Table wines are further classified by color, as red, white, or rose (pink); and by
character, as sweet or dry.
Red-wines are made from dark grapes, the skins of which are allowed to remain
in contact with the fermenting juice for a period of two to three weeks, depending on the
character and depth of color desired.
White wines may be made from white (that is, green) grapes or from dark grapes,
but in the latter case the grape skins and pressed juice do not come into contact.
Light wine (including light grape wine, and light white wine): It is grape wine having an
alcoholic content not in excess of 14 per cent by volume.
True rose wines are from dark grapes; their skins remain in contact with the juice
only until they have turned a pale pink.
1. Dry wine is wine in which the fermentation of the sugars is practically complete.
Most dry wines contain a small amount of sugar.
2. Sweet wine is wine in which the alcoholic fermentation has been arrested. Such
wines contain sufficient sugar for taste perception. Wines may be fortified by the
addition of brandy or wine spirits.
3. Fortified dry wine is dry wine to which brandy has been added out which
conforms in all other particulars to the standard of dry wine. Fortified sweet wine
is sweet wine to which wine spirits have been added.
4. Sparkling wines is “wine in which the after part of the fermentation is completed
in the bottle, the sediment being dislodged and its place supplied by wine or sugar
liquor and / or dextrose liquor, and which contains, in 100 cc. (20 ⁰c), not less
than 0.12 g f grape ash”. Such wine contains considerable carbon dioxide.

Other types of wines

1. Modified wine, ameliorated wine, corrected wine is the product made by the
alcoholic fermentation, with the usual cellar treatment of a mixture of the juice of
sound, ripe grapes with sugar and / gar dextrose, or a syrup containing not less
 than 65 per cent of the sugars, and in quantity not more than enough to raise the
alcoholic strength after fermentation to 11 per cent by volume.
2. Raising wine is the product made by the alcoholic fermentation of an infusion of
dried or evaporated grapes, or of a mixture of such infusion or raisins with grape
juice.

Sparkling grape wine

1. Sparkling grape wine (including sparkling wine, sparkling red wine, and sparkling
white wine):
2. It is grape wine made effervescent with carbon dioxide resulting solely from the
secondary fermentation of the wine within a closed container, tank, or bottle.
3. Champagne: It is a type of sparkling light white wine which derives its
effervescence solely from the secondary fermentation of the wine within glass
containers of not greater than one gallon capacity, and which possesses the taste,
aroma, and other characteristics attributed to champagne as made in the
champagne district of france.
4. Sparkling light white wine: It has the tastem aroma and characteristics generally
attributed to champagne but not otherwise conforming to the standard for
champagne may, in addition to but not in lieu of the class designation sparkling
wine, be further designated as champagne style or champagne type or American
(or New York State, California, etc.) Champagne-bulk process.

Carbonated grape wine

Carbonated grape wine (including carbonated wine, carbonated red wine, and
carbonated white wine) is grape wine made effervescent with carbon dioxide other than
that resulting solely from the secondary fermentation of the wine within a closed
container, tank, or bottle.

Citrus wine
1. Citrus wine or citrus fruit wine is wine produced by the normal alcoholic
fermentation of the juice of sound, ripe citrus fruit (including restored or
unrestored pure condensed citrus must), with or without the addition, after
fermentation, of pure condensed citrus must, and with or without added fortifying
 citrus spirits or alcohol, but without other addition or abstraction except as may
occur in cellar treatment.
2. Light citrus wine or light cotrus fruit wine is citrus wine having an alcoholic
content not in excess of 14 per cent by volume.
3. Natural citrus wine or natural citrus fruit wine is citrus wine containing no
fortifying citrus spirits of added alcohol.
4. Citrus wine derived wholly (except for sugar, water, or added alcohol) from one
kind of citrus fruit, shall be designated by the word wine qualified by the name
kof such citrus fruit, e.g., orange wine, grape fruit wine.

Fruit wine
1. Fruit wine: It is wine (other than grape wine or citrus wine) produced by the
normal alcoholic fermentation of the juice of sound, ripe fruit (including restored
or unrestored pure condensed fruit must), with or without added fortifying fruit
spirits or alcohol, but without other addition or abstraction except as may occur in
cellar treatment.
2. Berry wine: It I fruit wine produced from berries.
3. Light fruit wine: It is fruit wine having an alcoholic content not on excess of 14
per cent by volume.
4. Natural fruit wine; It’s fruit wine containing no fortifying fruit spirits and added
alcohol.
5. Fruit wine derived wholly (expect for sugar, water, or added alcohol) from one
kind of fruit shall be designated by the word wine qualified by the name of such
fruit, e.g., peach wine, blackberry wine.

Chemical composition of wine

Nearly one thousand components have been identified. Wine contains 85 to 90%
water. It also comprised ethyl acid resulting from yeasts fermentation. All wine
incorporate some acidity from organic acids among, which is tartaric acid characteristic
of grape. Acetic and propionic acids are the volatile acid found on sound wines, acetic
acid is the principal volatile acid of young wines but wines contain traces of propionic
acid in addition. Formic acid is usually found in diseased wines, together with acetic
acid. The mineral composition of wine is special as it contains potassium, calcium,
magnesium, sodium, iron, sulfates, phosphorus, all of which necessary to cover daily
needs of human beings. Potassium salts and sulfates are known to facilitate diuresis.
Wine also comprises polyols among which glycol which gives the sweet taste. Wine
contains a small amount of azoted substances as well as 20 amineted acids among which
proline can be found. It is surprising to notice that the concentration of amineted acids
among which proline can be found. It is surprising to notice that the wine contains
vitamins of the group B, and, above all vitamin P which reinforces the cell-wall of
capillary vessels, lessening the risks of hemorrhage and oedema. Wine also comprises
more specific components. The phenolic component is an element whose molecule
incorporates several phenolic functions among which are phenolic acids, anthocyanes and
tannin.

Regions of production
A large part of the world’s wine is produced in the countries located near the
mediterranean Sea. France leads the manufacture of wine, followed by Italy and Spain.
Portugal, Greece, thye Balkan states, and Germany; Algeria and other region of south
Aferica; Chile and Argentia. Canada and the United states produce considerable
quantities of wine.

The Manufacture of Red Wine:

Outline of the process.-Selected grapes of the proper maturity are crushed and
stemmed; treated with sulphur dioxide, or a sulphite, or pasteurized; and inoculated with
a starter containing a pure culture of yeast. After a short fermentation period the wine is
drawn off, placed in storage tanks for further for further fermentation, racked, stored for
aging, clarified, and packaged.

Details of process
Grape
The production of a fine wine may be regarded as commencing worth the
selection of the best variety of grape for use in its manufacture.

The quality of the grapes of a given variety will depend upon the conditions under
which they are grown-soil, climate, and other conditions.

Grapes should be gathered at the proper stage of maturity. In order is determine

the degree of maturity, representative bunches of grapes are picked, and the Balling
degree of their juice is determined. A reading is 21 to 23⁰ Balling is usually given by the
juice of the grapes when the get the optimum stage of maturity.

Handling the grapes- In gathering the grapes and transporting them to the winery,
the prime purpose should be to have them arrive in the very best condition possible. The
grapes should be pocked with care, placed in clean containers and protected from
deterioration. Careful supervision of the handling grapes is essential.

Crushing the grapes- In gathering the grapes and transporting them to the metal
used in the construction of this machinery and other equipment about the winery is
important. Iron and steel are used in some wineries but are undesirable for they may
cause clouding of the wine, forming so-called “ferric case”. The tin and copper dissolved
from bronze by grape juice, if sufficient in stainless steel, nickel or in cones should be
used in preference to iron, ordinary steel, and many bronzes.

If the grapes are not picked when cool, it is desirable to permit them to cool
overnight before they are crushed.

Treatment before fermentation- Grapes contain on their surfaces a varied flora of

microorganisms-molds, yeast, and bacteria. It is quite possible that the juice of crushed
grapes will produce a good wine without any special precautions, but a wine
manufacturer cannot afford to gamble in respect to the quality of his final product by
destroying or inhibiting the development of the microorganisms found on the grape and
by the ue of starters containing pure cultures of the specific yeast desired.

Sulphur dioxide or sulphites destroy or inhibit the growth of many undesirable

types of microorganisms- acetic acid bacteria, wild yeasts, and molds-with a minimum
amount of injury to the true wine yeast. Usually 2 to 6 oz., or twice the quantity of
potassium metabisulphite, are added per ton of crushed grapes, the quantity used
depending on the condition of the grapes-their maturity, the degree of contamination with
molds, the temperature of the crushed product, and other factors. The largest quantities
are used when the grapes are overripe, moldy, or relatively warm.
Pasteurization may be used in place of sulphites but is not usually considered to
be so desirable.
Fermentation
The selection of a yeast, the nutrient substances in the must (grape juice), the
concentration of the sugar the acidity, the oxygen supply, and the temperature are factors
that must be supervised in respect to fermentation. Saccharomyces ellipsoideus is the
yeast used for the fermentation of must.

A starter is prepared from a pure culture of the selected yeast. Pasteurized must is
used as the culture medium in preparing the starter, the magnitude of which should
represent 2 to 5 per cent of the volume of the crushed grapes being inoculated.

