Lecture No 10: Biofertilizers in organic farming

1. Biofertilizers
2. Different types of biofertilizers
3. Mass production of Biofertilizers
4. Application of Biofertilizers
5. Azolla as cattle feed

6. Constraints in Biofertilizer Technology

1. Biofertilizers

Biofertilizers are defined as preparations containing living cells or latent cells of

efficient strains of microorganisms that help crop plants’ uptake of nutrients by their
interactions in the rhizosphere when applied through seed or soil. They accelerate certain
microbial processes in the soil which augment the extent of availability of nutrients in a form
easily assimilated by plants.

 For a sustainable agriculture system, it is essential to use renewable inputs (fertilizer,

pesticides, water etc.) which can benefit the plant and cause no or minimal damage to
the environment.
 One of the energy efficient and pollution free method is to exploit the ability of certain
microorganisms like bacteria, algae and fungi to fix atmospheric nitrogen, solubilize
phosphorus, decompose organic material or oxidize sulphur in the soil.
 When they are applied in the soil, they enhance growth and yield of crops, improve soil
fertility and reduce pollution. They are known as “bio fertilizers”.
 Thus bio-fertilizers are living or biologically active products or microbial inoculants of
bacteria, algae and fungi (separately or in combination) which are able to enrich the soil
with nitrogen, phosphorus, organic matter etc.

Very often microorganisms are not as efficient in natural surroundings as one would
expect them to be and therefore artificially multiplied cultures of efficient selected
microorganisms play a vital role in accelerating the microbial processes in soil.
Use of biofertilizers is one of the important components of integrated nutrient management, as
they are cost effective and renewable source of plant nutrients to supplement the chemical
fertilizers for sustainable agriculture. Several microorganisms and their association with crop
plants are being exploited in the production of biofertilizers. They can be grouped in different
ways based on their nature and function.

S. No. Groups Examples

N2 fixing Biofertilizers
Azotobacter, Beijerinkia, Clostridium, Klebsiella, Anabaena,
1. Free-living
Nostoc,
2. Symbiotic Rhizobium, Frankia, Anabaena azollae
Associative
3. Azospirillum
Symbiotic
P Solubilizing Biofertilizers
Bacillus megaterium var. phosphaticum, Bacillus subtilis
1. Bacteria
Bacillus circulans, Pseudomonas striata
2. Fungi Penicillium sp, Aspergillus awamori
P Mobilizing Biofertilizers
Arbuscular Glomus sp.,Gigaspora sp.,Acaulospora sp.,
1.
mycorrhiza Scutellospora sp. & Sclerocystis sp.
2. Ectomycorrhiza Laccaria sp., Pisolithus sp., Boletus sp., Amanita sp.
Ericoid
3. Pezizella ericae
mycorrhizae
Orchid
4. Rhizoctonia solani
mycorrhiza
Biofertilizers for Micro nutrients
Silicate and Zinc
1. Bacillus sp.
solubilizers
Plant Growth Promoting Rhizobacteria
1. Pseudomonas Pseudomonas fluorescens

2. Different types of biofertilizers

2.1. Rhizobium

Rhizobium inoculant was first made in USA and commercialized by private enterprise
in 1930s and the strange situation at that time has been chronicled by Fred (1932).

Initially, due to absence of efficient bradyrhizobial strains in soil, soybean inoculation at

that time resulted in bumper crops but incessant inoculation during the last four decades by US
farmers has resulted in the build-up of a plethora of inefficient strains in soil whose replacement
by efficient strains of bradyrhizobia has become an insurmountable problem.

 Rhizobium is a symbiotic bacterium forming root nodules in legume plants.

 These nodules act as miniature nitrogen production factories in the fields.
 The nodule bacteria fix more nitrogen (N2) than needed by legume plant and the bacteria.
 The surplus fixed nitrogen is then secreted and fertilizes the soil.
 Rhizobium is more efficient than-free living nitrogen-fixing bacteria.
2.2. Azotobacter

Of the several species of Azotobacter, A. chroococcum happens to be the dominant

inhabitant in arable soils capable of fixing N2 (2-15 mg N2 fixed /g of carbon source) in culture
media. The bacterium produces abundant slime which helps in soil aggregation. The numbers
of A. chroococcum in Indian soils rarely exceeds 105/g soil due to lack of organic matter and
the presence of antagonistic microorganisms in soil.

 Azotobacter are aerobic free living nitrogen fixers.

 They grow in the rhizosphere (around the roots) and fix atmospheric nitrogen non-
symbiotically and make it available to the particular cereals.
 These bacteria produce growth promoting hormones which helps in enhancing growth
and yield of the plant.

2.3. Azospirillum

Azospirillum lipoferum and A. brasilense (Spirillum lipoferum in earlier literature) are

primary inhabitants of soil, the rhizosphere and intercellular spaces of root cortex of
graminaceous plants. They perform the associative symbiotic relation with the graminaceous
plants.
 The bacteria of Genus Azospirillum are N2 fixing organisms isolated from the root and
above ground parts of a variety of crop plants. They are Gram
negative, Vibrio or Spirillum having abundant accumulation of polybetahydroxybutyrate (70
%) in cytoplasm.

Five species of Azospirillum have been described to date A.

brasilense, A.lipoferum, A.amazonense, A.halopraeferens and A.irakense. The organism
proliferates under both anaerobic and aerobic conditions but it is preferentially micro-
aerophilic in the presence or absence of combined nitrogen in the medium.

Apart from nitrogen fixation, growth promoting substance production (IAA), disease
resistance and drought tolerance are some of the additional benefits due
to Azospirillum inoculation.

 These are aerobic free living nitrogen fixers which live in associative symbiosis.
 In this type of association bacteria live on the root surface of the host plant and do not
form any nodule with roots of grasses.
 It increases crop yield and its inoculation benefits crop.
 They also benefit the host plants by supplying growth hormones and vitamins.
 These bacteria are commonly used for the preparation of commercial inoculants (vaccines,
culture medium).

2.4. Cyanobacteria

Both free-living as well as symbiotic cyanobacteria (blue green algae) have been
harnessed in rice cultivation in India. A composite culture of BGA having
heterocystous Nostoc, Anabaena, Aulosira etc. is given as primary inoculum in trays,
polythene lined pots and later mass multiplied in the field for application as soil-based flakes
to the rice growing field at the rate of 10 kg/ha. The final product is not free from extraneous
contaminants and not very often monitored for checking the presence of desiredalgal flora.
 Blue green algae (BGA or cyanobacteria) like Nostoc and Anabaena are free living
photosynthetic organisms also capable of fixing atmospheric nitrogen.
 In the flooded rice fields blue green algae serves as a nitrogen biofertilizer.

