GOOD LABORATORY PRACTICES

Good laboratory practice (GLP) within which laboratory studies are planned, performed, monitored, recorded,
reported and achieved. These studies were undertaken to generate data by which the hazards and embodies a set
of principle that provides a framework risks to users, consumers and third parties, including the environment,
can be assessed for pharmaceuticals (only preclinical studies) ,agrochemicals, cosmetics, feed additives and
contaminants, novel foods, biocides, detergents etc.

Definition:-

“ GLP is a quality system concerned with the organizational process and conditions under which
non-clinical health and environmental safety studies are planned, performed, monitored, recorded, achieved
and reported “.

• Standard operating procedures (SOP’S)

• Statistical procedures for data evaluation.

• Instrumentation validation.

• Reagent /materials certification

• Analyst certification

• Laboratory facilities certification

• specimen/simple tracking

• Laboratory door and window should be kept closed when work is in progress .

• Protective laboratories clothing must be worn for work in the lab

• All technical procedure should to be performed in a way that minimizes the formation of aerosols and
droplets.

• After finishing work and before living the laboratory hand must be washed if infectious material have
been handled should be disinfected.

• Working space should kept clean and cleared up.

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 2. BIOFERTILIZERS

 Biofertilizer is a Natural organic fertilizer known that helps to provide all the nutrients required by the
plants and helps to increase the quality of the soil moss.Biofertilizer Contains a wide range of naturally
chelated plant nutrients and trace elements, carbohydrates, amino acids and other growth promoting
substances.
 Kelp acts as a soil conditioner by stimulating microbial activity in the soil which results in improved air
water relationships in soil, improved fertility and makes soil less prone to compaction and erosion.
Organic Growers who use kelp in their regular fertility program report increases in yield, quality, shelf-life
and resistance to environmental stresses such as drought, extreme heat, early frost, pest and disease
problem. This blend makes an excellent foliar fertilizer. Besides being a nutritionally complete fertilizer
(containing even calcium), the nutrients are readily absorbed by the leaf. This is because the nitrogen in
fish is in the form of amino acids which plants take in and use directly– unlike inorganic fertilizers in
which the nitrogen needs to be converted into a usable form first. Additionally, because the micro-
nutrients in the fish and in the kelp are in a naturally chelated form they are quickly and readily absorbed
into the leaf surface. Foliar applications on a regular basis can increase the health, vigor and yield of
plants due to this easily absorbed additional nutrients

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 Organic fertilizers differ from chemicals fertilizers in that they feed your plants while adding organic
material to the soil. Soils with lots of organic matter remain loose and airy, hold more moisture and
nutrients, foster growth of soil organisms, and promote healthier plant root development. If only chemicals
are added the soil gradually loses its organic matter and microbiotic activity. As organic matter is used up,
the soil structure deteriorates, becoming compact, lifeless and less able to hold water and nutrients. This
results in increased amounts of chemical fertilizers needed to feed plants. We also like organic fertilizers
because they're made from renewable resources; chemicals are not.
 The Biofertilizer, is a premium natural fertilizer composed just with certified organic ingredients special
of nutrient-poor Western soils. This organic fertilizers is unequaled in its ability to nourish the beneficial
micro-organisms in the soil greatly increasing the soil’s humus content and improving its ability to sustain
and nurture healthy, more colorful plants. Use by the handful when planting individual plants, broadcast
and mix it deeply into the soil when planting flower beds or spread it around established plants and scratch
it into the soil. It is also excellent for use in vegetable gardens, container plantings and as a compost-pile
activator
 Bio Fertilizer: The Best Economic Value: Proven, top-quality product. Stick with biofertilizer the one that
always has and always will give you top quality and the best value immediately for your investment and
much more profits at long term. Research winner. Growing trials conducted by an independent research
center at a professional greenhouse compared various soils amended with peat, coir,compost and blends.
The conclusion:-"Sphagnum peat can be considered the best overall performer as a soil amendment and
substrate: it is homogenous, easy to handle and has shown the best growth results; all of this at a highly
competitive price."
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 At present in India there is a gap of about 10 million tones of plant nutrients between removal bicrops and
replenishment through fertilizer. Supply of nutrient from organic manure has not so far been able to fill up
this gap and also in years to come nutrient supply from the source is unlikely to improve due to
competitive demand for alternate uses like fuel and fodder. Of late, biofertilizers are being promoted as an
important component in supplementing plant nutrient need of the country.

2.1 SYMBIOTIC RELATIONSHIP

A symbiotic relationship is a relationship between two entities which is mutually beneficial for the
participants of the relationship. Thus there is a positive-sum gain from cooperation. This is a term commonly
used in biology to explain the relationship between two entities that need each other to survive and prosper.
The bumblebee and the flower would be an example. The bumble bee extracts the flower's pollen for protein
and its nectar for energy. The bumblebee, while collecting these sources, inadvertently brushes pollen from
one flower to another to ensure the flower's reproduction process begins. The bumblebee needs the flower to
survive, the flower needs the bumblebee to survive. These are positive sum relationships. Other relationships
in biology, especially with respect to the food chain, are not so forgiving, being a zero sum relationship,
where one player clearly benefits from the other (typically consumes the other). In this type of relationship, it
is still essential that both players (as species not individuals) survive, or the dominant player will lose its food
supply, and therefore die. If the foxes kill all the chickens, the foxes die as they lose their source of survival. If
the predator kills all its prey, the predator will die.

2.2 NON-SYMBIOTIC RELATIONSHIP

In non-symbiotic relationship the species do live together nor are dependent on each-other, the relationship is
facultative or opportunistic but does not profit the organisms when together.

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Azotobacter, Azomonas, Azotococcus, Mycobacterium spp., Methylosinum trichosporium, Thiobacillus
ferooxidans, Chlorobium thiosulfatophilum, Chromatium vinosum. C. minutissinllm. Bacillus polymixa, B.
macerans, Enterobacter aerogenes (Aerobacter aerogenes), Escherichia intermedia, E. coli, Klebsiella spp.,
Rhodospirillum rubrum, Rhodomicrobium, Rhodopseudomonas, Clostridium spp., Desulfovibrio spp.
exemplify the bacteria (free living) actively participating in non-symbiotic biological nitrogen fixation.
Azotobacter spp. (aerobic) are the main nitrogen fixing free living bacteria. Clostridium spp., (anaerobic)
stand next.

The free living cyanobacteria are considered to be fairly important nitrogen-fixers. They may fix ten times as
much nitrogen as the other free living bacteria fix under suitable conditions. There are claims that the
cyanobacteria are mainly responsible for maintaining the fertility and productivity of rice fields, Anabaena,
Nostoc are the good examples.

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2.3 TYPES OF BIOFERTILIZERS

Biofertilizer can be divided into Nitrogen fixing bacteria, Phosphate solubilizing and mobilizing micro-
organism and compost etc. some of these micro-organism are also being reported to produce plant growth
promoting substance. These biofertilizer have an important role play in rain fed agriculture primarily because
they are low in cost input. In India about 70% of the cultivated area is under rain fed condition. Of this nearly
50% is under unsure rainfall condition.

i. Nitrogen Biofertilizers :-
This type of biofertilizers helps the agriculturists to determine the nitrogen level in the soil. Nitrogen is a
necessary component which is used for the growth of the plant. Plants need a limited amount of nitrogen for
their growth. The type of the crops also determines the level f nitrogen. Some crops need more nitrogen for
their growth while some crops need fewer amounts. The type of the soil also determines that which type of
biofertilizers is needed for this crop. Fr example, Azotobacteria is used for the non legume crops; Rhizobium
is needed for the legume crops. Similarly blue green algae are needed to grow rice while Acetobacter is used
to grow sugarcane. It means almost all the crops need different types of biofertilizers depending on their
needs.
The nitrogen-fixing organisms

All the nitrogen-fixing organisms are prokaryotes (bacteria). Some of them live independently of other
organisms - the so-called free-living nitrogen-fixing bacteria. Others live in intimate symbiotic associations
with plants or with other organisms (e.g. protozoa). Examples are shown in the table below

Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium)

Free living Symbiotic with plants
Aerobic Anaerobic Legumes Other plants
Clostridium (some)
Azotobacter
Desulfovibrio
Beijerinckia Frankia
Purple sulphur bacteria* Rhizobium
Klebsiella (some) Azospirillum
Purple non-sulphur bacteria*
Cyanobacteria (some)*
Green sulphur bacteria*