The optimum concentration of sugar is 22 ⁰ balling. The use of much higher

concentrations of sugar favors tends to inhibit the fermentation when present in
concentrations of 13 to 15 per cent by volume, a maximum of 13 per cent is usually
desirable. The concentration that actually inhibits fermentation depends in part on
decreasing with increasing temperature. The approximate concentration of the alcohol
that will be produced in the wine can be predetermined by multiplying the Balling
reading of the must by 0.575.

It is permissible to reduce the concentration of sugar in must by the addition of

water, another practice is to mix the juice with high sugar concentration with a juice of
low sugar concentration. Occasionally sugar may be added to must.

Grapes that have been permitted to become too mature are frequently of low
acidity. Fruit acid-tartaric, citric, or malic acids-may be added to restore the normal
acidity.

A large supply of oxygen is essential for the rapid multiplication of yeast cells and
the starting of the fermentation, as stated under yeast manufacture while the later stage
characterized by alcohol and carbon doxide production rather than growth proceeds best
under nearly anaerobic condition.

Approximately 6 hr after treating the crushed grapes with sulphur dioxide or

sulphite, the starter is added. Thereafter the contents of the fermentation, to facilitate
aeration, temperature equalization, and the extraction of color and tannin. Normally a
“cap” forms on the surface of the fermentation vat, which contains grape skins, pieces of
stem, seeds and other suspended matter. To mix the contents of the tank, one may punch
down the cap or pump juice from the bottom of the vat wvwr the surface of the must.

The amount of aeration produced by mixing the contents of the tank is determined
by the effectiveness of the procedure and by the frequency at which the operation is
repeated. Provided that the fermentation is slow at the beginning, or near the end of the
incubation period, the supply of oxygen may be increased by more frequent mixing of the
contents of the vat. However, the must should not be over aerated during fermentation,
for over aeration is likely to produce a wine of inferior quality, especially insofar as color
and flavor are concerned.

Fermentation should be carried out at carefully controlled temperatures. The

finest wines are produced usually at temperatures below 85⁰F (29.4⁰C). The
development of bouquet and aroma are favoured by maintaining the fermenting mut at
low temperatures, around 70 to 90⁰F (21.1 to 32.2⁰C) is satisfactory. When the
temperature rises to 85⁰F (29.4⁰C) or at the most to 90⁰F (32.2⁰C), the mash should be
artificially cooled. Temperatures above 95˚F (35˚C) are considered unsafe, while the
fermentation is inhibited usually at the temperature of 90 to 100˚F (36.1 to
37.8˚C).Fermentation cease at a temperature of 105˚F (40.5˚C) generally. Undesirable
bacteria develop at the higher temperatures. Accordingly, the quality of wine is impaired.
Obviously, at too low temperatures, the fermentation is too slow to be practical.

During the fermentation, records of the temperature and the balling degree should
be made at least twice a day, one set of observation being recorded on the side of the
fermentation vat in order that the progress of the wine may be followed.

After 3 to 5 days of active fermentation, sufficient tannin and a maximum of color

have been extracted from the skin of the grape. Extraction is facilitated by the agitation of
pomace (skin, seed and piece of stems) during fermentation, by the ethyl alcohol
produced from the grape sugar, the heat of fermentation, and the mechanical breaking up
of the skin.

The wine maker decides when the color and tannin are satisfactory and then
draws off the wine to separate it from the pomace. He does not wait to for all the sugars
to be fermented. At the time of drawing of the wine, the balling reading may be 0 to 4˚. It
is not considered advisable to mix the wine drawn off (“free run-wine”) with that
expressed from the pomace, for the latter is of lower quality.

Further fermentation
The free-run wine is placed in closed storage tanks, equipped with bungs that
allow the excess carbon dioxide to escape. An atmosphere of carbon dioxide over the
wine tends to inhibit the development of acetic acid bacteria and other aerobic types of
microorganisms. The fermentable sugar is usually consumed in 7 to 11 days at a
temperature of 70 to 85˚F (21.1 to 29.4˚C). If the after fermentation becomes becomes
sluggish before the sugar is utilized, the yeast may be activated by pumping over the
wine.

Racking
By racking is meant the drawing off of the wine from the lees or sediment.
Potassium bitartrate (KHC4H4O6) i.e, cream of Tartar, is found in the lees. This
substance is less soluble in alcohol than in water and precipitates out more rapidly at low
temperatures. Wine is racked to facilitate its clearing and to prevent undesirable flavours
from being extracted from the old yeast.

Storage and aging

Two important changes take place during storage and aging: clearing of the wine
and the development of flavour.

In a new wine there are present substances which, if not removed, will produce a
sediment and probably cloudiness. These substances include tartrates, certain proteins,
and other matter. Naturally these substances would be removed by racking and filtration
during a somewhat long storage and aging process, but the modern trend is to hasten the
removal of these substances by methods that involve flash pasteurization (to precipitate
certain proteins), cooling to room temperature and then to 24 to 27°F (-4.44 to -2.78°C.),
and holding at the latter temperature for a few days. Filtration is carried out in the cold.
Since the acid content of the wine is frequently reduced by the foregoing rapid process it
is customary to adjust the acidity with citric or tartaric acids, the former acid being
preferred. The wine is placed in tanks for aging.
 Wine storage tanks are generally constructed of white oak or redwood, white oak
being the better of the two. The tanks are completely filled with wine and sealed to
prevent the access of large quantities of oxygen which would favour the growth of acetic
acid bacteria and Mycoderma vini (wine flowers). Some sequently, the tanks should be
inspected regularly and kept filled with wine. Periodically the wine is racked. During
racking and filling, especially, carbon dioxide is lost while some oxygen is absorbed. A
small amount of oxygen accumulates in the headspace over the wine.

Flavor, which is due to a combination of taste and odor, is developed in wine as a

result of oxidative changes and ester formation.

Aging proceeds slowly until oxygen becomes available in small quantities. It is

inhibited by the presence of large quantities of carbon dioxide, by sulphur dioxide, and by
the exclusion of air. New wines stored in airtight bottles do not age properly.
Alcohols, aldehydes, tannins, and other substances present in the wine are
oxidizable. Alcohols may be converted to aldehyde and subsequently to acids by
oxidation. Aldehydes from acetals with alcohol.

Combinations of alcohols with acids give rise to esters, which are important in the
production of aroma or bouquet. Although opinions differ concerning the importance of
esters, it is recognized that the nature of volatile esters is of greater significance than the
quantity. The esters of acetic acid contribute much to the flavour and bouquet of wine.
The time required for aging varies with the type of wine and the conditions. A dry
wine may require at least 2 years. Some fine wines are aged for 5 or more years.

Clarification
Wines may clear naturally over a period of time, but resort is frequently made to
the use of findings followed by filtration, heating, refrigeration, or combinations of the
foregoing. Fining agents, which include such substances as casein, gelatine and tannin,
bentonite (of Wyoming origin, if possible), is in glass (fish protein from the
sturgeon),white of egg, and Spanish clay, are mixed with the wine carefully according to
direction, or preferably after laboratory tests have been carried out with small portions of
the wine and the fining agents. The improper use of some of the fining agents may, in
itself, be a cause of clouding of the wine. Filtration is carried out with filter aids.
 Packaging
The clarified wine is placed in oak barrels for bulk sale and in bottles or in
cans for unit sale. Bottles of small and medium size may be pasteurized for 30 min, at
140°F.

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

Antibiotics:
Anti-against, bios-life, they are chemical substances secreted by some
microorganisms which inhibits the growth and development of microbes. Most of them
are produced by actinomycetes, specially the genus Streptomyces.

History
Sir Alexander flemming-Penicillin from Penicillium notatum, P. chrysogenum-
antibacterial properties relation to gram +Ve bacteria. S.A. Waksman-Streptomycin by
Streptomyces griseus and his associate’s isolation of Actinomycin, streptothricin,
streptomycin, neomycin.

Classification of antibiotics:
 Based on 1. Microbial source, 2. Mode of action
 Classification based on microbial source has the drawback that it is much too
broad.
 Have more than one mode of action/attacks many site.
Garrod, Lambert and O’grady classified based on their chemical structure
1. Penicillin and related antibiotics
2. Amino glycosides antibiotics
3. Macrolide antibiotics
4. Tetracycline
5. Peptide antibiotics
6. Antifungal antibiotics
7. Chloramphenicol
8. Unclassified antibiotics
1. Penicillin and related antibiotics (A)
They are 6-aminopenicillinic acid, it have β-lactum ring in their structure
responsible for antibiotic activity. Eg. Natural penicillins, semi synthetic penicillin
cephalosporins, Non-toxic to mammals.
2. Amino glycoside antibiotic
Structure have amino sugar. Eg. Streptomycin, neomycin, kanamycin,
paromomycin, gentamicin, tobramycin.

3. Macrolide antibiotics
It have Macrocyclic lactone ring to which sugars are attached. Eg. Erythromycin,
oleandomycin, spiromycin

4. Tetracycline antibiotic
It derivatives of polycyclic naphthalene carboxamide. Eg. Tetracycline,
chlortetracycline, Oxytetracycline.