Once so much publicized as a biofertilizer for the rice crop, it has not presently attracted
the attention of rice growers all over India except pockets in the Southern States, notably Tamil
Nadu. The benefits due to algalization could be to the extent of 20-30 kg N/ha under ideal
conditions but the labour-oriented methodology for the preparation of BGA biofertilizer is in
itself a limitation. Quality control measures are not usually followed except perhaps for random
checking for the presence of desired species qualitatively.

2.5. Azolla

Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen
in association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of
sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots. Rice
growing areas in South East Asia and other third World countries have recently been evincing
increased interest in the use of the symbiotic N2 fixing water fern Azolla either as an alternate
nitrogen sources or as a supplement to commercial nitrogen fertilizers. Azolla is used as
biofertilizer for wetland rice and it is known to contribute 40-60 kg N/ha per rice crop.

 Azolla is a water fern inside which grows the nitrogen fixing blue green algae Anabaena.
 It contains 2-3% nitrogen when wet and also produces organic matter in the soil.
 The Azolla-Anabaena combination type biofertilizer is used all over the world.
 The only constraint in Azolla is that it is an aquatic plant and water becomes
limiting factor in growing it particularly in summer.
3. Phosphate solubilizing microorganism (PSM)

Several soil bacteria and fungi, notably species of Pseudomonas, Bacillus, Penicillium,
Aspergillus etc. secrete organic acids and lower the pH in their vicinity to bring about
dissolution of bound phosphates in soil. Increased yields of wheat and potato were
demonstrated due to inoculation of peat based cultures of Bacillus polymyxa and Pseudomonas
striata. Currently, phosphate solubilizers are manufactured by agricultural universities and
some private enterprises and sold to farmers through governmental agencies. These appear to
be no check on either the quality of the inoculants marketed in India or the establishment of
the desired organisms in the rhizosphere.

The transfer of nutrients mainly phosphorus and also zinc and sulphur from the
soil milleu to the cells of the root cortex is mediated by intracellular obligate fungal
endosymbionts of the genera Glomus, Gigaspora, Acaulospora,
Sclerocysts and Endogone which possess vesicles for storage of nutrients and arbuscles for
funneling these nutrients into the root system. By far, the commonest genus appears to
be Glomus, which has several species distributed in soil.

Availability for pure cultures of AM (Arbuscular Mycorrhiza) fungi is an impediment

in large scale production despite the fact that beneficial effects of AM fungal inoculation to
plants have been repeatedly shown under experimental conditions in the laboratory especially
in conjunction with other nitrogen fixers.

AM fungi

 Phosphorus is an important element required for plant growth.

 This element is also needed for nodulation by rhizobium.
 Some microorganisms are capable of solubilizing immobilized phosphorus making it
available to plants for absorption.
Mycorrhizal fungi

 Mycorrhizal fungi acts as biofertilizer and are known to occur naturally on roots of
forest trees and crop plants.
 Mycorrhizal fungi resist disease in plants. The plants also show drought and salinity
resistance. Plants can tolerate adverse soil, pH, high temperature and heavy metal
toxicity.
 In soils low in available nutrients there is an increased absorption of nutrients by plants
infected with Mycorrhiza.
 The fungus has the ability to dissolve and absorb phosphorus that plant roots cannot
readily absorb.

4. Silicate solubilizing bacteria (SSB)

Microorganisms are capable of degrading silicates and aluminum silicates. During the
metabolism of microbes several organic acids are produced and these have a dual role in silicate
weathering. They supply H+ ions to the medium and promote hydrolysis and the organic acids
like citric, oxalic acid, Keto acids and hydroxy carbolic acids which from complexes with
cations, promote their removal and retention in the medium in a dissolved state.

The studies conducted with a Bacillus sp. isolated from the soil of granite crusher yard
showed that the bacterium is capable of dissolving several silicate minerals under in
vitro condition. The examination of anthrpogenic materials like cement, agro inputs like super
phosphate and rock phosphate exhibited silicate solubilizing bacteria to a varying degree. The
bacterial isolates made from different locations had varying degree of silicate solubilizing
potential. Soil inoculation studies with selected isolate with red soil, clay soil, sand and hilly
soil showed that the organisms multiplied in all types of soil and released more of silica and
the available silica increased in soil and water. Rice responded well to application of organic
sliceous residue like rice straw, rice husk and black ash @ 5 t/ha. Combining SSB with these
residues further resulted in increased plant growth and grain yield. This enhancement is due to
increased dissolution of silica and nutrients from the soil.

Use of biofertilizers

• They are cheap, hence, reduced cost of cultivation.

• Free from pollution hazards

• They form an important association with other soil microbes and help in nutrient supply.
• Increase soil fertility

• Fixes atmospheric nitrogen.

• Quantity required decreased year by year

• Increase availability or uptake of nutrients through solubilization or increased absorption.

• Cyanobacteria secrete growth promoting substances like IAA, IBA, NAA, AA, Proteins,
Vitamins etc.

• Many agents secretes antibiotics which act as pesticides

• It improves physico-chemical properties of soil

• Harmless to human and animals

• Improves soil properties and sustaining soil fertility Lead to soil enrichment.

• Build up soil fertility in the long term.

• Reduces the use of chemical fertilizers.

• They are eco-friendly and pose no damage to the environment

As such there is no harmful impact of biofertilizers if it is used properly. some constraints:

• Specific to the plants

• Use within expiry date
• Rhizobium spp. culture doesn't work well in high nitrate tolerant strains of soybean.
• The acceptability of biofertilizers has been rather low chiefly because they do not produce
quick and spectacular responses.
• Require skill in production and application
• Difficult to store.

Liquid Biofertilizers

Biofertilizers are such as Rhizobium, Azospirillum and Phosphobacteria provide

nitrogen and phosphorous nutrients to crop plants through nitrogen fixation and phosphorous
solubilization processes. These Biofertilizers could be effectively utilized for rice, pulses,
millets, cotton, sugarcane, vegetable and other horticulture crops.

Biofertilizer is one of the prime inputs in organic farming not only enhances the crop
growth and yield but also improves the soil health and sustain soil fertility. At present,
Biofertilizers are supplied to the farmers as carrier based inoculants. As an alternative, liquid
formulation technology has been developed in the Department of Agricultural Microbiology,
TNAU, Coimbatore which has more advantages than the carrier inoculants.