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ii. Phosphorus biofertilizers :-
Phosphorus is needed for plant photosynthesis, maturation of fruits, and for reproduction. Phosphorus is a
naturally occurring element that helps promote healthy plant growth, and is found in all fertilizers, organic
and inorganic. The microorganisms in phosphorus biofertilizer solubilize fixed forms of phosphorus already
present in the soil, thus making it available for plants, states the Regional Biofertilizer Development Centre
website. If the soil is phosphorus deficient, the organisms will have a hard time extracting the trace amounts
in the soil, thus limiting the positive effects of the solution. Phosphorus biofertilizer is not crop specific;
therefore Rhizobium, Azotobacter, Azospirillum, and Acetobacter can be used for all crops.

iii. Compost Biofertilizers :-

Organic and synthetic fertilizers contain potassium, which helps the absorption of nitrogen and phosphorus in
plants. Enriched compost biofertilizer acts similar to phosphorus by speeding up the composting process and
enriching the nutrient values of the other two elemental fertilizers. Although enriched compost helps
maximize the nutrients available in the soil, it still requires the use of soil phosphorus for effectiveness. Low
levels of soil phosphorus make it hard for plants to absorb the vital nutrients from biofertilizers, thus making
them ineffective. According to the Regional Biofertilizer Development Centre website, enriched compost
biofertilizer comes in two varieties, Cellulolytic fungal culture and Phosphotika and Azotobacter culture. This
product is not crop specific; therefore, application can be used in any location.

Name Crops Suited Benefits Usually Remarks Types of Bacteria

Seen
Rhizobium Legumes like 10-35% yield Fodders give better results. Symbiotic bacteria
strains pulses, increase, 50- Leaves residual N
7
 groundnut, 200 kg N/ha. in the soil.
soybean
Azotobacter Soil treatment for 10-15% yield Also controls certain diseases. Free living
nonlegume increaseadds Bacteria
crops including 20-25 kg N/ha
dry land crops
Azospirillum Non-legumes like 10-20% yield A fodder gives Free living
maize, increase higher/enriches fodder Bacteria
barley, oats, response. Produces growth-
sorghum, promoting
millet, substances. It can be applied
Sugarcane, rice to legumes as co-inoculants
etc.
Phosphate Soil application 5-30% yield Can be mixed with rock Phosphate
Solubilizers for all increase phosphate. solubilizing
crops Bacteria
Blue-green Rice/wet lands 20 -30 kg N/ha, Reduces soil alkalinity, can be Symbiotic
algae and Azolla can used for fishes Bacteria
Azolla give biomass up as feed. They have growth
to 40-50 promoting
Tonnes and fix hormonal effects.
30-100 kg TNAU has developed high
N/ha yielding Azolla
Hybrids.
Mycorrhizae Many trees, some 30-50% yield Usually inoculated to Fungi
(VAM) crops, increase seedlings.
and some enhances uptake
ornamental of P. Zn,
plants S and Water

COMPARATION BETWEEN CHEMICAL AND BIOFERTILIZER

Factors Chemical Fertilizer Biofertilizers

8
Production Industrial, Centralized Biological, small scale or
Decentralized

Process Chemical Biological

Effect on
subsequent For nitrogen nil or low
Residual effect for nitrogen
Crop

Shelf life Long short for bacteria, long for BGA

Pollution effect exists due to indiscriminate use pollution free

Irrigation more useful to irrigated field useful for both irrigated

and dry farming

Cost high cost input low cost input

Soil health indiscriminate use deteriorates the soil improves soil health
health

ADVANTAGES

 Germination increase up to 20 percent. Improved seedling emergence and growth.

 Increase yield from 10 to 40 percent.

9
 Improve the quality of fruit and keeping quality.

 Saving of 25 to 35 percent inorganic fertilizers.

 Increase the availability and up take of N and P in plants.

 Improve the status of soil fertility maintain good soil health and crop productivity.

 Higher population of beneficial micro-organism in soil increase nutrient retention and availability

leading to improve yields.

 Improve nitrogen and phosphorus fertilizer efficiency.

 It is safe to handle and easy to apply.

 Leaves no harmful residues in plants or soil.

 Suppress harmful and pathogenic soil micro-organism.

 Composting waste matter and produce organic manure.

 They are compatible with organic manures, fertilizers and agro-chemicals.

 They are non- polluting and eco-friendly.

 Biofertilizers are also produce growth promoting substances.

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DISADVANTAGES

 Are short in supply.

 Are costly.

 Utilize petroleum (nitrogenous fertilizer).

 Damage the environment example about 10% of the ground water sample in Punjab contained

more nitrite than the maximum permissible limit prescribed by World Health Organization.

 Slow action.

 Other chemicals should not be mixed with the biofertilizers

 Biofertilizers are live product and require care in the storage.

 Biofertilizer packets need to be stored in cool and dry place away from direct sunlight and

heat.

 Right combinations of biofertilizers have to be used.

 Should use for the specified crop only. (Rhizobium)

APPLICATION

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A BRIEF STUDY OF BACTERIAL SPECIES

i. RHIZOBIUM

 It belongs to Rhizobiaceae family and fixed 50-100 kg atmospheric nitrogen per hectare. Obviously
most interest among biofertilizer surround Rhizobium inoculants because of their ability to fix
nitrogen in association with leguminous plants which result not only in meeting the nitrogen required
of the plant but also the symbiotic system leaves behind sizeable amount of air over a hectare of land
contain approximately 80000 tones of inert nitrogen which is not available to plants and animals as

12
 such but can be utilized by plants in association with bacteria having capability to fix atmospheric
nitrogen. Pulses, soybean and groundnut occupy nearly 25 million hectare in India and the bacteria
present in nodule of this plants fix the atmospheric nitrogen to meet their needs. However, the
rhizobium bacteria present in the nodules of these crops are not always efficient. Therefore, the
competitive, efficient bacteria are isolated, screened, selected and produced as carrier based inoculants
 This bacterium was isolated for the first time in 1838. It lives in soil and enter into symbiosis only
with leguminous plants by infecting their roots and forming nodules on them. It lives inside the
nodules and fixed atmospheric nitrogen for plants. There are man species of Rhizobium present in
the soil. Can you guess whether the same Rhizobium can form nodules and fix nitrogen with all the
leguminous plant. The answer is no. Rhizobium is very specific in choosing its symbiotic partners and
each Rhizobium species infect limited no of legumes.It means that Rhizobium of pea group will only
pea plant and fix nitrogen and not with soyabean plant. The soyabean Rhizobium will only infectand
form nodules on soyabean plantand not on groundnut plant. This shows that Rhizobium is specific for
each leguminous plant.
 Since nitrogenase is inactivated by O2, the fixation of N2 must occur under conditions which are
anaerobic at least locally. For anaerobes there is no problem. Facultative organisms such as purple
photosynthetic bacteria or Klesbsiella fix N2 only when anaerobic. Other organisms have protective
mechanisms. In Azotobacter, an obligate aerobe, the O2 concentration inside the cell is held down by
partial uncoupling of a highly active respiratory chain. This wastes carbohydrate, but if growth is
limited by absence of nitrogen compounds then this is justifiable. In cyanobacteria O2 is actually
generated by photosynthesis. Fixation of N2 occurs in special cells known as heterocysts which do
NOT photosynthesize but are devoted solely to N2 fixation.
.
Recommended for

Pulses: Chickpea, pea, lentil, blackgram, greengram, cowpea, pigeonpea.

Oil seeds: soybean, groundnut.
Fodders: berseem, lucern.

Increase in yield: 10-35%

ii. AZOTOBACTER

It belongs to azotobacteriaceae. The use of azotobacter help in saving 10 to 20 kg N/ha. It produces growth
promoting substances which improve seed germination and growth of extended root system. It produces
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polysaccharides which improve soil aggregation. Azotobacter suppresses the growth of saprophytic and
pathogenic micro-organism near the root system of crop plants. In a country like India where the application
of chemical fertilizer in rain fed cultivated area is low and in irrigated area its application is much less as
compared to many developed country its benefit have been reported.

Recommended for
Rice, wheat, millets, other cereals, cotton, vegetable, sunflower, mustard, flowers.