5. Peptide antibiotics
It have D & L forms of peptide linked amino acid. Eg. Bacitracin, gramicide,
polymyxine.

6. Antifungal antibiotics
Sub groups a. Polyenes: Large ring. Eg. Nystatin, conjugated double amphotericin
beta bond system. b. Other antifungal antibiotics. Eg. Clotrimazole griseofulvin

7. Chloramphenicol
Nitrobenzene derivative of dichloroacetic acid unclassified antibiotics. Eg.
Cycloserine, fusidic acid, novobiocin.

Penecillin
Penicillin are a group of β-lactam containing bacterial antibiotics. Being the first
among the antibiotics to be discovered, penicillin are historically important. The basic
structure of all the penicillin consist of a lactam ring and a thizolidine ring fused together
to form 6-aminopenicillanic acid.

Action of penicillin
 Natural penicillin (penicillin V and G) are effective against several Gram positive
bacteria. They inhibit the bacterial cell wall (i.e. peptidoglycan) synthesis and cause cell
death. Some persons (approximately 0.5-2% of populations) are allergic to penicillin.
Natural penicillin are ineffective against microorganisms that produce beta
lactamase, since this enzyme can hydrolyses penicillin e.g. Staphylococcus aureus.
Several semi-synthetic penicillin that are resistant to beta lactamase have been developed
and successfully used against a large number of Gram-negative bacteria. Cloxacillin,
ampicillin, floxacillin and azlocillin are some examples of semi synthetic penicillin.
These are quite comparable in action to cephalosporins. From the huge quantities of
penicillin produced by fermentation, about 40% are used for human healthcare, 15% for
animal healthcare and 45% for the preparation of semi-synthetic penicillin.

Organisms for penicillin production

In the early days, Penicillium notatum was used for the large scale production of
penicillin. Currently, Penicillium chrysogenum and its improved mutant strains are
preferred. Previously the penicillin production used to be less than 2 units/ml and with
the new strains, the production runs into several thousands of units/ml. One of the high
yielding strains wis Q176 is preferred by several penicillin manufacturers.
Genetic engineering for improved penicillin production

Some of the genes involved in penicillin biosynthesis by P. chrysogenum have

been identified. Genetic manipulations were carried out so as to substantially increase the
penicillin production. For instance, extra genes coding for the enzymes cyclase and
acyltransferase have been inserted into C. chrysogenum.
Biosynthesis of penicillin

L-α-Aminoadipic acid combines with L-cysteine, and then with L-valine to form
a tripeptide namely α-L-aminoadipylcysteinylvaline. This compound undergoes
cyclization to form isopenicillin which reacts with phenylacetyl CoA (catalyzed by the
enzyme acyltransferase) to produce penicillin G (benzyl penicillin). In this reaction,
aminoadipic acid gets exchanged with phenylacetic acid.

Regulation of biosynthesis:
Some of the biochemical reactions for the synthesis of penicillin and lysine are
common. Thus, L-α-aminoadipic acid is a common intermediate for the synthesis of
penicillin and lysine. The availability of aminoadipic acid plays a significant role in
regulating the synthesis of penicillin. Penicillin biosynthesis is inhibited by glucose
through catabolite repression. For this reason, penicillin was produced by a slowly
degraded sugar like lactose. The concentration of phosphate and ammonia also influence
penicillin synthesis.

Production process of penicillin

The lyophilized culture of spores is cultivated for inoculum development which is
transferred to preferment and then to fermenter. Penicillin production is an aerobic
process and therefore, a continuous supply of O2 to the growing culture is very essential.
The required aeration rate is 0.5-1.0 vvm. The pH is maintained around 6.5, and the
optimal temperature is in the range of 25-27°C. Penicillin production is usually carried
out by submerged process.

The medium used for fermentation consists of corn steep liquor (4-5%) dry
weight) and carbon source (usually lactose). An addition of yeast extract, soy meal or
whey is done for a good supply of nitrogen. Sometimes, ammonium sulfate is added for
the supply of nitrogen. Phenylacetic acid for phenoxyacetic acid) which serves as a
precursor for penicillin biosynthesis is continuously fed. Further, continuous feeding of
sugar is advantageous for a good yield of penicillin. It is estimated that approximately
10% of the metabolized carbon contributes to penicillin production, while 65% is utilized
towards energy supply and 25% for growth of the organisms. The efficiency of penicillin
production can be optimized by adequate supply of carbon source. Thus, by adding
glucose and acetic acid, the yield can be increased by about 25%.

For efficient synthesis of penicillin, the growth of the organism from spores must
be in a loose form and not as pellets. The growth phase is around 40 hours with a
doubling time of 6-8 hours. After the growth phase is stabilized, the penicillin production
exponentially increases with appropriate culture conditions. The penicillin production
phase can be extended to 150-180 hours.

Recovery of penicillin
As the fermentation is complete, the broth containing about 1% penicillin is
processed for extraction. The mycelium is removed by filtration. Penicillin is recovered
by solvent (n-butylacetate or methylketone) extraction at low temperature (<10°C) and
acidic pH (<3.0). By this way, the chemical and enzymatic (bacterial penicillinase)
degradations of penicillin can be minimized.

The penicillin containing solvent is treated with activated carbon to remove

impurities and pigments. Penicillin can be recovered by adding potassium or sodium
acetate. The potassium or sodium salts of penicillin can be further processed (in dry
solvents such as n-butanol or isopropanol) to remove impurities. The yield of penicillin is
around 90%.

As the water is totally removed, penicillin salts can be crystallized and dried
under required pressure. This can be then processed to finally produce the pharmaceutical
dosage forms. Penicillin G and H are the fermented products obtained from the fungus
Penicillium chrysogenum.

Production of 6-amino penicillanic acid

The penicillin G and H are mostly used as the starting materials for the production
of several synthetic penicillin containing the basic nucleus namely 6-amino penicillanic
acid (6-APA). About 10 years ago, only chemical methods were available for hydrolysis
of penicillin to produce 6-APA. Now a days, enzymatic methods are preferred.

Immobilized penicillin amidases enzymes have been developed for specific

hydrolysis of penicillin G and penicillin V. Penicillin salt of either G or V can be used for
hydrolysis by immobilized enzyme system. The pH during hydrolysis is kept around 7-8
and the product 6-APA can be recovered by bringing down the pH to 4. At pH 4, 6-amino
penicillanic acid gets precipitated almost completely in the presence of a water
immiscible solvent.
In general, the enzymatic hydrolysis is more efficient for penicillin V than for
penicillin G. However, penicillin G is a more versatile compound, as it is required for
ring expansion.

Aminoglycosides
Aminoglycosides are oligosaccharide (carbohydrate) antibiotics. They contain an
aminocyclohexanol moiety which is bound to other amino sugars by glycosidic linkages.
More than 100 aminoglycosides are known e.g. streptomycin, neomycin, kanamycin,
gentamicin, hygromycin, sisomicin. Aminoglycosides are very potent antibiotic and act
against Gram positive and Gram negative bacteria, besides mycobacteria. At the
molecular level, aminoglycosides and to 30S ribosome and block protein biosynthesis.
Prolonged used of aminoglycosides causes damages to kidneys and hearing impairment.

For the treatment of severe and chronic infections, aminoglycosides are the
antibiotics of choice. Streptomycin was the first aminoglycoside that was successfully
used to treat tuberculosis (i.e. against Mycobacterium tuberculosis). Usually,
aminoglycosides are regarded as reserve antibiotic, since resistance may develop easily.
Organisms for aminoglycoside production

Aminoglycoside antibiotics are produced by Actinomyces sp. Recombinant DNA

techniques have been used to produce hybrid aminoglycosides and for increasing the
fermentation yield.

Biosynthesis of aminoglycosides
All the ring structures in the molecules of aminoglycosides are ultimately derived
from glucose. Most of the biosynthetic pathways concerned with the formation of at least
some aminoglycosides have been elucidated.

Biosynthesis of streptomycin
More than 30 enzymatic steps have been identified. Glucose 6-phosphate obtained
from glucose takes three independent routes to respectively produce streptidine 6-
phosphate, L-dehydrostreptose and N-methylglucosamine. The former two compounds
condense to form an intermediate are which later combines with methylglucosamine to
produce dihydrostreptomycin-6-phosphate. This compound, in the next to couple of
reactions, gets converted to streptomycin.

Regulation of biosynthesis
Very little is known about the regulation of streptomycin synthesis. A compound
named as factor A (chemically isocapryloyl-hydroxymethyl- γ-butyrate) has been isolated
from streptomycin producing strains of S. griseus. Factor A promotes streptomycin
production. In fact, factor A mutants that cannot synthesize streptomycin have been
isolated. They can synthesize streptomycin on adding factor A. The nutrient sources
carbohydrates (glucose), ammonia and phosphate also regulate (by feedback mechanism)
streptomycin production.
Production process of streptomycin
The medium used for streptomycin usually consists of soy meal or soy flour or
corn syrup that can supply glucose at a slow rate (amylase activity is poor in
Streptomyces sp). The initial supply of nitrogen (NH3) and phosphate is also obtained
from soy meal. This is required since glucose, ammonia and phosphate in high quantities
inhibit streptomycin synthesis. The fermentation condition for optimal production of
streptomycin are temperature 27-30°C, pH 6.5-7.5, aeration rate 0.5-1.0 vvm. The
duration of fermentation process depends on the strain used and is between 6 to 8 days.