Benefits

The advantages of Liquid Bio-fertilizer over conventional carrier based Bio-fertilizers

are listed below:

 Longer shelf life -12-24 months.

 No contamination.
 No loss of properties due to storage upto 45º c.
 Greater potentials to fight with native population.
 High populations can be maintained more than 109 cells/ml upto 12 months to 24
months.
 Easy identification by typical fermented smell.
 Cost saving on carrier material, pulverization, neutralization, sterilization, packing and
transport.
 Quality control protocols are easy and quick.
 Better survival on seeds and soil.
 No need of running Bio-fertilizer production units throughout the year.
 Very much easy to use by the farmer.
 Dosages is 10 time less than carrier-based powder Bio-fertilizers.
  High commercial revenues.
 High export potential.
 Very high enzymatic activity since contamination is nil.

Characteritistics of different liquid Bio-fertilizers

Rhizobium

This belongs to bacterial group and the classical example is symbiotic nitrogen fixation.
The bacteria infect the legume root and form root nodules within which they reduce molecular
nitrogen to ammonia which is reality utilized by the plant to produce valuable proteins,
vitamins and other nitrogen containing compounds. The site of symbiosis is within the root
nodules. It has been estimated that 40-250 kg N / ha / year is fixed by different legume crops
by the microbial activities of Rhizobium. The percentage of nodules occupied, nodules dry
weight, plant dry weight and the grain yield per plant the multistrain inoculant was highly
promising Table-2 shows the N fixation rates.

Quantity of biological N fixed by Liqiud Rhizobium in different crops

Host Group Rhizobium Species Crops N fix kg/ha

Pea group Rhizobium leguminosarum Green pea, Lentil 62- 132
Soybean group R.japonicum Soybean 57- 105
Lupini Group R. lupine orinthopus Lupinus 70- 90
R.mellilotiMedicago
Alfafa grp.Group Melilotus 100- 150
Trigonella
Beans group R. phaseoli Phaseoli 80- 110
Clover group R. trifoli Trifolium 130
Moong, Redgram, Cowpea,
Cowpea group R. species 57- 105
Groundnut
Cicer group R. species Bengal gram 75- 117

Physical features of liquid Rhizobium

 Dull white in colour

 No bad smell
  No foam formation, pH 6.8-7.5

Azospirllium

It belongs to bacteria and is known to fix the considerable quantity of nitrogen in the
range of 20- 40 kg N/ha in the rhizosphere in non- non-leguminous plants such as cereals,
millets, Oilseeds, cotton etc. The efficiency of Azospirillium as a Bio-Fertilizer has increased
because of its ability of inducing abundant roots in several pants like rice, millets and oilseeds
even in upland conditions. Considerable quantity of nitrogen fertilizer up to 25-30 % can be
saved by the use of Azospirillum inoculant. The genus Azospirillum has three species viz., A.
lipoferum, A. brasilense and A. amazonense. These species have been commercially exploited
for the use as nitrogen supplying Bio-Fertilizers.

One of the characteristics of Azospirillum is its ability to reduce nitrate and denitrify.
Both A. lipoferum,and A. brasilense may comprise of strains which can actively or weakly
denitrify or reduce nitrate to nitrite and therefore, for inoculation preparation, it is necessary to
select strains which do not possess these characteristics. Azospirllium lipoferum present in the
roots of some of tropical forage grasses uch as Digitaria, Panicum, Brachiaria, Maize,
Sorghum, Wheat and Rye.

Physical features of liquid Azospirillum

 The colour of the liquid may be blue or dull white.

 Bad odours confirms improper liquid formulation and may be concluded as mere broth.
 Production of yellow gummy colour materials comfirms the quality product.
 Acidic pH always confirms that there is no Azospirillum bacteria in the liquid.

N2 fixing capacity of Azospirillum in the roots of several plants and the amount of N2
fixed by them.

Plant Mg N2 fixed /g of substrate

Oryza sativa (Paddy) 28
Sorghum 20
bicolour (Sorghum)
Zea mays (Maize) 20
 Panicum sp. 24
Cynodon dactylon 36
Setaria sp 12
Amaranthus spinosa 16

Production of growth hormones

Azospirillum cultures synthesize considerable amount of biologically active substances

like vitamins, nicotinic acid, indole acetic acids gibberellins. All these hormones/chemicals
help the plants in better germination, early emergence, better root development.

Role of Liquid Azospirillum under field conditions

 Stimulates growth and imparts green colour which is a characteristic of a healthy plant.
 Aids utilization of potash, phosphorous and other nutrients.
 Encourage plumpness and succulence of fruits and increase protein percentage.

Sign of non-functioning of Azospirillum in the field

 No growth promotion activity

 Yellowish green colour of leaves, which indicates no fixation of Nitrogen

Azotobacter

It is the important and well-known free-living nitrogen fixing aerobic bacterium. It is

used as a Bio-Fertilizer for all non-leguminous plants especially rice, cotton, vegetables
etc. Azotobacter cells are not present on the rhizoplane but are abundant in the rhizosphere
region. The lack of organic matter in the soil is a limiting factor for the proliferation
of Azotobacter in the soil.

Field experiments were conducted in 1992, 1993 and 1994 during the pre-kharif wet
seasons to find out the influence on rice grain yield by the combined use of N- fixing organisms
and inorganic nitrogen fertilizer which recorded increase in was yield.
Physical features of liquid Azotobacter

The pigmentation that is produced by Azotobacter in aged culture is melanin which is

due to oxidation of tyrosine by tyrosinase an enzyme which has copper. The colour can be
noted in liquid forms. Some of the pigmentation are described below-

 A. chroococcum: Produces brown-black pigmentation in liquid inoculum.

 A. beijerinchii: Produces yellow- light brown pigementation in liquid inoculum
 A. vinelandii: Produces green fluorescent pigmentation in liquid inoculum.
 A. paspali: Produces green fluorescent pigmentation in liquid inoculum.
 A. macrocytogenes: Produces, pink pigmentation in liquid inoculum.
 A. insignis: Produces less, gum less, grayish-blue pigmentation in liquid inoculum.
 A. agilies: Produces green-fluorescent pigmentation in liquid inoculum.