Increase in yield: 20 to 30%

iii. PHOSPHATE SOLUBILIZING BACTERIA

Phosphorus is one of the most important plant nutrients and may be critical nutrient the optimum growth of
plants. Most of our soils are in available forms of phosphorus required phosphate application. The
proliferation of efficient strain of phosphate solubilization micro-organism. In the Rhizosphere of crops will
render insoluble soil phosphate available to plants due to production and secretion of organic acid by them.
The use of this biofertilizer will also increase the availability of phosphate from rock phosphate applied
directly even to neutral to alkaline soil or when used for preparation of phosphor-compost. Phosphate
solubilizing micro-organism include efficient strain of bacteria, fungi, yeast and actinomycetes in that order

 Increase in yield: 10 to 20%

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2.4 MECHANISM OF NITROGEN FIXATION

 Nitrogen is a part of vital organic compounds in microrganisms, such as amino acids, proteins and
DNA. The gaseous form of nitrogen (N2) makes up 78% of the troposphere. One might think this
means we always have plenty of nitrogen available, but unfortunately it does not work that way.
Nitrogen in the gaseous form cannot be absorbed and used as a nutrient by plants and animals; it must
first be converted by nitrifying bacteria, so that it can enter food chains as a part of the nitrogen cycle.
 During the conversion of nitrogen cyanobacteria will first convert nitrogen into ammonia and
ammonium, during the nitrogen fixation process. Plants can use ammonia as a nitrogen source.
 Nitrogen fixation is carried out according to the following reaction:
N2 + 3 H2 -> 2 NH3
 After ammonium fixation, the ammonia and ammonium that is formed will be transferred further,
during the nitrification process. Aerobic bacteria use oxygen to convert these compounds.
Nitrosomonas bacteria first convert nitrogen gas to nitrite (NO2-) and subsequently Nitrobacter convert
nitrite to nitrate (NO3-), a plant nutrient.
 Nitrification is carried out according to the following reactions:
2 NH3 + 3O2 - > 2 NO2 + 2 H+ + 2 H2O
2 NO2- + O2 -> 2 NO3-

15
  Plants absorb ammonium and nitrate during the assimilation process, after which they are converted
into nitrogen-containing organic molecules, such as amino acids and DNA. Animals cannot absorb
nitrates directly. They receive their nutrient supplies by consuming plants or plant-consuming animals.
 When nitrogen nutrients have served their purpose in plants and animals, specialized decomposing
bacteria will start a process called ammonification, to convert them back into ammonia and water-
soluble ammonium salts. After the nutrients are converted back into ammonia, anaerobic bacteria will
convert them back into nitrogen gas, during a process called denitrification.
 Denitrification is carried out according to the following reaction:
NO3- + CH2O + H+ -> ½ N2O + CO2 + 1½ H2O
 Finally, nitrogen is released into the atmosphere again. The whole process starts over after release.

Role of nitrogen in the biosphere

The growth of all organisms depends on the availability of mineral nutrients, and none is more important than
nitrogen, which is required in large amounts as an essential component of proteins, nucleic acids and other
cellular constituents. There is an abundant supply of nitrogen in the earth's atmosphere - nearly 79% in the
form of N2 gas. However, N2 is unavailable for use by most organisms because there is a triple bond between
the two nitrogen atoms, making the molecule almost inert. In order for nitrogen to be used for growth it must
be "fixed" (combined) in the form of ammonium (NH 4) or nitrate (NO3) ions. The weathering of rocks
releases these ions so slowly that it has a neglible effect on the availability of fixed nitrogen. So, nitrogen is
often the limiting factor for growth and biomass production in all environments where there is suitable climate
and availability of water to support life.

Nitrogen fixation reactions

 N2 + 6 e- + 8H+ ---> 2 NH4+ (ammonium ion)

 NO3- + 2e- + 2H+ -----------> NO2- + H2O

(nitrate ion) (nitrite ion)

16
 NO2- + 6e- + 2H+ ----------> NH4+ + 2 H20

 NO2- + 6e- + 2H+ ----------> NH4+ + 2 H2O

 2 NO + O2 ---------------> 2NO2

 2 NO2 + H2O -------> HNO3 + HNO2

 HNO3 --------> H+ + NO3- (nitrate ions)

 HNO2 --------> H+ + NO2- (nitrite ions)

Types of Nitrogen fixation processes

The nitrogen molecule (N2) is quite inert. To break it apart so that its atoms can combine with other atoms
requires the input of substantial amounts of energy.

Three processes are responsible for most of the nitrogen fixation in the biosphere:

• Atmospheric fixation by lightning

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• Industrial fixation
• Biological fixation by certain microbes — alone or in a symbiotic relationship with some plants and
animals

N2 fixed (1012 g per year, or 106 metric tons

Type of fixation
per year)
Non-biological
Industrial about 50
Combustion about 20
Lightning about 10
Total about 80
Biological
Agricultural land about 90
Forest and non-agricultural land about 50
Sea about 35
Total about 175

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 Structure and Operation of Nitrogenase

Nitrogenase contains the two proteins molybdoferredoxin and azoferredoxin. These must be supplied with
reducing equivalents by other proteins that vary. Here we consider nitrogenase from Klebsiella, a close
relative of E. coli where the accessory proteins are flavodoxin and pyruvate flavodoxin reductase. In most
bacteria electrons are passed from NAD(P)H or pyruvate to ferredoxin, an FeS protein. If iron is in short
supply ferredoxin is replaced by flavodoxin, a flavoprotein. In Klebsiella there is no ferredoxin and
flavodoxin (NifF protein) is used all the time. Azoferredoxin transfers electrons from reduced flavodoxin (or
ferredoxin) to molybdoferredoxin.

Molybdoferredoxin is an alpha2/beta2 tetramer. The alpha and beta subunits are similar but distinct and are
encoded by genes nifK and nifD. Each tetramer contains 2 Mo and several FeS groups. The molybdenum is
part of a low molecular weight cofactor containing Mo bound to an Fe7S8 cluster and to homocitrate. This
MoFe cofactor is unique to nitrogen fixation and distinct from the Mo-pterin cofactor of other Mo proteins
(e.g. nitrate reductase, xanthine oxidase). Azoferredoxin is a dimer of identical subunits encoded by nifH and
contains a single Fe4S4 group per dimer. Azoferredoxin is modified by the NifM protein. Molybdoferredoxin
from one genus can often interact with azoferredoxin from another genus to give active enzyme. These two
proteins have several alternative names:

Molybdoferredoxin = component I, MoFe protein, or "nitrogenase"

Azoferredoxin = component II, Fe protein, or nitrogenase reductase

Nitrogenase is not very fast (the turnover number is around 50 moles/min per mole of Mo) and so about 2-5%
of the total cell protein is nitrogenase. The reaction N2 + 3H2 Æ 2NH3 actually releases energy. However, the
activation energy needed to break the NºN triple bond is very high and in practice energy, as ATP, is
consumed by NifH protein (azoferredoxin). If there is an excess of azoferredoxin then ATP tends to be

19
wasted. In Klebsiella nifHDK form an operon that keeps the ratio of components constant.

The Nif (nitrogen fixation) proteins are often referred to by their gene names:

NifJ = Pyruvate Flavodoxin Reductase

nifF = Flavodoxin

nifH = Azoferredoxin

nifM = processing of NifH protein

nifK,D = Molybdoferredoxin

nifB,N,E,V,W,Z = MoFe cofactor synthesis

nifY = MoFe cofactor insertion

nifQ = Molybdenum uptake

nifA,L,R = regulation

nifU,S = metal center biosynthesis

nifX,T = function unknown (not necessary, at least under normal conditions)

Mechanism of Nitrogenase
Nitrogenase will reduce many small molecules with triple bonds in addition to nitrogen. Oxygen, which is
triple-bonded inactivates nitrogenase. Carbon monoxide, another triply bonded molecule is a competitive
inhibitor.

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme
20
called nitrogenase.[1] The formula for BNF is:

N2 + 8 H+ + 6 e− → 2 NH3 + H2

Proposed Steps In Nitrogenase Mechanism :-

The mechanism is largely based on work with non-protein MoFe complexes, some of which will fix N2
chemically (but very inefficiently).

 Mo in active site is reduced from Mo6+ to Mo5+ to Mo4+ by sequential electron transfer from
Azoferredoxin.

 Semi-activated Nitrogenase can reduce easy substrates such as Acetylene.

 Further transfer of two electrons activates the Fe of the MoFe cofactor in the active site, which carries
2[H].