Recovery of streptomycin

Streptomycin or other aminoglycosides are basic in nature. They can be recovered

by weak cationic exchange resins in an ion-exchange column. Treatment with activated
carbon is often necessary to remove impurities. Streptomycin can be precipitated in the
form of sulfate salt.

Production of vitamins microbial fermentation

Vitamin B12
The disease, pernicious anemia characterized by low levels of hemoglobin
decreased number of erythrocytes and neurological manifestations, has been known for
several decades. It was in 1926 some workers reported the liver extracts could cure
pernicious anemia. The active principle was later identified as vitamin B12, a water
soluble B-complex vitamin.

Occurrence
Vitamin B12 is present in animal tissue at a very low concentration (e.g 1 ppm in
the liver). It occurs mostly in the coenzyme forms-methylcobalamin and
deoxyadenosylcobalamin. Isolation of vitamin B12 from animal tissue is very expensive
and tedious.

Chemistry
Vitamin B12 (cyanocobalamin) is a water soluble vitamin with complex structure.
The empirical formula of cyanocobalamin is C63H90N14O14PCO. The structure of
vitamin B12 consists of a corrin ring with a central cobalt atom. The corrin ring is almost
similar to the tetrapyrrole ring structure found in other porphyrin compunds e.g. heme
(with Fe) and chlorophyll (with Mg).

The corrin ring has four pyrrole units. Cobalt present at the centre of the coring
ring is bonded to the four pyrrole nitrogens. Cobalt also binds to dimethylbenzimidazole
and aminoisopropanol. Thus, cobalt atom present in vitamin B12 is in a coordination
state of six.

Biosynthesis
Vitamin B12 is exclusively synthesized in nature by microorganisms. The
biosynthesis of B12 is comparable with that of chlorophyll and hemoglobin. Many of the
reactions in the synthesis of vitamin B12 are not yet fully understood.

Commercial production of vitamin B12

Vitamin B12 is commercially produced by fermentation. It was first obtained as a
byproduct of Streptomyces fermentation in the production of certain antibiotics
(Streptomycin, chloramphenicol, or neomycin). But the yield was very low. Later, high
yielding strains were developed. And at present, vitamin B12 is entirely produced by
fermentation. It is estimated that the world’s annual production of vitamin B12 is around
15000 kg.

High concentrations of vitamin B12 are detected in sewage sludge solids. This is
produced by microorganisms. Recovery of vitamin B12 from sewage sludge was carried
out in some parts of United States. Unlike most other vitamins, the chemical synthesis of
vitamin B12 is not practicable, since about 20 complicated reactions steps need to be
carried out, fermentation of vitamin B12 is the only choice.

Microorganisms and yields of vitamin B12

Several microorganisms can be employed for the production of vitamin B12, with
varying yields. Glucose is the most commonly used carbon source. The most commonly
used microorganisms are Propionibacterium freudenreichii, Pseudomonas denitrificans,
Bacillus megaterium and Streptomyces olivaceus.

Genetically engineered strains for vitamin B12 production

 By employing modern techniques of genetic engineering, vitamin B12 production
can be enhanced. A protoplast fusion technique between Protaminobacter rubber and
Rhodopseudomonas spheroids resulted in a hybrid strain called Rhodopseudomonas
protamicus. The new strain can produce as high as 135 mg/l of vitamin B12 utilizing
carbon source.

Production of vitamin B12 using Propionibacterium sp.

Propionibacterium freudeneichii and P. shermanii and their mutant strains are
commonly used for vitamin B12 production. The process is carried out by adding cobalt
in two phases.
Anaerobic phase:
This is a preliminary phase that may take 2-4 days. In the anaerobic phase 5’-
deoxyadenosylcobinamide is predominantly produced.

Aerobic phase:
Vitamin B12 is present in animal tissue at a very low concentration (e.g 1 ppm in
the liver). It occurs mostly in the coenzyme forms-methylcobalamin and
deoxyadenosylcobalamin. Isolation of vitamin B12 from animal tissue is very expensive
and tedious. Vitamin B12 is present in animal tissue at a very low concentration (e.g 1
ppm in the liver). It occurs mostly in the coenzyme forms-methylcobalamin and
deoxyadenosylcobalamin. Isolation of vitamin B12 from animal tissue is very expensive
and tedious. In recent years, some fermentation technologists have successfully clubbed
both an anaerobic and aerobic phases to carry out the operation continuously in tow
reaction tanks. The bulk production of vitamin B12 is mostly done by submerged
bacterial fermentation with beet molasses medium supplemented with cobalt chloride.

Recovery of vitamin B12:

The cobalamins produced by fermentation are mostly bound to the cells. They can
be solubilized by heat treatment at 80-120°C for about 30 minutes at pH 6.5-8.5. The
solids and mycelium are filtered or centrifuged and the fermentation broth collected. The
cobalamins can be converted to more stable cyanocobalamins. This vitamin B12 is
around 80% purity and can be directly used as a feed additive. However, for medical use
(particularly or treatment of pernicious anemia), vitamin B12 should be further purified
(95-98% purity).

Production of vitamin B12 using Pseudomonas sp

Pseudomonas denitrificans is also used for large scale production of vitamin B12
in a cost-effective manner. Starting with a low yield (0.6 mg/l) two decades ago, several
improvements have been made in the strains of P. denirificans for tremendous
improvement in the yield (60 mg/l). Addition of cobalt and 5,6-dimethylbenzimidazole to
the medium is essential. The yield of vitamin B12 increases when the medium is
supplemented with betain (usual source being sugar beet molasses).

Carbon sources for vitamin B12 production

Glucose is the most commonly used carbon source for large scale manufacture of
vitamin B12. Other carbon sources like alcohols (methanol, ethanol, isopropanol) and
hydrocarbons (alkanes, decane, hexadecane) with varying yields can also be used. A
yield of 42 mg/l of vitamin B12 was reported using methanol as the carbon source by the
microorganism Methanosarcina barkeri, in fed-batch culture system.

Riboflavin (B2)
Riboflavin (B2) is a water soluble vitamin, essential for growth and reproduction
in man and animals. Deficiency of riboflavin in rats causes growth retardation, dermatitis
and eye lesions. In human vitamin B2 deficiency results in cheilosis (fissures at the
corner of mouth), glossitis (purplish tounger) and dermatitis. Riboflavin exerts its
biochemical functions through the coenzymes namely flavin adenine dinucleotide (FAD)
and flavin mononucleotide (FMN).

Occurrence
Riboflavin occurs in milk and milk products, meat, eggs, liver and kidney. While
in milk and eggs, it is present in free form, in others foods it is found in the form of
flavoproteins (i.e. coenzymes of riboflavin bound to proteins).

Chemistry
 Riboflavin contains 6, 7-dimethyl isoalloxazine (a heterocyclic 3 ring structure)
attached to D-ribitol by a nitrogen atom. The isoalloxazine ring participates in the
oxidation reduction reactions brought out by the coenzymes (FAD and FMN).

Biosynthesis
The biosynthetic pathway of riboflavin, elucidated for the microorganisms
Ashbya gossypii and Eremothecium ashbyii. The over production of riboflavin in these
organism takes place mainly due to the constitutive nature of the riboflavin synthesizing
enzymes iron which inhibits the production of vitamin B2 in clostridia and yeasts has no
effect on A. gossypii and E. ashbyii.

Commercial production of riboflavin

There are three processes employed for the large scale production of riboflavin.
The worldwide requirement of riboflavin is estimated to be around 2500 tons per year.

1. Biotransformation:
About 50% of the world’s requirement of riboflavin is produced by
biotransformation followed by chemical synthesis. For this purpose, glucose is first
converted to D-ribose by mutant strains of Bacillus pumilus. The D-ribose so produced is
converted to riboflavin by chemical reactions.

2. Chemical synthesis:
Approximately 20% of the world’s riboflavin is produced by direct chemical
synthesis.

3. Fermentation:
At least one third of world’s riboflavin requirements are met by direct
fermentation processes.

Microorganisms and yields of riboflavin

Several microorganisms (bacteria, yeasts and fungi) can be employed for the
production of riboflavin. In the acetone-butanol fermentation, employing the organisms
Clostridium acetobutylicum and C. butylicum riboflavin is formed as a byproduct.
Commercial production of riboflavin is predominantly carried out by direct fermentation
using the ascomycetes. The two plant pathogens namely Ashbya gossypii and
Eremothecium shbyii are most commonly employed due to high yield. Among these tow
organism A. gossypii is preferred as it is more stable with a high producing capacity of
riboflavin.

Genetically engineered strains for riboflavin production:

High yielding strains of Ashbya gossypii have been developed by genetic
manipulations. Such strains can yield as high as 15g/l riboflavin.

Production process of riboflavin

Industrial production of riboflavin is mostly carried out with the organism,
Ashbya gossypii by using simple sugars such as glucose and corn steep liquor. Glucose
can be replaced by sucrose or maltose for the supply of carbon source. In recent years,
lipids such as corn oil, when added to the medium for energy purpose, have a profound
influence on riboflavin production. Further, supplementation of the medium with yeast
extract, peptones, glycine, inositol, purines (not pyrimidines) also increases the yield of
riboflavin.