Role of liquid Azotobacter in tissue culture

The study was conducted by Dr. Senthil et al (2004) on sugarcane variety CO 86032 in
Tissue culture Laboratories of Rajashree Sugars and Chemicals Ltd, Varadaraj nagar, Theni,
Tamilnadu. The liquid bioinoculants were provided by Dr. Krishnan Chandra, Regional
Director, RCOF, Bangalore to evaluate their growth promoting effects on sugarcane
micropropagation. He recorded Biometric observations like Plant height, leaf length, width,
root length, no of roots. Chemical parameters –Protein, Carbohydrates, N, P,K total biomass
and concluded as follows:

 The performance of Azotobacter liquid inoculant was comparatively better than all the
treatments in 10 % MS medium followed Azospirillum.
 The performance of Azotobacter liquid inoculant was comparatively better than all the
treatments followed by Azosopirillum for the growth of the polybag sugarcane
seedlings.

Role of liquid Azotobacter as a Bio-control agent

Azotobacter have been found to produce some antifungal substance which

inhibits the growth of some soil fungi like Aspergillus, Fusarium, Curvularia,
Alternaria, Helminthosporium, Fusarium etc.
Acetobacter

This is a sacharophillic bacteria and associate with sugarcane, sweet potato and sweet
sorghum plants and fixes 30 kgs/ N/ ha year. Mainly this bacterium is commercialized for
sugarcane crop. It is known to increase yield by 10-20 t/ acre and sugar content by about 10-
15 percent.

Effect of liquid Acetobacter diazotrophicus on sugarcane

In South India use of Azospirillum and Phospho-bacterium on the cash crop sugarcane
is a regular practice for the past few years with a saving of nearly 20 % of chemical nitrogen
and phosphate applications. Now, it has been reported that a bacterium Acetobacter
diazotrophicus which is present in the sugarcane stem, leaves, soils have a capacity to fix up
to 300 kgs of nitrogen. This bacterium first reported in brazil where the farmers cultivate
sugarcane in very poor sub-soil fertilized with Phosphate, Potassium and micro elements alone,
could produce yield for three consecutive harvests, without any nitrogen fertilizer. They have
recorded yield 182- 244 tonnes per ha. This leads to the assumption that active nitrogen fixing
bacteria has associated within the plant.

Liquid biofertilizers application methodology

There are three ways of using Liquid Bio-fertilizers

1. Seed treatment
2. Root dipping
3. Soil application

Seed Treatment

Seed Treatment is a most common method adopted for all types of inoculants. The seed
treatment is effective and economic. For small quantity of seeds (up to 5 kgs quantity) the
coating can do in a plastic bag. For this purpose, a plastic bag having size (21” x 10”) or big
size can be used. The bag should be filled with 2 kg or more of seeds. The bag should be closed
in such a way to trap the airs as much as possible. The bag should be squeezed for 2 minutes
or more until all the seed are uniformly wetted. Then bag is opened, inflated again and shaked
gently. Stop shaking after each seed gets a uniform layer of culture coating. The bag is opened
 and the seed is dried under the shade for 20-30 minutes. For large amount of seeds coating can
be done in a bucket and inoculant can be mixed directly with hand. Seed Treatment
with Rhizobium, Azotobacter, Azospirillum, along with PSM can be done.

The seed treatment can be done with any of two or more bacteria. There is no side
(antagonistic) effect. The important things that has to be kept in mind are that the seeds must
be coated first with Rhizobium, Azotobacter or Azospirillum. When each seed get a layer of
above bacteria then PSM inoculant has to be coated as outer layer. This method will provide
maximum number of each bacteria required for better results. Treatments of seed with any two
bacteria will not provide maximum number of bacteria on individual seed.

Root dipping

For application of Azospirillum/ /PSM on paddy transplating/ vegetable crops this

method is used. The required quantity of Azospirillum/ /PSM has to be mixed with 5-10 litres
of water at one corner of the field and the roots of seedlings has to be dipped for a minimum
of half-an-hour before transplantation.

Soil application

Use 200ml of PSM per acre. Mix PSM with 400 to 600 kgs of Cow dung FYM along
with ½ bag of rock phosphate if available. The mixture of PSM, cow dung and rock phosphate
have to be kept under any tree or under shade for overnight and maintain 50% moisture. Use
the mixture as soil application in rows or during leveling of soil.

Dosage of liquid Bio-fertilizers in different crops

Recommended Liquid Bio-fertilizers and its application method, quantity to be used for
different crops are as follows:

Recommended Bio- Application Quantity to be

Crop
fertilizer method used
Field crops, Pulses, Chickpea, pea,
Groundnut, soybean, beans, Lentil,
Rhizobium Seed treatment 200ml/acre
lucerne, Berseem, Green gram, Black
gram, Cowpea and pigeon pea
Cereals- Wheat, oat, barley Azotobacter/Azospirillum Seed treatment 200ml/acre
Rice Azospirillum Seed treatment 200ml/acre
Oil seeds-Mustard, Sesamum,
Azotobacter Seed treatment 200ml/acre
Linseeds, Sunflower, castor
Millets
Pearl millets, Finger millets, kodo Azotobacter Seed treatment 200ml/acre
millet
Maize and Sorghum Azospirillum Seed treatment 200ml/acre
Forage crops and Grasses
Bermuda grass, Sudan grass, Napier Azotobacter Seed treatment 200ml/acre
Grass, ParaGrass, StarGrass etc.
Other Misc. Plantation Crops-
Azotobacter Seedling treatment 500ml/acre
Tobacco
Tea, Coffee Azotobacter Soil treatment 400ml/acre
Rubber, Coconuts Azotobacter Soil treatment 2-3 ml/plant
Agro-Forestry/Fruit Plants
All fruit/agro-forestry (herb,shrubs,
annuals and perennial) plants for fuel 2-3 ml/plant at
Azotobacter Soil treatment
wood fodder, fruits, gum, spice, nursery
leaves, flowers, nuts and seeds
puppose
Leguminous plants/ trees Rhizobium Soil treatment 1-2 ml/plant

Mass production of Bacterial Biofertilizer

Azospirillum Rhizobium Phosphobacteria Azotobacter

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 a greater number of bacterial cells and support the
survival of cells for longer periods of time.

 The mass production of carrier based bacterial biofertilizers involves three stages.
 Culturing of microorganisms
 Processing of carrier material
 Mixing the carrier and the broth culture and packing

Culturing of Microorganisms

 Although many bacteria can be used beneficially as a biofertilizer the technique of mass
production is standardizedfor Rhizobium, Azospirillum, Azotobacter and phosphobacteria.
The media used for mass culturing are as follows:
 Rhizobium: Yeast extract mannitol broth.