 N2 binds end on to the Fe[H]2 complex and releases H2.

 The bound N2 is reduced to HN=NH by sideways transfer of 2e- (plus 2H+) from the active site
Mo4+.

 Conversion of N2H2 to 2NH3 requires two further 2e- steps, but partial activation of the enzyme is
sufficient (i.e. ATP is no longer needed to hype up the redox potential) since only step requires
extreme reducing power.

This mechanism also explains why acetylene, C2H2, is a non-competitive inhibitor of N2 fixation. Acetylene
reduction discharges nitrogenase before it ever reaches full activation. Although N2 fixation wastes reducing
power when H2 is evolved, most N2 fixing bacteria contain hydrogenase which uses gaseous H2 to reduce
NAD(P). Hence they recycle the hydrogen at least partly.

21
Regulation of Nitrogen Fixation

All the nif genes in Klebsiella are clustered and coordinately regulated. E. coli to which the nif genes of
Klebsiella have been transferred can fix N2. In both the original Klebsiella and the E. coli nitrogenase is
expressed only in the absence of both O2 and NH3 in the growth medium. Other organic N-sources will also
repress nitrogenase. The better the N-source the greater the repression.

The nif genes are regulated by the nifLA operon. The nitrogen regulators NtrC (= GlnG), and NtrB determine
whether or not the nifLA operon is expressed (depending on the presence of ammonia or organic nitrogen). In
the absence of ammonia or organic nitrogen the NtrC protein is phosphorylated by the NtrB protein. NtrC-P
then binds to the upstream region of the nifLA operon and activates transcription.

NtrA (= GlnF = RpoN = s54) is the nitrogen sigma factor, which is needed for expression of the nifLA operon
and the nif structural genes. NtrA is an alternative sigma factor used by RNA polymerase to recognize many
genes involved in nitrogen metabolism which are not recognized by the standard sigma factor. The nifA gene
encodes a protein required for switching on all of the nif genes except the regulatory genes nifLA themselves.
if NifA protein is made, its function is to activate the other nif genes. The nifL gene is required for O2
repression. In the absence of NifL protein, nitrogenase is made in the presence of O2 (but is inactivated by
O2). When oxygen is present, the NifL protein binds to NifA and prevents it from activating the other nif
genes.

Alternative Nitrogen Fixation Systems

When Mo is absent some N-fixing bacteria, such as Azotobacter, make an alternative nitrogenase in which
vanadium is used instead of Mo. This is encoded by a duplicate set of vnf genes which make VFe cofactor as
well as the corresponding nitrogenase proteins. Mo, if available represses the vnf system which is less
efficient. If vanadium is also absent Azotobacter can make a third nitrogenase which uses only iron - the even
less efficient anf system. The sequences of the nif, vnf and anf genes are very similar.

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2.5 INDUSTRIAL ANALYSIS OF BIOFERTILIZER

India is one of the important countries in biofertilizer production and consumption in the world. The present
production capacity of different biofertilizers production units in the country is about 4500 tones per
annum .The maximum production capacity is in agro industries corporation followed by state agriculture
universities and private sector. Among the different states the maximum production capacity is in Tamil Nadu
followed by MP, UP, Gujarat and Maharashtra. The actual production of biofertilizers during 1994-1995 was
about 2000-2500 tones.
IFFCO :-
 Established in 1967
 Backed by five mega plants
 More than 39824 members societies
 More than 70 lakh metric tonnes of Fertilizer Production per annum.
Products
 Urea,NPK,DAP,NP and Biofertilizer

NFL :-
 NFL, a Schedule A and Mini Ratna Company, is the second largest producer of
Nitrogenous Fertilizers in the Country with 15.8% share in domestic production
of Urea achieved in the country during 2009-10.
 They produce Nitrogen Biofertilizers and Phosphorous Biofertilizers.
RCF :-
 RCF is one of the leading producers of Fertilizers in India.

23
  Sujala, Suphala 15:15:15, Suphala 20:20:0, Ujjwala, Microla and Biola are its
major fertilizers.
 RCF pioneered the manufacture of basic chemicals such as Methanol, Sodium
Nitrate, Sodium Nitrite, Ammonium bicarbonate, Methylamines, Dimethyl
Formamide, Dimethylacetamide in India.
 Today R.C.F is the only manufacture of DMF in India

Main biofertilizer producing companies are :-

GSFC :-
 GSFC today stands for superior quality with many of its products being ISO 9001
certified.

 Urea, Ammonium Sulphate, DAP, APS,

NPK, Bio-fertilizers, Water Soluble Fertilizers,
Gypsum, Melamine, Caprolactam, Nylon-6,
Methyl Ethyl Ketoxime, Cyclohexanone,
Sulphuric Acid, Oleum, Anhydrous Ammonia,
Argon Gas, Bio-Fuels,
Plant Tissue Culture seeds .

BVFCL :-
 The Brahmaputra Valley Fertilizer Corporation
Limited located on the bank of the river Dilli
in the south-western border of Dibrugarh
District in Assam is the first factory of its kind
in India to use associated natural gas as basic
raw material for producing nitrogenous
fertilizer.
 BVFCL has only one finished product ie Prilled
Urea (Brand name : Mukta Urea).
 Ammonia is used as a intermediate product
which is used to make Urea.

24
 MFL :-

 Established in 1966, Madras Fertilizers Limited is a Public Sector Undertaking

under administrative control of the Department of Fertilizers, Ministry of Chemicals and Fertilizers.
Madras Fertilizers Limited has been serving the Nation for the past 40 years since plant
commissioning in 1971 and is proud to be part of Green Revolution.
• Products :-

Chemical fertilizers

Urea

NPK - Complex (17:17:17)

(14:28:14)

(19:19:19)

(20:20:0:13)

NK Mixture (20:0:10)

MOP (Imported)

DAP (Imported)

Biofertilizers

Azospirillum (Paddy)

Azospirillum (Other crops)

Azospirillum (Plantation Crops)

Rhizobium (Groundnut)

Rhizobium (Pulses)

Phospho Bacteria (All Crops)

NP Bio (All Crops)

25
  Based on the area under different crops and dose of biofertilizers to be applied ,the national
biofertilizers development centre (NBDC) Ghaziabad and biotech consortium India ltd (BCIL)
have estimated the total requirement of biofertilizers to be about 5.07 and 3.44 lakh tones. The
recent estimated potential demand of different kind of biofertilizers by government of Tamil Nadu
is Rhizobium 35000 tones, Azospirillum 482 thousand tones, Azotobacter 162.61 thousand tones,
blue green algae 267.72 thousand tones, Azolla 20.38 thousand tones and phosphate solubilizer
275.51 thousand tones. The total of all these amounts come out to be 12.44 lakh tones which is
significantly higher than the estimates of NBDC and BCIL, mainly because they did not indicate
phosphate solubilizer and there estimates for Azospirillum were also low.
 Production technology of biofertilizers is relatively simple and its installation cost is very low
compare to chemical fertilizer plants. Organized promotional and marketing strategy for
successful biofertilizers business is very important, most of the biofertilizer units lack in this
respect. Fertilizer company like GSFC has more than 200 farm information centres-cum-depots
suitated in remote areas. These are well manage by experienced agricultural graduates to provide
the technical know-how, conduct the demonstration and give before and after sale service to the
farmers. In order to provide biofertilizers upto village level, GSFC has established its own
distributor network. MLF and SPIC have also well organized themselves in this respect.