It is essential to carefully sterilize the medium for good yield of riboflavin. The
initial pH of the culture medium is adjusted to around 6-7.5. The fermentation is
conducted at temperature 26-28°C with an aeration rate 0.3 vvm. The process is carried
out for about 5-7 days by submerged aerated fermentation.

Riboflavin fermentation by Eremothecium ashbyii is comparable to that described

above for Ashbya gossypii. Candida sp can also produce riboflavin, but this fermentation
process is extremely sensitive to the presence of iron. Consequently, iron or steel
equipment cannot be used. Such equipment has to be lined with plastic material.

Fermentation through phases

Some studies have been carried out to understand the process of fermentation of
riboflavin particularly by ascomycetes. It is now accepted that the fermentation occurs
through three phases.

Phase I:
 This phase is characterized by rapid growth of the organism utilizing glucose. As
pyruvic acid accumulates, pH becomes acidic. The growth of the organism stops as
glucose gets exhausted in phase I, there is no production of riboflavin.

Phase II:
Sporulation occurs in this phase and pyruvate concentration decreases.
Simultaneously, there is an accumulation of ammonia (due to enhanced deaminase
acitivity) which makes the medium alkaline. Phase II is characterized by a maximal
production of riboflavin. But this mostly in the form of FAD and a small portion of FMN.

Phase III:
In this last phase, cells get disrupted by a process of autolysis. This allows release
of FAD, FMN and free riboflavin into the medium.

Recovery:
Riboflavin is found in fermentation broth and in a bound form to the cells. The
latter can be released by heat treatment i.e. 120°C for about 1 hour. The cells can be
discarded after filtration or centrifugation. The filtrate can be further purified and dried as
per the requirements.

Other carbon sources for riboflavin production

Besides sugars, other carbon sources have also been used for riboflavin
production. A pure grade of riboflavin can be prepared by using Saccharomyces sp.
Utilizing acetate as sole carbon source. Methanol utilizing organism Hansenula
polymorpha was found to produce riboflavin. The other carbon sources used with limited
success for riboflavin production are aliphatic hydrocarbons (organism Pichia
guilliermoudii) and n-hexadecane (organisms-Pichia miso).

Lecture 16
16. Microbes in food industry – Single Cell Protein, Baker’s and Brewer’s yeast, Dairy
products – cheese and yoghurt

Commercial Production method of Single Cell Protein - Spirulina

 After the process of fermentation is over, the exhausted bacteria can be separated
from the broth by filtration. This cell mass has a number of names, such as “microbial
biomass” or “single cell protein” (SCP).
 Microbial biomass is a biproduct of all fermentation processes but in some cases it is
actually the sole target product.
 Bacterial cells have a high content of protein, but are low in fat and cholesterol. This
explains the names “single cell protein” (SCP) or “microbial protein”.

Solution to world food problem

Definition - Purified, dried microorganisms used as source of protein. The term
single cell protein (SCP) was coined at Massachusetts Institute of Technology (MIT) by a
group of scientists in 1966. On an average, the microbial biomass contains about 45 to
55% protein, although in some bacteria the protein content is as high as 80%.The biomass
also contains other essential nutrients as well, and as such it is an ideal supplement to
conventional food supply.

Advantages of SCP
• It has high protein and low fat content.
• It is good source of vitamins particularly B-complex. eg. Mushrooms and Yeasts
• It can be produced through-out the year.
• Waste materials are used as substrate for the production of these proteins. It
reduces the environmental pollution and helps in recycling of materials.
• SCP organisms grow faster and produce large quantities of SCP from relatively
small area of land and time.
• During the production of SCP biomass, some organisms produce useful bye
products such as organic acids

Criteria for selection of microbes as SCP

• Must be one that is edible and can serve as a feedstock for humans and/or
livestock
• Organisms must grow rapidly and vigorously
• Culture of the organism should involve the use of relatively simple growth units
and inexpensive nutrient sources (e.g., commercial crop fertilisers)
 • Ideally, the organism could be grown in open culture, or at least as an enrichment
culture

As compared with traditional methods of producing proteins for food or feed,

large scale production of microbial biomass has the following advantages:
b. Microorganisms in general have a high rate of multiplication.
c. Microbes have high protein content.
d. They can utilize a large number of different carbon sources, some of which are
waste products
e. Strains with high yield and good composition can be selected or produced
relatively easily.
f. Microbial biomass production is independent of seasonal and climatic variation.
g. In India, at CFTRI (Central Food Technology Res. Institute), Mysore, research is
being conducted on the use of blue green alga, Spirulina as a supplement to diet
(food and feed). The, alga is cultured, dried, powdered and then used in the form
of one gram tablets. It contains 60% protein, essential vitamins and unsaturated
fatty acids.

Disadvantages
 Not suitable for human consumption because they are rich in Chlorophyll.
(Except Spirulina)
 It has low density i.e. 1-2 gm dry weight/litre of substrate.
 There is lot of risk of contamination during growth.
 These have high nucleic acid (RNA) content.
 Filamentous fungi show slow growth rate than yeasts and bacteria.
 Some strains produce mycotoxins and hence they should be screened.
 Risk of contamination is very high during the production process.
 Recovering the cells is a bit problematic.
 Endotoxin production should be carefully tested.

Examples of bacteria used as food

Methylophilus methylotrophus grown on methanol, and proposed as animal feed.
70% protein. Problems with commercial viability due to subsidisation of alternative
animal feeds.
Examples of Yeasts and Fungi used as food
• The filamentous fungi such as Chaetomium celluloliticum – grows on cellulose
waste,
• Fusarium graminearum –grows on starch
• Paecilomyces varioti – grows on sulphur liquor. 50 – 55 % protein.
• Yeasts such as Candida utilis (Torula yeast), Candida lipolytica – grow on
Ethanol
• Saccharomyces cervicea – grows on molasses, consists of high protein with good
balance of amino acids and rich in B – complex vitamins. It is more suitable as
poultry feed.
• Torula yeast as a food is obtained through fermentation using molasses as
substrate. It has high protein – carbohydrate ratio than forages. It is rich in lysine
but poor in methionine and cysteine.

The raw materials that can be used for single-cell protein manufacture include
whey, sulphate waste liquors, hydrocarbon waste from the pet petroleum industry, and
the vats used to produce alcoholic beverages. The production process involves growth of
the organisms in large fermenting tanks with forced aeration for vigorous cell-growth.
Manufacture process used by British Petroleum Industry for single-cell protein from
hydrocarbons is represented in Figure.

Diagrammatic Representation of Commercial Production of Single Cell from

Hydrocarbons
Flow diagram of cultivation of algae in sewage oxidation ponds

Single Cell Protein SCP Process

Regardless of the type of substrate or organism employed, the production of SCP
involves following basic steps:
a. Preparation of suitable medium with suitable carbon source
b. Prevention of contamination of medium and the plant
 c. Production of the desired micro organism
d. Separation of microbial biomass and its processing

The medium for SCP production varies according to the microorganism. Among
other things, the medium must contain a carbon source for cultivating the heterotrophic
microorganisms, although green algae (Chlorella, Scenedesmus, Spirulina, etc.) can be
cultivated autographically without a dissolved carbon source.

Algae as SCP
Algae as a source of SCP for human consumption has been receiving worldwide
attention. They have been reported from quite different environments: fresh water
streams, brackishwater, sea, tidal ponds and saline ponds. Spirulina contains around 60%
protein and can play an important role in human nutrition as a protein supplement.
Spirulina is used for food from time immermorial by tribes living around Chad
Lake in Africa. The predominant species of phytoplankton of the lake is Spirulina
platensis. The dried mat of Spirulina in the form of biscuits is used to prepare the sauce
that accompanies millet meal eaten by the kannebou population, the predominant ethnic
group of Chad Republic.

Benefits from Spirulina SCP

 Mass cultivation of Spirulina offers several advantages over Chlorella and
Scenedemus as given below:
 Being a filamentous alga, Spirulina can be harvested by simple and less expensive
methods such as nylon or cotton cloth filter.
 Filaments of Spirulina float on water surface due to presence of gas vacuoles.
Hence, there is no problem of harvesting unlike Chlorella and Scenedesmus.
 There is least chance of contamination in growth tanks of Spirulina as it grows at
high alkaline pH 8-11.
 Heat drying is sufficient for Spirulina as it has thin cell wall, whereas spray
drying is required for Chlorella and Scenedesmus which is expensive
 Researches done by UNIDO programme in Mexico (1980), Mexican National
Institute of Nutrition, Hyderabad (1988, 1990) under Indian Council of Medical
Research on several aspects of possible adverse changes in multigeneration
feeding tests on laboratory animals and humans have shown no adverse effects.
  Spirulina is highly digestive (85-95%) due to thin wall and low nucleic acid
contents (4%). It contains high percentage of digestible proteins (62-72%),
vitamins, amino acids and other nutrients (Table). The aminogram of Spirulina is
comparable to the FAD, milk and egg protein pattern.

Mass Cultivation of Spirulina SCP

At present two types of farms for mass production of Spirulina SCP are under
operation. A third type (i.e. enclosed system using transparent tube, biocoil or
photobioreacter) is under development.