Growth on Congo red yeast extract mannitol agar medium

Mannitol - 10.0 g
K2 HPO4 - 0.5 g
Mg So4 7H2 O - 0.2 g
NaCl - 0.1 g
Yeast extract - 0.5 g
Agar 20.0 g
Distilled water 1000.0 ml

 Add 10 ml of Congo red stock solution (dissolve 250 mg of Congo red in 100ml water) to
1 liter after adjusting the PH to 6.8 and before adding agar.
 Rhizobium forms white, translucent, glistening, elevated and comparatively small colonies
on this medium. Moreover, Rhizobium colonies do not take up the colour of congo red dye
added in the medium. Those colonies which readily take up the congo red stain are not
rhizobia but presumably Agrobacterium, a soil bacterium closely related to Rhizobium.
 Azospirillum: Dobereiner's malic acid broth with NH4Cl (1g per liter).
Composition of the N-free semisolid malic acid medium

Malic acid - 5.0g

Potassium hydroxide - 4.0g
Dipotassium hydrogen
- 0.5g
orthophosphate
Magnesium sulphate - 0.2g
Sodium chloride - 0.1g
Calcium chloride - 0.2g
Fe-EDTA (1.64% w/v
- 4.0 ml
aqueous)
Trace element solution - 2.0 ml
BTB (0.5% alcoholic
- 2.0 ml
solution)
Agar - 1.75 g
Distilled water - 1000 ml
pH - 6.8
Trace element solution
Sodium molybdate - 200 mg
Manganous sulphate - 235 mg
Boric acid - 280 mg
Copper sulphate - 8 mg
Zinc sulphate - 24 mg
Distilled water - 200 ml

Waksman medium No.77 (N-free Mannitol Agar Medium for Azotobacter)

Mannitol : 10.0 g
Ca CO3 : 5.0 g
K2HPO4 : 0.5 g
Mg SO4.7H2O : 0.2 g
NaCl : 0.2 g
Ferric chloride : Trace
MnSO4.4H2O : Trace
N-free washed Agar : 15.0 g
pH : 7.0
Distilled Water : 1000 ml

Phosphobacteria: Pikovskaya’s Broth

Glucose : 10.0 g
Ca3(PO4)2 : 5.0 g
(NH4)2SO4 : 0.5 g
KCl : 0.2 g
MgSO4. 7H2O : 0.1 g
MnSO4 : Trace
FeSO4 : Trace
Yeast Extract : 0.5 g
Distilled Water : 1000 ml

The broth is prepared in flasks and inoculum from mother culture is transferred to
flasks. The culture is grown under shaking conditions at 30±2°C as submerged culture. The
culture is incubated until maximum cell population of 1010 to 1011 cfu/ml is produced. Under
optimum conditions this population level could be attained with in 4 to 5 days for Rhizobium;
5 to 7 days for Azospirillum; 2 to 3 days for phosphobacteria and 6-7 days for Azotobacter. The
culture obtained in the flask is called starter culture. For large scale production of inoculant,
inoculum from starter culture is transferred to large flasks/seed tank fermentor and grown until
required level of cell count is reached.

Inoculum preparation

 Prepare appropriate media for specific to the bacterial inoculant in 250 ml, 500 ml, 3
litre and 5 litre conical flasks and sterilize.
 The media in 250 ml flask is inoculated with efficient bacterial strain under aseptic
condition
 Keep the flask under room temperature in rotary shaker (200 rpm) for 5- 7 days.
 Observe the flask for growth of the culture and estimate the population, which serves
as the starter culture.
  Using the starter culture (at log phase) inoculate the larger flasks (500 ml, 3 litre and 5
litre) containing the media, after obtaining growth in each flask.
 The above media is prepared in large quantities in fermentor, sterilized well, cooled and
kept it ready.
 The media in the fermentor is inoculated with the log phase culture grown in 5 litre
flask. Usually 1 -2 % inoculum is sufficient, however inoculation is done up to 5%
depending on the growth of the culture in the larger flasks.
 The cells are grown in fermentor by providing aeration (passing sterile air through
compressor and sterilizing agents like glass wool, cotton wool, acid etc.) and given
continuous stirring.
 The broth is checked for the population of inoculated organism and contamination if
any at the growth period.
 The cells are harvested with the population load of 109 cells ml-1 after incubation
period.
 There should not be any fungal or any other bacterial contamination at 10-6 dilution
level
 It is not advisable to store the broth after fermentation for periods longer than 24 hours.
Even at 4o C number of viable cells begins to decrease.

Processing of carrier material

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

Preparation of carrier material

 The carrier material (peat or lignite) is powdered to a fine powder so as to pass through 212
microns IS sieve.
 The pH of the carrier material is neutralized with the help of calcium carbonate (1:10 ratio)
, since the peat soil / lignite are acidic in nature ( pH of 4 - 5). The neutralized carrier
material is sterilized in an autoclave to eliminate the contaminants.

Mixing the carrier and the broth culture and packing

Inoculant packets are prepared by mixing the broth culture obtained from fermentor with sterile
carrier material as described below:

Preparation of Inoculants packet

 The neutralized, sterilized carrier material is spread in a clean, dry, sterile metallic or
plastic tray.
 The bacterial culture drawn from the fermentor is added to the sterilized carrier and
mixed well by manual (by wearing sterile gloves) or by mechanical mixer. The culture
suspension is to be added to a level of 40 – 50% water holding capacity depending upon
the population.
 The inoculant packet of 200 g quantities in polythene bags, sealed with electric sealer
and allowed for curing for 2 -3 days at room temperature ( curing can be done by
spreading the inoculant on a clean floor/polythene sheet/ by keeping in open shallow
 tubs/ trays with polythene covering for 2 -3 days at room temperature before
packaging).

Schematic representation of mass production of bacterial biofertilizers

Specification of the polythene bags

 The polythene bags should be of low density grade.

 The thickness of the bag should be around 50 – 75 micron.
 Each packet should be marked with the name of the manufacturer, name of the product,
strain number, the crop to which recommended, method of inoculation, date of
manufacture, batch number, date of expiry, price, full address of the manufacturer and
storage instructions etc.,

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 / g of inoculant at the time of
preparation and107 cells/ g on dry weight basis before expiry date.