PRODUCTION STATISTICS OF BIOFERTILIZER

Name of
S.No the
Capacity/Production (in MT)
. PSU/Coo
p.
2002-03 2003-05 2005-06 2007-08 2009-10
Capacit Productio Capacit Productio Capacit Productio Capacit Productio Capacit Productio
y n y n y n y n y n
KRIBHC
1. 250.00 296.03 450.00 602.71 450.00 560.43 450.00 775.17 550.00 737.8
O
26
2. IFFCO 240.00 133.23 240.00 136.50 240.00 339.14 240.00 431.12 240.00 394.27
3. NFL 100.00 186.10 100.00 115.25 100.00 130.39 100.00 143.95 100.00 165.39
4. MFL 400.00 195.54 400.00 125.92 400.00 213.00 400.00 234.84 100.00 228.21
5. FACT 150.00 6.00 150.00 4.80 150.00 3.75 150.00 8.34 150.00 3.91
6. RCF 150.00 54.68 150.00 58.43 150.00 39.15 150.00 84.95 150.00 105.00
7. BVFCL 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 30.00 7.06

REGI

South

North

East

West

North-

27
 3 MATERIALS AND METHODS

REQUIREMENTS :-

28
 GLASSWARES

Petri Plates
Test Tubes
Conical Flasks
Beakers
Spreader
Glass Rod
Measuring Cylinder

EQUIPMENTS

Autoclave
Laminar Air Flow
Incubator
Hot Air Oven

3.1 STRAIN SELECTION

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

RHIZOBIUM

Rhizobium is a genus of Gram-negative soil bacteria that fix nitrogen. Rhizobium forms an endosymbiotic
nitrogen fixing association with roots of legumes and Parasponia. The bacteria colonize plant cells within root
nodules; here the bacteria convert atmospheric nitrogen to ammonia and then provide organic nitrogenous
compounds such as glutamine or ureides to the plant. The plant provides the bacteria with organic compounds
made by photosynthesis
29
Identifying characterstics

 Strictly aerobic.
 Rod shaped cells.
 0.5-0.9 um x 1.2-3.0 um in size.
 Non-spore forming.
 Cells stain Gram negative.
 Fastgrowing.
 Contains the enzyme nitrogenase.
 Granules of polyhydroxybutyrate common.
 Mobile by a single polar flagellum or two to six peritrichous flagella.
 Forms root nodules with legumes and undergoes nitrogen fixation.
 Chemoorganotrophic.
 Colonies are white pigmented.
 Colonies are circular, convex, semi-translucent, raised and mucilaginous.
 Host specific.
 The percent G+C ranges from 59-64%.

Physiological properties

 Nature : Chemoheterotrophic, Symbiotic with legume.

 C source : supplied by legume through photosynthesis, mono and disaccharide.
 N source : fixed from atmosphere.
 Respiration : aerobic.
 Growth : fast ( Rhizobium), slow (Bradyrhizobium)
 Doubling time : fast grower 2-4 hours slow grower 6-12 hours.
 Growth media : YEMA

30
Taxonomy of Rhizobia

All rhizobium are root nodulating bacteria and are gram negative, non spore forming medium sized rod
shaped cells that contain the enzyme complex called nitrogenase and are typically motile. Their cellular
morphology and biochemical characteristics are very similar to those of nonsymbiotic nitrogen-fixing bacteria
called Azotobacter. The primary distinguishing characteristic of Rhizobium species is their ability to nodulate
leguminous plants. Rhizobia are predominantly aerobic chemoorganotrophs and grow well in the presence of
oxygen and utilize a wide range of relatively simple carbohydrates and amino compounds. (With the
exception of a few strains, they have not been found to fix N in the free-living form except under special
conditions.) Optimal growth of most strains occurs at a temperature range of 25-30 C and at a pH of 6.0-7.0.
Despite their usual aerobic metabolism, many strains are able to grow well under microaerophillic conditions
at oxygen tensions of less than 0.1 atm. Rhizobium is surrounded by a slimy capsule made of
exopolysaccharide, which protects it from drying out. And also helps the bacterium stick to root hairs during
various stages of its life cycle.

Table of important Species :-

S.No Rhizobium sps. Cross inoculation group Legume types

1 R.leguminosarum Pea group Pisum, Vicia, Lens

2 R.phaseoli Bean group Phaseolus

3 R.trifolii Clover group Trifolium

4 R.meliloti Alfalfa group Melilotus, Medicago,

Trigonella

5 R.lupini Lupini group Lupinus

6 R.japonicum Soybean group Glycine

7 R.sps Cowpea group Vigna, Arachis

31
Nodule formation and functioning

Infection threads grow to the nodule, infect its central tissue and release the rhizobia in these cells where they
differentiate morphologically into bacteroids and fix nitrogen from the atmosphere into a plant usable form,
ammonium (NH4+), utilizing the enzyme nitrogenase. In return the plant supplies the bacteria with
carbohydrates, proteins, and sufficient oxygen so as not to interfere with the fixation process.
Leghaemoglobins, plant proteins similar to human Hemoglobins help to provide oxygen for respiration while
keeping the free oxygen concentration low enough not to inhibit nitrogenase activity. Recently, it was
discovered that a Bradyrhizobium strain forms nodules in Aeschynomene without producing Nod factors,
suggesting the existence of alternative communication signals other than Nod factors.[

The legume–rhizobium symbiosis is a classic example of mutualism—rhizobia supply ammonia or amino

acids to the plant and in return receive organic acids (principally as the dicarboxylic acids malate and
succinate) as a carbon and energy source—but its evolutionary persistence is actually somewhat surprising.
Because several unrelated strains infect each individual plant, any one strain could redirect resources from
nitrogen fixation to its own reproduction without killing the host plant upon which they all depend. But this
form of cheating should be equally tempting for all strains, a classic tragedy of the commons. There are two
competing hypotheses for the mechanism that maintains legume-rhizobium symbiosis (though both may occur
in nature). The sanctions hypothesis suggests that plants police cheating rhizobia. Sanctions could take the
form of reduced nodule growth, early nodule death, decreased carbon supply to nodules, or reduced oxygen
supply to nodules that fix less nitrogen. The partner choice hypothesis proposes that the plant uses pre-
nodulation signals from the rhizobia to decide whether to allow nodulation and chooses only non-cheating
rhizobia. There is evidence for sanctions in soybean plants, which reduce rhizobium reproduction (perhaps by
limiting oxygen supply) in nodules that fix less nitrogen. Similarly, wild lupine plants allocate less resources
to nodules containing less-beneficial rhizobia, limiting rhizobial reproduction inside. This is consistent with
the definition of sanctions just given, although called "partner choice" by the authors. However, other studies
have found no evidence of plant sanctions and instead support the partner choice hypothesis.

AZOTOBACTER

32
Azotobacter is a genus of usually motile, oval or spherical bacteria that form thick-walled cysts and may
produce large quantities of capsular slime. They are aerobic, free-living soil microbes which play an important
role in the nitrogen cycle in nature, binding atmospheric nitrogen, which is inaccessible to plants, and
releasing it in the form of ammonium ions into the soil. Apart from being a model organism, it is used by
humans for the production of biofertilizers, food additives and some biopolymers. The first representative of
the genus, Azotobacter chroococcum, was discovered and described in 1901 by the Dutch microbiologist and
botanist Martinus Beijerinck. Azotobacter are Gram-negative bacteria. They are found in neutral and
alkaline soils, in water and in association with some plants.

Taxonomy

Scientific classification
Domain: Bacteria
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Pseudomonadales
Family: Pseudomonadaceae/Azotobacteraceae
Genus: Azotobacter

The Azotobacter genus was discovered in 1901 by the Dutch microbiologist and botanist Martinus Beijerinck,
who was one of the founders of environmental microbiology. He selected and described the species
Azotobacter chroococcum – the first aerobic, free-living nitrogen fixer.

In 1903, Lipman described Azotobacter vinelandii, and a year later Azotobacter beijerinckii LIPMAN, 1904,
which he named in honor of Beijerinck. In 1949, Russian microbiologist Nikolai Krasilnikov identified the
species of Azotobacter nigricans KRASIL'NIKOV, 1949 which was divided in 1981 by Thompson Skerman into
two subspecies of Azotobacter nigricans subsp. nigricans and Azotobacter nigricans subsp. achromogenes; in
33
the same year, Thompson and Skerman described the Azotobacter armeniacus THOMPSON AND SKERMAN, 1981.
In 1991, Page and Shivprasad reported a microaerophilic and air-tolerant type Azotobacter salinestris PAGE
AND SHIVPRASAD 1991 which was dependent on sodium ions.[64]

Earlier, representatives of the genus were assigned to the family Azotobacteraceae PRIBRAM, 1933, but then
were transferred to the family Pseudomonadaceae based on the studies of nucleotide sequences 16S rRNA. In
2004, a phylogenetic study revealed that Azotobacter vinelandii belongs to the same clade as the bacterium
Pseudomonas aeruginosa,[65] and in 2007 it was suggested that the genera Azotobacter, Azomonas and
Pseudomonas are related and might be synonyms.