I. Semi-natural lake system.

Sosa Texcoco Lake (Mexico) and Lake Chand (Africa) offer an ideal environment
for the natural growth of Spirulina. The product is expensive but of low quality due to
contamination and pollution by uncontrolled natural factors. SCP of these lakes is good
for fish and animal feed.

II. Artificially built cultivation system.

In artificially built cultivation system, waste water or clean water can be used for
mass cultivation. The diluted sewage, liquid effluents from agro industries or biogas
plants are used after removal of solids and incorporation of nutrients. Fertilized sea water
is also used in waste water system. The microalgae thus produced can be used as feed in
aquaculture, animal feed and source of fine chemicals. In clean water system, low cost
medium with nutrients (urea, magnesium sulphate, sodium bicarbonate, rock phophsate
and FYM extract) promote the growth of spirulina

a) Clean water system

This system is more expensive because of construction of artificial cultivation
farms. These have shallow raceway ponds circulated with paddle wheels and high quality
of nutrients. pH of the water must be initially maintained to 8.5. It is a self pH adjusting
alga which elevates the pH between 10-10.5. At this pH levels there is the least chance of
contamination.

In India, food grade Spirulina is cultivated at two main centres, one at Shri
A.M.M. Murugappa Chettiar Research Central (MCRC), Madras, and the other at Central
Food Technology and Research Institute (CFTRI), Mysore. Madras centre is the biggest
food grade Spiulina farm in India. Its annual production capacity is of about 75 tonnes.
The products are marketed in India and abroad as health food, baby food and
multivitamin tablets.

(b) Waste water system

This system is applicable in highly populated countries like India where wastes
are generated in high quantities and pose environmental problem. In this system, human
and animal wastes and sewage are used for growth of Spirulina. The wastes are added
into the digester to settle down the solid particles. The liquid effluent is used as a source
of the nutrients and added in artificially constructed ponds. As desired NaNO3 and
NaHCO3 are also mixed. S. platensis is found to grow better in sewage amended with
NaCO3 and nutrients in different proportion and also in diluted sewage.
When full growth of Spirulina is over, it is screened from the pond and added to
aquaculture to feed fish or dried in a small solar drier for human food.
This system is most suitable for third world countries where wastes are the major
sources of pollution.

Growth Requirements for Spirulina

Algal tanks
Generally, circular or rectangular cemented tanks are constructed. The circular
tanks are more preferred over the rectangular one because of ease in handling. Size may
be according to convenience and yield needed. Depth should be about 25cm. Open tanks
are suitable for tropical and subtropical regions.

Light
Low light intensity is required at the beginning to avoid photolysis. Spirulina
exposed to high light intensity is lysed.
Temperature
Optimum temperature is required for growing spirulina rages from 35-40°C.
pH
The pH ranging from 8.5 to 10.5 optimum for growth of spirilina. Initially, culture
should be maintained at pH 8.5 which automatically is elevated to 10.5.

Agitation.
 Agitation of culture is very necessary to get good quality and better yield. The
culture is agitated by brush, paddle power, pipe pumps, wind power, rotators, etc.

Harvesting.
The filaments of Spirulina float on surface of water forming thick mat. Therefore,
it can be harvested by fine mesh steel screens, nylon or cotton cloths, etc.

Drying.
As it has a thin wall, sun drying is the most suitable and economical.
Yield
An average yield of 8-12 g Spirulina powder/m2/day has been obtained in India
and other countries. This is equivalent to 20 tonnes /ha/annum. In warmer climate, the
yield can increased to about 20 g/m2/day.
Dried Spirulina powder is packed in aluminium bags or sealed in bottles and sent
to market.

Uses of Spirulina Single Cell Protein

a) As protein supplemented food.
b) As health food.
c) In the therapeutic and natural medicine.
d) In cosmetics.

Baker’s Yeast biomass production:

The living cells of aerobically grown Saccharomyces cerevisiae are collectively
referred to as Baker’s yeast. Baker’s yeast is commercially available either as a dried
powder i.e. dry yeast with about 95% dry weight or in the form of cakes (about 25-30%
dry weight). These commercially available yeast preparations can be used in bread
making.

Production of baker’s yeast

The medium for baker’s yeast production contains molasses, ammonium salts,
vitamins, phosphates and antifoam agents. Sugarcane or sugar beet molasses with a sugar
concentration of 45-50% is usually preferred. Baker’s yeast production is carried out by
an aerobic fed-batch process.
The Manufacturing of Yeast
The manufacturing process for yeast can be likened to farming – it involves
preparation, seeding, cultivation and harvesting. In the commercial production of yeast,
molasses is used to provide this sugar source.

Quality Assurance
In all the yeast processes, utmost care is taken to produce a product of the highest
possible quality and purity. Samples are routinely checked by the laboratory and frequent
cleaning and sterilization of the equipment are conducted to assure the proper standards
are met.

Preparation
Before feeding molasses to the yeast cells, it must be clarified and sterilized. This
is done in order to assure the final yeast color. The sterilizing also prevents bacteria and
other organisms from being introduced during manufacturing. The molasses is then
diluted with water, adjusted for acidity, heated until almost boiling and filtered through
heavy clothes.

Seeding
The seed yeast is a carefully maintained laboratory culture so as to avoid
contamination by “wild” yeast present in the air. Yeast seeds are selected with care
according to the type of yeast to be produced and the specific characteristics desired. All
cultures are laboratory pure; all transfers are made with absolute sterility; all vessels are
completely sterilized. The “seed yeast” is placed in small flasks where it is allowed to
grow. It is then transferred in a series of steps from these small flasks to tanks of about
1,000 gallons in volume. Now known as “stock yeast”, it is separated from the alcohol
generated by the fermentation and stored in refrigerated tanks for the subsequent
fermentation cultivation.

Cultivation
 The cultivation or advancement of the fermentation process is accomplished in
large 40,000-gallon vessels. The “stock yeast” is fed measured quantities of molasses and
large quantities of air. The temperature is carefully controlled and acidity (pH) frequently
adjusted through the addition of ammonium salts. This process is continued until the
yeast achieves the capacity of these 40,000-gallon fermenting tanks. The yeast is then
harvested.

Harvesting
The harvesting of yeast is nothing more than concentrating the yeast cells by
passing the fermented liquid through large centrifugal pumps called “separators”. The
result is an off-white liquid called “cream yeast”. Further processing is dependent on the
type of yeast desired.

Fermented Dairy products

Cheese
Cheese production is the largest dairy industry in the world. There are around
1000 types of different cheeses. They are broadly of two types: unripened cheese
(Cottage cheese with low fat, cream cheese with high fat) and ripened cheeses (hard
cheese ex: chedder, blue cheese, soft cheese ex: limburger, camembert). Irrespective of
the type of the cheese all of them are invariably made from the casein of milk, that is
produced after separating the whey (liquid portion of milk). Milk from different animals
can be used ex: sheep, cow, goat, buffalo.

Historical perspective;
The use of animal stomachs for carrying liquids is centuries old. When milk was
transported in this fashion, the formation of solids was observed. The solids were
concentrated after draining liquids. These solids were salted and consumed later. A good
example of food preservation, long long ago!. We now know that this solid portion is the
cheese. It is produced by the combined action of enzymes (rennet) of the stomach living
and the bacterial contamination.

Production process
 As already stated, cheese is produced from milk. This is carried out by a process
of dehydration wherein casein and fats are concentrated 5-15 fold. Cheese production is
very complicated, and broadly involves four stages – acidification of milk, coagulum
formation, separation of curd from whey and ripening of cheese
Acidification of milk: By employing lactic acid bacteria (Streptococus lactis,
Lactobacillus lactis) the sugar of milk (lactose) can be converted to lactic acid. This
lowers the pH to around 4.6, and thus acidifies milk.

Coagulum formation:
When the acidified milk is treated with rennet (i.e., the enzyme chymosin of
animal or fugal origin), casein gets coagulated. Casein mainly consists of three
components insoluble ∂ and ß caseins and a ѓ casein that keeps them in soluble state. By
the action of chymosin, ѓ casein is degraded. Consequently, ∂ and ß caseins and the
degraded product of ѓ casein combine to form a coagulum (curd). This process of
coagulation is dependent on calcium ions.

Separation of curd from whey:

When the temperature of the coagulum is raised to around 40 0C; the coagulum
(curd) and whey (fluid portion) get separated. The separated curd is cut into blocks,
drained and pressed into different shapes.

Ripening of cheese:
The flavor of raw cheese (with rubber texture) such as cheddar is bland. Ripening
imparts flavours, besides making changes in its texture. The procedures adopted for
ripening (or maturation) are highly variable depending on the type of cheese to be
prepared. The blocks of curd separated are subjected to the action of proteases and/or
lipases. Alternatively, they may be inoculated with certain fungi (Ex: Penicillium
roquefortii). The hydrolysis of proteins and fats (either by enzymes or microorganisms)
results in certain compounds which imparts flavor to the cheese. Mild hydrolysis of fats
(or cheese), usually carried out by lipases or Aspergilus niger or Mucor maihai results in
butyric acid formation with characteristic flavor

Sources of chymosin for cheese production

There are several sources of rennet (chymosin enzyme) for cheese production.
These include calves, adult cows, pigs and fungal sources. Fortunately, the fungal (Mucor
meihei) sources of chymosin are almost comparable to the animal sources and are widely
used in some countries. However some people always prefer animal rennet used cheese
due to its slight superior flavor.