Mass production of Mycorrhizal biofertilizer

 The commercial utilization of mycorrhizal fungi has become difficult because of the
obligate symbiotic nature and difficulty in culturing on laboratory media. Production of AM
inoculum has evolved from the original use of infested field soils to the current practice of
using pot culture inoculum derived from the surface disinfected spores of single AM fungus
on a host plant grown in sterilized culture medium. Several researches in different parts of the
world resulted in different methods of production of AM fungal inoculum as soil based culture
as well as carrier based inoculum. Root organ culture and nutrient film technique provide scope
for the production of soil less culture.

As a carrier based inoculum, pot culture is widely adopted method for production. The
AM inoculum was prepared by using sterilized soil and wide array of host crops were used as
host. The sterilization process is a cumbersome one and scientists started using inert materials
for production of AM fungi. The researchers tried use of perlite, montmorillonite clay etc., In
TNAU vermiculite was tried as substrate for the replacement of soil sterilization, which
resulted in the best method of inoculum production.
Method of production

1. Tank for mass 2. Sprinkling of water in tank 3. Making of furrows to sow

multiplication of AM with vermiculite maize seeds

6. Vermiculite contained
4. Sowing the seeds in 5. View of the maize sown AM
raised AM infected maize
furrows pit
plants

 A trench (1m x 1m x 0.3m) is formed and lined with black polythene sheet to be used
as a plant growth tub.
 Mixed 50 kg of vermiculite and 5 kg of sterilized soil and packed in the trench up to a
height of 20 cm
 Spread 1 kg of AM inoculum (mother culture) 2-5 cm below the surface of vermiculite
 Maize seeds surface sterilized with 5% sodium hypochlorite for 2 minutes are sown
 Applied 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench at the
time of sowing seeds. Further 10 g of urea is applied twice on 30 and 45 days after
sowing for each trench
 Quality test on AM colonization in root samples is carried out on 30th and 45th day
  Stock plants are grown for 60 days (8 weeks). The inoculum is obtained by cutting all
the roots of stock plants. The inoculum produced consists of a mixture of vermiculite,
spores, pieces of hyphae and infected root pieces.
 Thus within 60 days 55 kg of AM inoculum could be produced from 1 sq
meter area. This inoculum will be sufficient to treat 550 m2 nursery area having 11,000
seedlings.

AM fungi

Nursery application: 100 g bulk inoculum is sufficient for one metre square. The inoculum
should be applied at 2-3 cm below the soil at the time of sowing. The seeds/cutting should be
sown/planted above the VAM inoculum to cause infection.

For polythene bag raised crops: 5 to 10 g bulk inoculum is sufficient for each packet. Mix
10 kg of inoculum with 1000 kg of sand potting mixture and pack the potting mixture in
polythene bag before sowing.

For out –planting: Twenty grams of VAM inoculum is required per seedling. Apply inoculum
at the time of planting.

For existing trees: Two hundred gram of VAM inoculum is required for inoculating one tree.
Apply inoculum near the root surface at the time of fertilizer application.

Mass production and field application of cyanobacteria

Blue green algal inoculation with composite cultures was found to be more effective than single
culture inoculation. A technology for mass scale production of composite culture of blue green
algae under rice field condition was developed at TNAU and the soil based BGA inoculum
could survive for more than 2 years.

At many sites where algal inoculation was used for three to four consecutive cropping seasons,
the inoculated algae establish well and the effect persisted over subsequent rice crop.
Technologies for utilizing nitrogen fixing organisms in low land rice were the beneficial role
of blue green algal inoculation in rice soils of Tamil Nadu.
The blue green algal inoculum may be produced by several methods viz., in tubs, galvanized
trays, small pits and also in field conditions. However the large-scale production is advisable
under field condition which is easily adopted by farmers.

I. Multiplication in trays

 Big metallic trays (6’x 3’x 6”lbh) can be used for small scale production
 Take 10 kg of paddy field soil, dry powder well and spread
 Fill water to a height of 3”
 Add 250 g of dried algal flakes (soil based) as inoculum
 Add 150 g of super phosphate and 30 g of lime and mix well with the soil
 Sprinkle 25 g carbofuran to control the insects
 Maintain water level in trays
 After 10 to 15 days, the blooms of BGA will start floating on the water sources
 At this stage stop watering and drain. Let the soil to dry completely
 Collect the dry soil based inoculum as flakes
 Store in a dry place. By this method 5 to 7 kg of soil based inoculum can be obtained.

II. Multiplication under field condition

Materials

 Rice field
 Super phosphate
 Carbofuran
 Composite BGA starter culture
Procedure

Select an area of 40 m2 (20m x 2m) near a water source which is directly exposed to sunlight.
Make a bund all around the plot to a height of 15 cm and give it a coating with mud to prevent
loss of water due to percolation.

 Plot is well prepared and levelled uniformly and water is allowed to a depth of 5-7.5
cm and left to settle for 12 hrs.
 Apply 2 kg of super phosphate and 200 g lime to each plot uniformly over the area.
 The soil based composite starter culture of BGA containing 8-10 species @ 5 kg / plot
is powdered well and broadcasted.
 Carbofuran @ 200 g is also applied to control soil insects occurring in BGA.
 Water is let in at periodic intervals so that the height of water level is always maintained
at 5 cm.
 After 15 days of inoculation, the plots are allowed to dry up in the sun and the algal
flakes are collected and stored.

Observations

The floating algal flasks are green or blue green in colour. From each harvest, 30 to 40 kg of
dry algal flakes are obtained from the plot.

Method of inoculation of BGA in rice field

Blue green algae may be applied as soil based inoculum to the rice field following the method
described below.

 Powder the soil based algal flakes very well.

 Mix it with 10 kg soil or sand (10kg powdered algal flakes with 10 kg soil / sand).
 BGA is to be inoculated on 7-10 days after rice transplanting.
 Water level at 3-4” is to be maintained at the time of BGA inoculation and then for a
month so as to have maximum BGA development.
Observation

A week after BGA inoculation, algal growth can be seen and algal mat will float on the water
after 2-3 weeks. The algal mat colour will be green or brown or yellowish green.

Mass production and field application of Azolla

Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen
in association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of
sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots. Rice
growing areas in South East Asia and other third World countries have recently been evincing
increased interest in the use of the symbiotic N2 fixing water fern Azolla either as an alternate
nitrogen sources or as a supplement to commercial nitrogen fertilizers. Azolla is used as
biofertilizer for wetland rice and it is known to contribute 40-60 kg N ha-1 per rice crop. The
agronomic potential of Azolla is quite significant particularly for rice crop and it is widely used
as biofertilizer for increasing rice yields. Rice crop response studies with Azolla biofertilizer in
the People’s Republic in China and in Vietnam have provided good evidence
that Azolla incorporation into the soil as a green manure crop is one of the most effective ways
of providing nitrogen source for rice.