Azotobacter agilis
Azotobacter armeniacus
Azotobacter sp. AR
Azotobacter beijerinckii
Azotobacter chroococcum
Azotobacter sp. DCU26
Azotobacter sp. FA8
Azotobacter nigricans
Azotobacter paspali
Azotobacter salinestris
Azotobacter tropicalis
Azotobacter vinelandii

Identifying characterstics

 Aerobic but can grow under reduced oxygen conditions.

 Large rod of varying length down to coccoid morphology.
 2 um or more in diameter; may be almost the size of yeasts.
 Can occur singly, in chains, or in clumps.
 No endospores are formed, but do form thick-walled cysts.
 Gram negative.
34
  May be mobile by peritrichous flagella or non-mobile.
 Non-symbiotic nitrogen fixers.
 Copious amounts of capsular slime may be formed.
 Catalyse positive.
 Can produce a water soluble pigment, either yellow-green, fluorescent or red-violet/brownish-
black.
 The percent G+C = 63 - 66 %.
 Optimum temperature range between 20 and 30°C.

Growth range is between pH 5.5 - 8.5, but optimum pH is 7.0 – 7.5.

Physiological properties

Azotobacter respire aerobically, receiving energy from redox reactions, using organic compounds as electron
donors. Azotobacter can use a variety of carbohydrates, alcohols and salts of organic acids as sources of
carbon and can fix at least 10 micrograms of nitrogen per gram of glucose consumed. Nitrogen fixation
requires molybdenum ions, but they can be partially replaced by vanadium ions. The source of nitrogen can be
nitrates, ammonium ions or amino acids. The optimal pH for the growth and nitrogen fixation is 7.0–7.5, but
growth is sustained in the pH range from 4.8 to 8.5.[23] Azotobacter can also grow mixotrophically, in a
nitrogen-free medium containing mannose; this growth mode is hydrogen dependent. Hydrogen is available in
the soil, thus this growth mode may occur in nature.[24]

While growing, Azotobacter produce flat, slimy, paste-like colonies with a diameter of 5–10 mm, which may
form films in liquid nutrient media. The colonies can have dark brown, green and other colors, or may be
colorless, depending on the species. The growth is favored at a temperature of 20–30 °C

Benefits :-
 It improves seed germination and plant growth
 Azotobacter are tolerant to high salts.
 It can benefit crops by Nitrogen fixation, growth promoting substances, fungi static substances.
 Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its
growth.

35
  It is poor competitor for nutrients in soil and hence its growth promoting substances, fungistatic
substance.
 It thrives even in alkaline soils.
 Azotobacter is less effective in soils with poor organic matter content.

Functions of Azotobacter :-

Azotobacter naturally fixes atmospheric nitrogen in the rhizosphere. There are different strains of
Azotobacter each has varied chemical, biological and other characters. However, some strains have higher
nitrogen fixing ability than others.. Azotobacter uses carbon for its metabolism from simple or compound
substances of carbonaceous in nature. Besides carbon, Azotobacter also requires calcium for nitrogen fixation.
Similarly, a medium used for growth of Azotobacter is required to have presence of organic nitrogen, micro-
nutrients and salt in order to enhance the nitrogen fixing ability of Azotobacter.

Azotobacter also produces fixation of Thiomin, Riboflavin, Nicotin, indol acitic acid and giberalin. When
Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls
plant diseases due to above substances produced by Azotobacter.

Azotobacter in Soil

Soils are habited by a very large number of microbial species. The co-existence of the relative populations of
each one of the species is determined by ecological factors prevailing in the soil. These various species
survive in soil while maintaining a balance of population is between various microbial species within certain
limits. A normal population of Azotobacter could be about than 10 thousand to 1 lakh/g of soil and it is mostly
influenced by other micro-organisms present in soil. There are some micro-organism which stimulate the
Azotobacter population in soil thereby increasing the nitrogen fixation by Azotobacter. On the other hand
there are some micro-organisms which adversely affect the Azotobacter population and hence nitrogen
fixation process is hampered. For example cephallosporium is most commonly found organisms in soil which
restricts the growth of Azotobacter.

Importance

36
Nitrogen fixation plays an important role in the nitrogen cycle. Azotobacter also synthesize some biologically
active substances, including some phytohormones such as auxins, thereby stimulating plant growth. They also
facilitate the mobility of heavy metals in the soil and thus enhance bioremediation of soil from heavy metals,
such as cadmium, mercury and lead. Some kinds of Azotobacter can also biodegrade chlorine-containing
aromatic compounds, such as 2,4,6-trichlorophenol. The latter was previously used as an insecticide,
fungicide and herbicide but later found to have mutagenic and carcinogenic effects.

Applications

Owing to its ability to fix molecular nitrogen and therefore increase the soil fertility and stimulate plant
growth, Azotobacter are widely used in agriculture. Particularly in nitrogen biofertilizers such as
azotobacterin. They are also used in production of alginic acid (E400), which is applied in medicine as an
antacid, in the food industry as an additive to ice cream, puddings and creams,[61] and in the biosorption of
metals.

MOONG BEAN

Mung bean, also known as green bean, mung, mongo, moong, moog dal, mash bean, munggo or
monggo, green gram, golden gram, and green soy, is native to Bangladesh, India and Pakistan. It is
the seed of Vigna Radiata and when split, the bean is known as moong dal in India. In fact, the
English word ‘mung’ has been derived from the Hindi word ‘moong’. The dal is green in color along
with the husk and yellow when the husk is removed. Mung beans are small and ovoid in shape.
They comprise of one of the several species to have been moved from the genus Phaseolus to
Vigna.

Mung beans are commonly used in the preparation of Chinese dishes, where they are called lǜ dòu.
They are also used extensively in Thailand, Taiwan, Japan, Korea, Pakistan, India, and Southeast
Asia. In Vietnam, mung beans are called đậu xanh. Generally, these beans are eaten either whole
or as bean sprouts. They are also used to make soups and desserts. The starch extracted from

37
them is used to make jellies and noodles. Mung beans are known to be very healthy and packed
with a variety of nutrients. Read on to know the health benefits of eating mung beans, along with
their nutritional value.

38
 Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

(unranked): Rosids

Order: Fabales

Family: Fabaceae

Genus: Vigna

Species: V. radiata

Binomial name

Vigna radiata

Health and Nutrition Benefits Of Eating Moong bean

39
 Mung bean sprouts contain rich quantities of Vitamin A, B, C and E. They are also known to be an
excellent source of many minerals, such as calcium, iron and potassium
 The bean is popular as the perfect food for reducing weight. It is recommended as a food replacement
in many slimming programs, as it has a very low fat content. It is a rich source of protein and fiber,
which helps one to lower the high cholesterol level in the blood system.
 The high fiber content of mung beans yields complex carbohydrates, which aid digestion. Complex
carbs are also effective in stabilizing blood sugar and prevent its rapid rise after meal consumption,
apart from keeping body’s energy at a balanced level. Those who suffer from diabetes or high
cholesterol are recommended frequent consumption of mung bean.
 In Chinese medicine, mung bean sprouts are considered as a cooling food, containing anti-cancer
properties. Herbalists use them for all hot, inflammatory conditions, ranging from systematic
infections to heat stroke and even hypertension.
 Aids in weight loss ie Moong dal or green beans provide great source of complex carbohydrates, fiber
and protein. Also, they are an excellent source of molybdenum and folic acid. They provide a good
nutrition for diet ers since they are low in fat.
 Lowers cholesterol levels, regular consumption of green beans helps to reduce cholesterol since they
are rich in fiber. According to recent studies, high fiber in green beans keeps blood sugar from rising
after mealtime. This makes green beans a great choice for people with insulin resistance, or
hypoglycemia.
 Promote heart health, Green beans have antioxidants properties; folic acid, fiber, magnesium and
vitamin B6 which help promote heart health. Vitamin B6 and folic acid lower homocysteine levels in
the body, which is essential in a metabolic process known as methylation cycle. A high level of
homocysteine in the blood is attributed to heart attack, peripheral vascular disease and stroke.
 Provides resistance against infectious diseasesand also, green beans contain thiamin (vitamin B-1),
vitamin-C and vitamin-B6 (pyridoxine). Regular consumption of diets rich in Vitamin-B6 help you
develop resistance against contagious agents that cause diseases.
 Contain anti-cancer properties, in Chinese medicine, green beans are popularly used for hot,
inflammatory conditions, such as hypertension. They were considered to have anti-cancer properties
and were also used as a cooling diet.
 Prevent age related muscular diseases, they provide an excellent source of folates, a great nutrition
during preconception. It prevents neural-tube defects during pregnancy.
 Since moong dal or green bean is a low carb diet, studies show that it may have negative effects on
health, especially with regards to weight loss.