Commercial cheese production is a modification of ancient practice, with careful

control of fermentation. Cheese is the product made from the curd of the milk of cows
and other animals. The curd is obtained by the coagulation of the milk casein with an
enzyme (usualy rennin), an acid (usually lactic acid) and with or without further
treatment of the curd by heat, pressure, salt and ripening (usually with selected
microorganisms). All cheese types begin with curd making and then involve various
treatment of curd or whey.
There are over 800 kinds of cheeses. However there are only about 18 distinct types of
natural cheeses.

Types
• Roquefort cheese – ripened by Penicillium roqueforti also known as Blue cheese.
• Swiss cheese – Propionic bacteria, Swiss having eye like structure, due to
production of carbondioxide by propionic bacteria
• Camemberg cheese – Penicillium camembergi – Also known as soft cheese
(Amount of moisture is more so it cannot store for a longer time)

Cottage cheese
Cottage cheese is a soft cheese generally coagulated with lactic acid rather than
renin. The curd is not pressed, aged or ripened. Starting with pasteurized skim milk rather
than whole milk the curd forming operations have many similarities to the early stages of
all cheese making.

Swiss cheese
This is a hard type cheese, characterized by large holes or eyes and a sweet nutty
flavor obtained through the activities of Propionibacterium shermanii. This organism
follow the growth of lactic acid organisms and ferments and fermetns lactic acid to
propionic acid and carbondioxide. The propionic acid contributes to the nutty flavor and
the carbondioxide gas collects in pockets within the ripening curd and forms the eyes.
Swiss starter, lactic acid organisms including Lactobacillus bulgaricus, which
produces lactic acid through the rather high cooking processing.
Yoghurt
Yoghurt is produced by fermenting whole milk employing a mixed culture of
Lactobacillus bulgaricus and Streptococcus thermophilus. While L. bulgaricus produces
acetaldehyde that imparts a characteristic taste, S. thermophilus results in the formation
of lactic acid to give acid flavor. In addition, both these bacteria produce extracellular
polymers that increase the viscosity of the fermented milk. Yoghurt is very delicious and
in fact frozen yoghurt is becoming popular as an alternative to icecream.

Koumiss
Koumiss is similar to kefir, but is made from mare’s milk. Mare’s milk does not
coagulate at the electric point of casein and hence koumiss, which contains about 0.7 –
1.8 % lactic acid and 1-2.5 percent ethanol is not a curdled product. The microorganisms
involved in kumiss fermentation are Lactobacillus bulgaricus and lactose fermenting
yeasts. The major end products are lactic acid, ethanol and carbondioxide. Koumiss also
fizzes and effervesces like kefir. Kousmiss is considered to be a therapeutic drink.

Bulgarian milk
Bulgarian milk is an extremely high acid and acrid product. It is made from whole
milk. The milk is heated at 85 0C for 30 minutes, cooled to 37 -38 0C and inoculated
with 2% milk starter made from Lactobacillus bulgaricus. The milk is incubated until the
acidity reaches at least 1.4% and is then cooled to 7 0C. In some cases, the acidity
reaches at least 1.4% and is then cooled to 7 0C. In some cases, the acidity of the product
may be as high as 4%.
Acidophilus milk
Acidophilus milk or reform yogurt is the product obtained by fermenting milk
with an authentic culture of L. acidophilus. The therapeutic value of milk containing
viable L. acidophilus in controlling intestinal disorders.

Traditional acidophilus milk is an extremely sour product. To stimulate growth to

L. acidophilus, the milk needs to be heated at 120 0C for 15 min, which denatures and
release some peptides from proteins in the milk. After heat treatment, the milk is cooked
to 37 -38 0C, inoculated with 5% milk starter, and incubated at the same temperature for
18 – 25 hours. It is cooled to 70C after the acidity reaches 1.0 per cent and bottled.

Leben and Dahi

 Laben is a concentrated yogurt product and the condensing amy be accomplished
by hanging the fermented curd in a cloth bag which allows the whey to drain out as the
curd shrink and is squeezed out by its own weight. The predominant flora of leben consist
of Streptococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus and lactose
fermenting yeasts. The product is made from sheep’s cow’s goat’s or buffalo’s milk or a
mixture of two or more of these milks. A portion is saved from the previous days
production is used as a starter.

Dahi is the Indian equivalent of yogurt. Milk is brought to the boil to destroy
pathogenic and spoilage microorganisms. The microorganisms involved in dahi
fermentation include Streptococcus lactis, Streptococcus thermophilus, Lactobacillus
bulgaricus, Lactobacillus helveticus, Lactobacillus plantarum and lactose fermenting
yeasts.

Vilia
Vilia or filia is a finish (Finland) fermented milk. This product is made from fresh
unheated cow’s milk or pasteurizd milk. The starters used consist of a mixture of
Streptococcus lactis and Streptococcus cremoris. The milk is tempered to 17 -18 0C,
inoculated with starter, and held at this temperature until a smooth uniform coagulum is
formed. The product has a stringy texture pleasant acid taste and good diacetyl flavor.
Vilia may be eaten plain or with sugar or cinnamom.

Lecture 17

17. Biofuels – alcohol and biodiesel production

MICROBIAL FUELS – METHANE, HYDROGEN, ETHANOL AND ALGAL

BIODIESEL

Biofuels
Energy may be considered as a form of mate which is interconvertible. The
modern man is mostly dependent on three sources for his energy needs coal, natural gas
and oils collectively referred to as fossil fuels or fossil energy sources. The fear of
depletion of global fossil fuels has forced man to look for suitable alternative energy
sources such as solar, hydro, tidal and wind power and most recently nuclear energy. In
addition to these advances in biotechnology have helped to fruitfully utilize the energy
from biological systems.

Production of biogas from biomass

Biogas is a mixture of gases composed of methane (50-60%), carbon dioxide (15-
40%), nitrogen (4%) and hydrogen sulfide (1%) with trace of hydrogen, oxygen and
carbon monoxide. There are many other common names for biogas – gobar gas sewage
gas, klar gas and sludge gas. Biogas is a gaseous fuel and serves as a good source of
energy of various purposes.
1. It can be used for cooking purposes (on combustion).
2. Gobar gas can generate electricity.
3. It can be purified to yield good grade methane. Methane gas is extensively used as
a fuel for domestic and industrial purposes. It is employed for the generation of
electrical, mechanical and heat energy.

It is estimated that under ideal conditions, 10 kg of biomass can produce 3 m3 of

biogas. This biogas can provide 3 hour cooking, 3 hour lighting or 24 hours refrigeration
(with 80% methane) is around 8500 Cal/m3.

Biogas production in different countries

Biogas production significantly contributes to world’s energy gas systems, with
an estimated 7 million units. Government of China encourages and offers subsidies for
construction of biogas units. As a result, the cost of a biogas plant is cheaper than a
bicycle in china. Governments in many developing countries also encourage installation
of biogas plants. In India, Government provides 25% subsidy, besides encouraging banks
to offer loans for construction of biogas plants. They are becoming popular in rural areas.
Pakistan also has a good number of gobar gas plants.

Substrates for biogas

The usual substrates for biogas production are the waste products of animal
husbandry, industries, agriculture and municipalities. In India and other developing
countries, cattle dung (gobar) is most commonly used. The major raw material for biogas
in China is pig dung. The concentration of organic dry matter or total solid (TS) is useful
for grading the industrial, agricultural and municipal wastes. Thus, low grade (<1% TS),
medium grade (1-5% TS), high grade (5-20% TS) and solid (20-40% TS) wastes are
available. Solid or high grade wastes are preferred as substrates for biogas production.

In general, most of the substrates used in biogas plant s contain adequate

quantities of almost all the essential nutrients required for microbial growth. If necessary
nitrogen, phosphorus and trace elements are added. A carbon nitrogen ration (C/N) less
than 40:1 is preferred for optimal biogas formation. Water hyacinth (Eichornia crasipes)
an aquatic weed with huge biomass is a good source (raw material for methane
production. With high C/N ratio and low lignin content, the yield of methane is high.
Another aquatic weed Azolla is equally important for methane generation.

Microbial production of methane (biogas)

Methane is the most abundant constituent of biogas. It can also be directly used
for various domestic and industrial purposes. The microbial generation of methane,
appropriately referred to as methanogenesis from biomass occurs in four phases.

1. Hydrolytic phase:
Certain facultative anaerobic bacteria hydrolyses the complex organic materials of
the biomass (cellulose, starch, protein lipids)to low molecular weight soluble products
and some organic acids.

2. Acidifying phase:
This phase is characterized by more formation of organic acids, besides H2, CO2
and alcohol.
3. Acetogenic phase:
Acetogenic bacteria convert alcohol into acetate. These bacteria also generate
acetate from H2 and CO2.