The utilization of Azolla as dual crop with wetland rice is gaining importance in
Philippines, Thailand, Srilanka and India. The important factor in using Azolla as a biofertilizer
for rice crop is its quick decomposition in soil and efficient availability of its nitrogen to rice.
In tropical rice soils the applied Azolla mineralizes rapidly and its nitrogen is available to the
rice crop in very short period. The common species of Azolla are A. microphylla, A.
filiculoides, A. pinnata, A. caroliniana, A. nilotica, A. rubra and A. mexicana.

I. Mass multiplication of Azolla under field conditions

A simple Azolla nursery method for large scale multiplication of Azolla in the field has been
evolved for easy adoption by the farmers.

Materials

 One cent (40 sq.m) area plot

 Cattle dung
  Super phosphate
 Furadan
 Fresh Azolla inoculum

Procedure

 Select a wetland field and prepare thoroughly and level uniformly.

 Mark the field into one cent plots (20 x 2m) by providing suitable bunds and irrigation
channels.
 Maintain water level to a height of 10 cm.
 Mix 10 kg of cattle dung in 20 litres of water and sprinkle in the field.
 Apply 100 g super phosphate as basal dose.
 Inoculate fresh Azolla biomass @ 8 kg to each pot.
 Apply super phosphate @ 100 g as top dressing fertilizer on 4th and 8th day
after Azolla inoculation.
 Apply carbofuran (furadan) granules @ 100 g/plot on 7th day after Azolla inoculation.
 Maintain the water level at 10 cm height throughout the growth period of two or three
weeks.
 Observations
 Note the Azolla mat floating on the plot. Harvest the Azolla, drain the water and record
the biomass.

II. Method of inoculation of Azolla to rice crop

The Azolla biofertilizer may be applied in two ways for the wetland paddy. In the first method,
fresh Azolla biomass is inoculated in the paddy field before transplanting and incorporated as
green manure. This method requires huge quantity of fresh Azolla. In the other
method, Azolla may be inoculated after transplanting rice and grown as dual culture with rice
and incorporated subsequently.

A. Azolla biomass incorporation as green manure for rice crop

 Collect the fresh Azolla biomass from the Azolla nursery plot.
 Prepare the wetland well and maintain water just enough for easy incorporation.
  Apply fresh Azolla biomass (15 t ha-1) to the main field and incorporate the Azolla by
using implements or tractor.

B. Azolla inoculation as dual crop for rice

 Select a transplanted rice field.

 Collect fresh Azolla inoculum from Azolla nursery.
 Broadcast the fresh Azolla in the transplanted rice field on 7th day after planting (500
kg / ha).
 Maintain water level at 5-7.5cm.
 Note the growth of Azolla mat four weeks after transplanting and incorporate
the Azolla biomass by using implements or tranctor or during inter-cultivation
practices.
 A second bloom of Azolla will develop 8 weeks after transplanting which may be
incorporated again.
 By the two incorporations, 20-25 tonnes of Azolla can be incorporated in one hectare
rice field.

4. Application of Biofertilizers

1. Seed treatment or seed inoculation

2. Seedling root dip

3. Main field application

Seed treatment

One packet of the inoculant is mixed with 200 ml of rice kanji to make a slurry. The seeds
required for an acre are mixed in the slurry so as to have a uniform coating of the inoculant
over the seeds and then shade dried for 30 minutes. The shade dried seeds should be sown
within 24 hours. One packet of the inoculant (200 g) is sufficient to treat 10 kg of seeds.

Seedling root dip

This method is used for transplanted crops. Two packets of the inoculant is mixed in 40 litres
of water. The root portion of the seedlings required for an acre is dipped in the mixture for 5 to
10 minutes and then transplanted.

Main field application

Four packets of the inoculant is mixed with 20 kgs of dried and powdered farm yard manure
and then broadcasted in one acre of main field just before transplanting.

Rhizobium

For all legumes Rhizobium is applied as seed inoculant.

Azospirillum/Azotobacter

In the transplanted crops, Azospirillum is inoculated through seed, seedling root dip and soil
application methods. For direct sown crops, Azospirillum is applied through seed treatment and
soil application.

Phosphobacteria

Inoculated through seed, seedling root dip and soil application methods as in the case
of Azospirillum.
Combined application of bacterial biofertilizers.

Phosphobacteria can be mixed with Azospirillum and Rhizobium. The inoculants should be
mixed in equal quantities and applied as mentioned above.

Points to remember

 Bacterial inoculants should not be mixed with insecticide, fungicide, herbicide and
fertilizers.
 Seed treatment with bacterial inoculant is to be done at last when seeds are treated with
fungicides.
Biofertilizers recommendation (one packet - 200 g)

Total requirement
Crop Seed Nursery Seedling dip Main field
of packets per ha
Rice 5 10 5 10 30
Sorghum 3 - - 10 13
Pearl millet 3 - - 10 13
Ragi 3 - 5 10 18
Maize 3 - - 10 13
Cotton 3 - - 10 13
Sunflower 3 - - 10 13
Castor 3 - - 10 13
36
Sugarcane 10 - - 46
(3 split)
24
Turmeric - - - 24
(2 split)
Tobacco 1 3 - 10 g/pit 14
Papaya 2 - - 10 -
Mandarin
2 - - 10 g/pit -
Orange
Tomato 1 - - 10 14
Banana - - 5 10 g/pit -

Rhizobium (only seed application is recommended)

Total requirement of packets

Crop
per ha
Soybean 5
Groundnut 5
Bengalgram 5
Blackgram 3
Greengram 3
 Redgram 3
Cowpea 3

Phosphobacteria
The recommended dosage of Azospirillum is adopted for phosphobacteria inoculation; for
combined inoculation, both biofertilizers as per recommendations are to be mixed uniformly
before using.

5. Azolla – The best feed for cattle and poultry

Azolla is a free floating water fern that floats in water and fixes nitrogen in association
with the nitrogen fixing blue green algae, Anabaena azollae. Azolla is considered to be a
potential biofertilizer in terms of nitrogen contribution to rice. Long before its cultivation as a
green manure, Azolla has been used as a fodder for domesticated animals such as pigs and
ducks. In recent days, Azolla is very much used as a sustainable feed substitute for livestock
especially dairy cattle, poultry, piggery and fish.