40
i. CULTURING OF MICROORGANISMS

Although many bacteria can be used beneficially as a biofertilizer the technique for production of Rhizobium
and Azotobacter are discussed here. The growth mediums used for mass culturing of different bacterial
biofertilizers used by us are Yema Medium and Jensen Medium.

ii. INOCULUM PREPARATION

 Prepare appropriate medias for specific to the bacterial inoculant ( Rhizobium and Azotobacter) in
25 mL, 50 mL, 100 mL and 250 mL conical flasks and sterilize.
 The media in 50 mL flask is inoculated with efficient bacterial strain of Rhizobium and Azotobacter
under aseptic conditions.
 Keep flasks under room temperature in Rotary Shaker (200 rpm) for 2- 4 days.
 Observe flasks for growth in culture and estimate the population, which serves as the starter culture.
 Using the starter culture (at log phase) inoculate the larger flasks (100 mL, 200 mL and 250 mL)
containing the media.
 After obtaining growth in each flask,the above media for Rhizobium and Azotobacter is prepared in
large quantities and kept ready for use.
 The broth is checked for the population of inoculated organism and contamination if any at the growth
period.

41
iii. STREAKING ON PLATES

For Rhizobium

In this step streaking of Rhizobium is done using an inoculation loop on its respective media (Yema media).
After streaking is done the petriplates are kept in the Incubator for about 24-48 hrs at 37 degree centigrade.
Now when after 48 hrs bacterial colonies become visible, one of the colony is picked up and put in the bottles
containing media in order to scale up the culture.

For Azotobacter

Streaking of Azotobacter is also done using inoculation loop on the media (Jensen media).After the
completion of streaking, the plates are kept in the Incubator for 24-48 hrs at 37 degree centigrade.Bacterial
colonies are picked up and and scaling up of culture is done.
42
iv. PROCESSING OF CARRIER MATERIAL
The use of ideal carrier material is necessary for the production of good quality biofertilizer. Peat soil, lignite,
vermiculite, charcoal, press mud, farmyard manure and soil mixture can be used as carrier materials. The
neutralized peat soil/lignite are found to be better carrier materials for biofertilizer production The following
points are to be considered in the selection of ideal carrier material.
• Cheaper in cost
• Should be locally available
• High organic matter content
• No toxic chemicals
• Water holding capacity of more than 50%
• Easy to process, friability and vulnerability.

v. PREPARATION OF CARRIER MATERIAL

VERMICOMPOST

Vermicompost is an organic manure (bio-fertilizer) produced as the vermicast by earth worm feeding on
biological waste material; plant residues. This compost is an odorless, clean, organic material containing
adequate quantities of N, P, K and several micronutrients essential for plant growth. Vermicompost is a
preferred nutrient source for organic farming. It is eco-friendly, non-toxic, consumes low energy input for
composting and is a recycled biological product.

43
 vi. STERILIZATION OF CARRIER

Sterilization of carrier material is essential to keep high number of inoculant bacteria on carrier
for long storage period. Gamma-irradiation is the most suitable way of carrier sterilization, because the
sterilization process makes almost no change in physical and chemical properties of the material. In brief,
carrier material is packed in thin-walled polyethylene bag, and then gamma-irradiated at 50 kGy (5 Mrads).
Another way of carrier sterilization is autoclaving. Carrier material is packed in partially opened, thin-walled
polypropylene bags and autoclaved for 60 min at 121 ºC. It should be noted that during autoclaving, some
materials changes their properties and produce toxic substance to some bacterial strains.

vii. BINDING OF CULTURE WITH CARRIER

In this step the Carrier (vermicompost) in mixed with respective bacterial cultures (Rhizobium and
Azotobacter) with the help of a Glass Rod until a thick slurry is formed. This thick slurry is now placed
evenly in trays which are kept for drying.The process of drying may take 2-3 days as these trays are kept in
sunlight.Complete drying is necessary as this slurry has to be beaten to convert into powdered form.

viii. SERIAL DILUTION METHOD FOR COUNTING OF BACTERIAL

COLONIES

Serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is
constant, resulting in a geometric progression of the concentration in a logarithmic fashion.
44
 Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments
resulting in concentration curves with a logarithmic scale.

Besides the more conventional uses described above, serial dilution may also be used to reduce the
concentration of microscopic organisms or cells in a sample.

A tenfold dilution for each step is called a logarithmic dilution or log-dilution and a 3.16-fold (100.5-fold)
dilution is called a half-logarithmic dilution or half-log dilution.

Materials

 Buffer used to dissolve the sample

 The sample
 Multiple tubes
 Pipette or graduated cylinder
 Stirring rod

Procedure

 Shake the solution by hand or use the stirring rod to swirl the solution. Make sure the solution is uniformly
mixed.
45
 Take half of the solution out to a new tube and add equal amount of buffer into it to dilute the solution
concentration to half of the original solution.
 Take half of the newly made solution to another new tube and add equal amount of buffer into it to dilute
the solution concentration to one quarter of the original solution.
 Continue the process until reaching the desired concentration of the solution.
 In this procedure we take 10 (-2,-4,-6,-8 -10) and take the tube for measuring are 10 (-4 and -6) and
spread on media plates.
 Keep plate in incubator for 1 to 2 days for result and then count the colonies.

Analysis

Serial dilution is a process of solution preparation. Therefore, analysis is not necessary.

Result

On YEMA media for Rhizobium

Measuring plate 10(-4) = 10 colonies

Plate 10(-6) = 7colonies

On Jensen media for Azotobacter

Measuring plate 10(-4) = 17 colonies

Plate 10(-6) = 11 colonies

46
 ix. SEED PREPRATION FOR INOCULATION

Moong dal seeds are taken and dipped in water for about two days.After two days all the seeds are taken out
of water and tied in piece of cloth for a 12-24 hrs.The cloth is watered 3-4 times to keep it wet.Now after 24
hrs sprouts are seen.

x. FIELD TREATMENT

Moong Dal seeds are taken and grown for two days until sprouts come out of them. Now pots are taken , are
filled by soil, vermicompost containing Rhizobium and Azotobacter cultures is mixed and Moong Dal seeds
are sown into these pots.These pots need to be watered everyday.

47
 XI. STORAGE OF BIOFERTILIZER PACKET
• The packet should be stored in a cool place away from the heat or direct sunlight.
• The packets may be stored at room temperature or in cold storage conditions in lots in plastic crates or
polythene/gunny bags.
• The population of inoculant in the carrier inoculant packet may be determined at 15 days interval. There
should be more than 109 cells per gram of inoculant at the time of preparation and107 cells per gram on dry
weight basis before expiry date.

3.2 MEDIA USED FOR BIOFERTILIZER PRODUCTION

All micro organisms require water, source of energy carbon, nitrogen, minerals element and possibly vitamins
plus oxygen if aerobic. On a small scale it is relatively simple to devise a medium containing pure compound,
but the resulting medium although supporting satisfactory growth may be un suitable for use in large scale
process.
On a large scale one must normally use sources of nutrients to create a medium that will meet all of the
following criteria:-
1. It should produce the maximum yield of product or biomass per gram of substrate used.
2. It should permit the maximum rate of product formation.
3. There should be minimum yield of an undesired product.
4. It should be of a consistent quality and be available throughout the year.

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The constituents of a medium must satisfy the element requirement for cell biomass and metabolite
production, there must be an adequate supply of energy for biosynthesis and cell maintenance. Most of the
fermented products formed by organism are produce as a result of their response to the environmental
condition such as nutrients, growth hormones and ions. The qualitative and quantitative nutritional
requirements of cell are always determine to optimize growth and product formation. Nutrients required
by microbial cells are classified as:-
i. Macronutrients and it includes carbon, oxygen, nitrogen, hydrogen, sulfur, phosphorous,
magnesium and potassium.
ii. Micronutrients and it includes trace elements, vitamins, growth hormones and certain metabolic
precursors.

Microorganisms need following components for their growth :-

WATER: - water is the major component of all fermentation media and is needed in many of the ancillary
services such as heating, cooling, cleaning and rinsing. Clean water of consistent composition is therefore
required in large quantities from reliable permanent sources. When assessing the suitability of water
supply it is important to consider pH, dissolved salts and effluent contamination.