4. Methanogenic phase:
This is the actual phase of methane gas formation. The methanogenic bacteria
(e.g. Methanobacterium omelianskii, M. formicicum, M. byantii, Methanosarcina
barkeri) convert acetate and CO2 and H2 into methane.
CH3COOH CH4 + CO2
4H2 + CO2 CH4 + 2H2O
 Some other substrates like formate and methanol can also be converted to
methane.
4HCOOH CH4 + 3CO2 + 2H2O
4CH3OH 3CH4 + CO2 + 2H2O
The overall reaction of methane formation from glucose as the starting material
may be represented as follows.
C6H12O6 3CH4 + 3CO2

The complex polysaccharides particularly lignin and cellulose due to their

inefficient conversion, limit methane production. In the normal process of
methanogenesis, approximately 50% of the complex polysaccharides contribute to
methanogenesis.

Process of biogas production (biogas plant)

Biogas production from biomass is an anaerobic process. The anaerobic digestion
is usually carried out by using air tight cylindrical tanks which are referred to as
anaerobic digesters. A digester may be made up of concrete bricks and cement or steel,
usually build underground. The digester has an inlet attached to mixing tank for feeding
cow dung. The methanogenic bacteria from another digester are also added with cow
dung. The digester is attached to a movable gas holding or storage tank with a gas outlet.
The used slurry (spent cow dung) comes out from the digester through an outlet. This can
be used as manure. The anaerobic digester described above, is a low technology gobar
gas plant, commonly used for domestic purposes in rural areas in India. The process of
digestion usually takes about 2-3 weeks when cow dung is used as the substrate.
Landfill sites for methane production

Landfill sites are low cost digesters built underground for the digestion of solid
wastes (of industries and municipalities). As the anaerobic digestion of solid organic
material occurs, methane gas is generated. It can be recovered by boring has wells into
the top of the landfill.

Factors affecting biogas (methane) production

The factors affecting methane production, with special reference to biogas plant
are briefly described.
1. Temerature and pH:
The ideal temperature is 30-40°C, while the pH is 6-8 for good yield.

2. Slurry composition:
The ratio between solid and water composition in the slurry should be around 1:1.
A carbon nitrogen ration of 30:1 in the slurry results in optimal methane production.
Good mixing and solubilization of the organic constituents is required.

3. Anaerobic conditions:
The digester should be completely airtight, so as to create suitable anaerobic
conditions.

4. Presence of inhibitors:
Ammonium sulfate and antibiotics inhibit methane production. Agricultural
wastes, pig and chicken manure (generating ammonia) and wastes from paper (rich in
sulfate) inhibit biogas production.

Advantages of biogas production

By using a simple technology, agricultural, industrial and municipal wastes can be
converted into a biofuel. The left over residue after biogas formation can be used as
fertilizers. Thus, the waste materials that would cause environmental pollution are
fruitfully utilized for biogas and fertilizer production.

Limitations for large scale production of methane

Although production of methane as a constituent of biogas is ideally suited for
domestic purpose. Many people argue against the large scale commercial production of
methane for the following reasons.
1. Methane is abundantly available in the natural oil and gas fields (produced by the
same mechanism of methanogenesis, over a period of years).
2. Microbial production of methane is more expensive than its isolation from the
natural gas.
3. Methane production by gasification of coal is more economical than its
production from biomass.
4. Being a gaseous fuel, it is quite difficult as well s expensive to store, transport and
distribute methane.
 5. Methane is unsuitable for use as a fuel in automobiles. This is because it is very
difficult to convert the gaseous methane into liquid state.

Despite the limitations listed above production of methane from a wide range of
biodegradable materials (particularly the wastes) is still attractive. This is due to the fact
that the biomass used for methane generation is renewable, in contrast to the permanent
depletion of naturally produced methane (in the gas and oil fields).

Hydrogen
Hydrogen is a simple molecule which can be easily collected, stored (as a gas or
liquid) and transported. It is highly combustible and can be used as a fuel or for the
production of electricity. Hydrogen, on mixing with oxygen, provides around 30,000
calories per gram as compared to 11,000 and 8,000 calories per gram of gasoline and coal
respectively. Further, use of hydrogen is environmental friendly, since it is a non-
pollutant.
Hydrogen is truly a versatile fuel. It can be used for automobiles, aeroplanes,
helicopters, buses, cars and scooters. Liquid hydrogen is considered to be an ideal fuel for
subsonic and supersonic aircrafts world over.

Production of biohydrogen
There are mainly two ways of generating biohydrogen by photosynthetic bacteria
and by fermentation.

By photosynthetic bacteria
Biological production of hydrogen can be achieved by photolysis of water by
photosynthetic algae and bacteria, a phenomenon referred to as biophotolysis. Certain
microalgae and cyanobacteria (e.g. Chlorella, Chlamydomonas, Scenedesmus,
Microsystis, Oscillatoria and Anabaena) can generate molecular hydrogen. Water is the
source of raw material.
H2O O2 + H+ + e-
H+ + H+ H2

The action of hydrogenase can be inhibited by creating oxygen pressure. This

condition favours release of free hydrogen. Isolated chloroplasts along with the bacterial
enzyme hydrogenase have also been used for production of hydrogen.
By fermentation
It is possible to produce hydrogen from glucose, by bacterial action. However, the
yield is less and uneconomical. Hydrogen can also be generated by anaerobic
fermentation, by a process comparable to that of methane production. This is also not
economical, besides being low in efficiency. Photosynthetic bacterium Rhodospirillum
can be used to produce hydrogen from organic wastes.

By legume crops
The leguminous plant converts N2 to NH3 and H2. This reaction is catalyzed by
the enzyme nitrogenase. In the normal circumstances, this H2 gas, byproduct of nitrogen
metabolism is lost in the soil. It is estimated that from a soybean crop in one hectare field,
about 30 billion m3 hydrogen is generated and lost annually. As such, there are no
methods available to trap such huge quantities of hydrogen produced in agricultural
fields.

Algal Bio-Fuel
Algae fuel or algal biofuel is an alternative to liquid fossil fuels that uses algae as
its source of energy rich oils. Like fossil fuel, algae fuel releases CO2 when burnt, but
unlike fossil fuel, algae fuel and other biofuels only release CO2 recently removed from
the atmosphere via photosynthesis as the algae or plant grew. The energy crisis and the
world food crisis have ignited interest in algal culture (farming algae) for making
biodiesel and other biofuels using land unsuitable for agriculture. Among algal fuels
attractive characteristics are that they can be grown with minimal impact on fresh water
resources can be produced using saline and waste water has a high flash point and are
biodegradable and relatively harmless to the environment if spilled. Algae cost more per
unit mass than other second generation biofuel crops due to high capital and operating
costs, but are claimed to yield between 10 and 100 times more fuel per unit area.

Fuels:
Algae can be converted into various types of fuel, depending on the technique and
the part of the cells used. The lipid or oily part of the algae biomass can be extracted and
converted into biodiesel through a process similar to that used for any other vegetable oil
or converted in a refinery into “drop-in” replacements for petroleum based fuels.
Alternatively or following lipid extraction, the carbohydrate content of algae can be
fermented into bioethanol or butanol fuel.

Bio-Butanol:
Butanol can be made from algae or diatoms using only a solar powered bio-
refinery. This fuel has an energy density 10 per cent less than gasoline and greater than
that of either ethanol or methanol.

Methane:
Methane, the main constituent of natural gas can be produced from algae in
various methods, namely gasification, pyrolysis and anaerobic digestion. In gasification
and pyrolysis methods methane is extracted under high temperature and pressure.
Anaerobic digestion is a straight forward method involved in decomposition of algae into
simple components then transforming it into fatty acids using microbes like acidific
bacteria followed by removing any solid particles and finally adding methanogenic
bacteria to release a gas mixture containing methane. A number of studies have
successfully shown that biomass from microalgae can be converted into biogas via
anaerobic digestion.
The following species are employed,
• Botrycoccus braunii
• Chlorella
• Dunaliella tertiolecta
• Gracilaria
• Pleurochrysis carterae
• Sargassum

Algal cultivation:
Algae grow much faster than food crops and can produce hundreds of times more
oil per unit area than conventional crops such as rapeseed, palms, soybeans or Jatropha.
As algae have a harvesting cycle of 1-10 days, their cultivation permits several harvests
in a very short time frame, a strategy differing from that associated with annual crops. In
addition, algae can be grown on land unsuitable for terrestrial crops, including arid land
and land with excessively saline soil, minimizing competition with agriculture. Most
research on algae cultivation has focused on growing algae in clean but expensive photo
bioreactors or in open ponds which are cheap to maintain but prone to contamination.

Fuel production:
After harvesting the algae, the biomass is typically processed in a series of steps,
which can differ based on the species and desired product; this is an active area of
research.

Dehydration:
Often, the algae are dehydrated and then a solvent such as hexane is used to
extract energy-rich compounds like triglycerides from the dried material. Then, the
extracted compounds can be processed into fuel using standard industrial procedures.

Hydro-thermal liquefaction:
An alternative approach called hydrothermal liquefaction employs a continuous
process that subjects harvested wet algae to high temperatures and pressures-350°C
(662°F) and 3000 pounds per square inch (21000 kPa). Products include crude oil which
can be further refined into aviation fuel, gasoline or diesel fuel.

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