Azolla contains 25 – 35 per cent protein on dry weight basis and rich in essential amino
acids, minerals, vitamins and carotenoids including the antioxidant b carotene. Cholorophyll a,
chlorophyll b and carotenoids are also present in Azolla, while the cyanobiont Anabaena
azollae contains cholorophyll a, phycobiliproteins and carotenoids. The rare combination of
high nutritive value and rapid biomass production make Azolla a potential and effective feed
substitute for live stocks.

Inputs required

Azolla fronds, Polythene sheet, Super phosphate and Cow dung.

Methodology
 The area selected for Azolla nursery should be partially shaded. The convenient size
for Azolla is 10 feet length, 2 feet breadth and 1 feet depth. The nursery plot is spread with a
polythene sheet at the bottom to prevent water loss. Soil is applied to a depth of 2 cm and a
gram of super phosphate is applied along with 2 kg of vermicompost or cow dung in the nursery
for quick growth. Azolla mother inoculum is introduced @ 5 kg/plot.

The contents in the plot are stirred daily so that the nutrients in the soil dissolve in water
for easy uptake by Azolla. Azolla is harvested fifteen days after inoculation at the rate of 50-
80 kg / plot. One third of Azolla should be left in the plot for further multiplication. Five kg
cow dung slurry should be sprinkled in the Azolla nursery at ten days intervals. Neem oil can
be sprayed over the Azolla at 0.5 5 level to avoid pest incidence.

Animal Dosage / day

Adult cow , Buffalo, Bullock 1.5-2 kg
Layer, Broiler birds 20 – 30 grams
Goat 300 – 500 grams
Pig 1.5 – 2.0 kg
Rabbit 100 gram

Value of the technology

The egg yield is increased in layer birds due to Azolla feeding. The Azolla fed birds
register an overall egg productivity of 89.0 per cent as against 83.7 per cent recorded by the
birds fed with only concentrated feed. The average daily intake of concentrated feed is
considerably low (106.0 g) for birds due to Azolla substitution as against 122.0 g in the control
birds. More impotantly Azolla feeding shows considerable amount of savings in the
consumption of concentrated feed (13.0 %) leading to reduced operational cost. By considering
the average cost of the concentrated feed as Rs. 17/ Kg, a 13.0 % saving in the consumption
ultimately leads to a feed cost savings of 10.0 paise /day/ bird and hence a layer unit
maintaining 10,000 birds could cut down its expense towards feed to a tune of rs.1000/day.

Benefits
The Azolla feeding to layer birds increase egg weight, albumin, globulin and carotene contents.
The total protein content of the eggs laid by the Azolla fed birds is high and the total carotene
content of Azolla eggs (440 g 100 g-1 of edible portion)is also higher than the control. The
rapid biomass production due to the high relative growth rate, increased protein and carotene
contents and good digestability of the Azolla hybrid Rong ping favour its use as an effective
feed supplement to poultry birds.

Effect of Azolla hybrid Rong Ping on the nutritional value of egg

Parameters Azolla egg Control percentage increase over control

Egg weight (g) 61.20 57.40 6.62
Albumin (g /100 g of edible
3.9 3.4 14.70
portion)
Globulin (g /100 g of edible
10.1 9.5 6.31
portion)
Total protein (g/ 100 g of
14.0 12.9 8.52
edible portion)
Carotenes (µg / 100 g of
440 405 8.64
edible portion)

Application

In Indian conditions, agriculture is very much coupled with poultry farming. Azolla is an
important low cost input, which plays a vital role in improving soil quantity in sustainable rice
farming. The twin potentials as biofertilizer and animal feed make the water fern Azolla as an
effective input to both the vital components of integrated farming, agricultural and animalo
husbandry.

Limitation

Azolla is a water fern and requires a growth temperature of 35-38º C. The multiplication of
Azolla is affected under elevated temperature. Hence adopting this technology in dry zones
where the temperature exceeds 40ºc is difficult.

Achievements

Azolla hybrid Rong ping had been selected to supply to the tribal population. Azolla mother
inoculum nursery was laid out in villages with the help of Krishi Vigyan Kendra, TNAU,
Coimbatore and Krishi Vigyan Kendra, Karamadai, women entrepreneurs were selected and
one day training was imparted to them on the cultivation of Azolla. Wet biomass (Starter
inoculm) was supplied at free of cost @ 10 kg/women entrepreneur during the training so as to
enable them to initiate commercial Azolla cultivation in their backyards.

Azolla multiplication plots had been laid out in Narasipuram. Azolla mass production
training was conducted to the SHG in Narasipuram village with the help of Kalaimagal Arts
and Science College, Narasipuram, Sappanimadai (tribal village) and Avinashilingam KVK,
Karamadai. With the help of Avinashilingam KVK, Karamadai Azolla trainings were
conducted to women volunteers and we have established Azolla village in Karamadai. The
Avin milk producers union Coimbatore and the poultry owners association, Namakkal have
been contacted and explained the importance of Azolla as feed supplement.

The Milk Producers Union also involved in the training and marketing of Azolla. They
are purchasing Azolla fronds from the village level Azolla growers both under wet and dry
conditions. Around 400 rural women and 370 tribal people have been trained on the cultivation
of Azolla through this project. The Azolla laboratory and the Azolla germplasm center at AC&
RI, TNAU, Coimbatore helped us in the maintenance of germplasm by providing the mother
inoculum. The Animal Husbandry Unit at AC&RI, TNAU, Coimbatore helped us in
standardizing the Azolla and concentrated feed mixing ratio.

Azolla mass multiplication in pits Feeding Azolla to Rabbit

Feeding Azolla to Poultry Feeding Azolla to Livestock

Inoculating Super phosphate and Cow dung in Azolla pit

Potential use of soil microbes in sustainable crop production.
The beneficial soil micro-organisms sustain crop production either as biofertilizers [19] or
symbiont [17]. They perform nutrient solubilisation which facilitate nutrient availability and
thereby uptake [20, 21]. It improves the plant growth by advancing the root architecture [26].
Their activity provides several useful traits to plants such as increased root hairs, nodules and
nitrate reductase activity and [36]. Efficient strains of Azotobacter, Azospirillum,
Phosphobacter and Rhizobacter can provide significant amount of available nitrogen through
nitrogen cycling [22]. The biofertilizers produced plant hormones, which include indole acetic
acid (IAA), gibberellins (GA) and cytokinins (CK) [25, 44]. Biofertilizers improve
photosynthesis performance to confer plant tolerance to stress [44] and increase resistance to
pathogens [45] thereby resulting in crop improvement [18].