ENERGY SOURCES: - energy from growth comes from either the oxidation of medium components or
from light. Most industrial microorganisms are chemorganotrophs, therefore the commonest source of
energy will be the carbon source such as carbohydrates, lipids and proteins. Some microorganisms can
also use hydrocarbon or methanol as carbon sources.

CARBON SOURCES:
a) Carbohydrates: - it is common practice use carbohydrates as the carbon source in microbial
fermentation processes. Following carbohydrates are used as carbon sources :-
• Starch – Starch is obtained from maize grain and other cereals, potatoes and cassava.
• Sucrose – Sucrose is obtained from sugarcane and sugar beet.
• Glucose
• Molasses
• Corn stew liquor
• Lactose
b) Oils and fats :- Vegetable oils (olive, maize, cottonseed, linseed, soyabean)

49
 c) Hydrocarbons and their derivatives: - Hydrocarbons and their derivatives might have potential
role in higher value products such as intermediates, pharmaceuticals, fine chemicals and
agricultural chemicals.

NITROGEN SOURCES :- most industrially used microorganisms can utilize inorganic or organic sources of
nitrogen. Inoganic nitrogen maybe supplied as ammonia gas, ammonium salt or nitrate. Ammonia has been
used for pH control and as the.

MINERALS: - all microorganisms require certain mineral elements for growth and metabolism. In many
media, Mg, P, K, S, Ca and Cl are essential components, of and because of the concentration required, they
must be added as distinct component. Others such as Co, Cu, Fe, Mn, Mo and Zn are essential but are usually
present as impurities in other major ingredients.

GROWTH FACTORS: - some microorganisms cannot synthesize a full component of a cell component and
therefore require performed compounds called growth factors. The growth factors most commonly require
vitamins but there maybe also be need for specific amino acids, fatty acids or sterols. Many of natural carbon
and nitrogen sources used in media formulations contain all or some of the required growth factors.

BUFFERS: - the buffers are used to control the pH of media. Buffers like sodium acetate,K2PO4 etc are also
included in the media as raw materials.

YEMA Media (Yeast Extract Mannitol Agar Media) for Rhizobium :-

Composition

Ingredients gms / litre

0.5
Magnesium sulphate 0.2
Sodium chloride 0.1
Mannitol 7.0
Yeast Extract 1.0
Agar 20.0
Distilled Water 1000ml

Jensen’s Media for Azotobacter:-

Composition

Ingredients gms / litre

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Sucrose 20.000
Dipotassium phosphate 1.000
Magnesium sulphate 0.500
Sodium chloride 0.500
Ferrous sulphate 0.100
Sodium molybdate 0.005
Calcium carbonate 2.000
Agar 15.000

4. OBSERVATION

5. RESULT AND CONCLUSION

Biofertilizers improve soil fertilitily and enhance nutrient uptake and water uptake in deficient soil thereby
aiding in better establishment of plants. Biofertilizers also secrete growth substances and antifungal chemicals
as well as improve seed germination and root growth. The dual effects of phosphorus mobilizing fungi and
specific nitrogen fixing bacteria can cater to the needs of current coffee plantation sector. Thus the use of
biofertilizers will effectively enrich the soil and will cost less then chemical fertilizers, which harm the
environment and deplete non renewable energy sources.

Biofertilizers have definite advantage over chemical fertilizers. Chemical fertilizers supply over nitrogen
whereas Biofertilizers provide inaddition to nitrogen certain growth promoting substances like hormones,
vitamins, amino-acids etc, crops have to be provided with chemical fertilizers repeatedly to replenish the loss
of nitrogen utilized for crop growth. On the other hand biofertilizers supply the nitrogen continuously
throughout the entire period of crop growth in the field under favourable conditions. Continuous use of
chemical fertilizers adversely effect the soil structure whereas biofertilizers when applied to soil improve the
soil structure. The deleterious effects of chemical fertilizers are that they are toxic at higher doses,
biofertilizers however have no toxic effects.

It may be borne in mind but biofertilizers are no substitute for chemical fertilizers. At present the use of
chemical fertilizers is far below the recommended level. Therefore the aim and object of spread of
biofertilizer technology as a industry has to build up efficiency in use of chemical fertilizers supplemented by
low cost inoculants to the extent possible.

QUALITY CONTROL REQUIRED FOR BIOFERTILIZER

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 • Colors: Carrier based, the color depending upon the color of the carrier.
• Total viable count: Total number of viable cells during the entire period of shelf life should be at
minimum of 1x10^8 g of carrier on dry mass basis.
• Contamination: The product should be free from all types of contamination upto 105 dilutions.
• pH: The pH should be between 6.5 and 7.5.
• Expiry period: Maximum of 6 months from the date of manufacture.
• Carrier and its particle size: Peat lignite, Peat soil, Humus are similar material in the form of powder
capable of passing through 150-212 micron in sieve.
• Moisture: Carrier should be in moist form with 30-40% moisture.

3.3 PRECAUTIONS

 Surface Sterilization of LAF is very important.

 Wearing gloves, mask and apparen is necessary to avoid contamination.
 Nails should be properly cut.
 Biofertilizer packets need to be stored in cool and dry place away from direct
sunlight and heat.
 Right combinations of biofertilizers have to be used.
 As Rhizobium is crop specific, one should use for the specified crop only.
 Other chemicals should not be mixed with the biofertilizers.
 While purchasing one should ensure that each packet is provided with
necessary information like name of the product, name of the crop for which
intended, name and address of the manufacturer, date of manufacture, date
of expiry, batch number and instructions for use.
 The packet has to be used before its expiry, only for the specified crop and
by the recommended method of application.
 Biofertilizers are live product and require care in the storage
 Both nitrogenous and phosphatic biofertilizers are to be used to get the best
results.
 It is important to use biofertilizers along with chemical fertilizers and organic
manures.

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  Biofertilizers are not replacement of fertilizers but can supplement plant
nutrient requirements.

EQUIPMENTS

In biofertilizers production industry equipment are the major infrastructure which involve 70% of capital
investment. Any compromise on the usage of the following mentioned equipments may finally decline in the
quality of biofertilizers. After studying the principle behind the use of all instruments, some of the instrument
can be replaced with a culture room fitted with a U.V. lamp Autoclave, Hot air oven, Incubator and sealing
machine are indigenously made with proper technical specification. The correct use of equipment will give
uninterrupted introduction with quality inoculums.
 Autoclave
 Laminar Air Flow
 Incubator
 pH Strips
 Inoculation Loop
 Spirit Lamp
 Hot air Oven

Autoclave

53
It is aapparatus in which materials are sterilized by air free saturated steam ( under pressure ) at a temp above
00 oC . If the steam pressure inside the autoclave is increased to 15 psi, the temp will rise to 121 oC . This is
sufficient to destroy all vegetative cell. Normally all growth medium sterilized under autoclave .

Laminar Air flow

LAF chamber provide a uniform flow of sterilized air. This uniform flow of Air will prevent settling of
particle in the work area. Air borne contamination is avoided in the chamber. Culture transfer and inoculation
can be done here.

Incubator

54
Incubator providing controlled condition (light, temp, humidity) required for the growth and development of
microorganism. Multiplication of starter culture can be done in this instrument.

Hot Air Oven

Hot air oven is meant for sterilizing all glass ware materials. Dry heat is used in this apparatus to sterilised the
materials. Normally 180 oC is used for 2 hrs for sterilizing glass ware

BIBLOGRAPHY

• Phelps, A. S., and Wilson, P. W., Proc. Sot. Exp. Biol. and Med., 47, 473 (1941).
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• Lee, S. B., Burris, R. H., and Wilson, P. W., J. Bad., 43,60 (1942).

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• Allen. O.P. and E.K. Allen. 1981. The Leguminosae. University of Wisconsin Press.
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Science. Eds. R.J. Sammerfield and A.H. Bunting p69. Royal Botanic Gardens, Kew England.
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Postgate, J (1998). Nitrogen Fixation, 3rd Edition. Cambridge University Press, Cambridge
UK.
• Moir, JWB (editor) (2011). Nitrogen Cycling in Bacteria: Molecular Analysis. Caister Academic Press
• The Nature and Properties of Soil" by Nyle C. Brady and Ray R. Weil, Prentice Hall, Inc., 1996,
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Education, Vol. 26
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WEBSITES

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