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.
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 |
| 1. |
Free-living |
Azotobacter, Beijerinkia, Clostridium, Klebsiella, Anabaena, Nostoc, |
| 2. |
Symbiotic |
Rhizobium, Frankia, Anabaena azollae |
| 3. |
Associative Symbiotic |
Azospirillum |
| P Solubilizing Biofertilizers |
| 1. |
Bacteria |
Bacillus megaterium var. phosphaticum, Bacillus subtilis
Bacillus circulans, Pseudomonas striata |
| 2. |
Fungi |
Penicillium sp, Aspergillus awamori |
| P Mobilizing Biofertilizers |
| 1. |
Arbuscular mycorrhiza |
Glomus sp.,Gigaspora sp.,Acaulospora sp.,
Scutellospora sp. & Sclerocystis sp. |
| 2. |
Ectomycorrhiza |
Laccaria sp., Pisolithus sp., Boletus sp., Amanita sp. |
| 3. |
Ericoid mycorrhizae |
Pezizella ericae |
| 4. |
Orchid mycorrhiza |
Rhizoctonia solani |
| Biofertilizers for Micro nutrients |
| 1. |
Silicate and Zinc solubilizers |
Bacillus sp. |
| Plant Growth Promoting Rhizobacteria |
| 1. |
Pseudomonas |
Pseudomonas fluorescens |
2. Different types of biofertilizers
| Rhizobium |
|
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Rhizobium
is a soil habitat bacterium, which can able to colonize the legume
roots and fixes the atmospheric nitrogen symbiotically. The morphology
and physiology of Rhizobium will vary from free-living condition to
the bacteroid of nodules. They are the most efficient biofertilizer as
per the quantity of nitrogen fixed concerned. They have seven genera and
highly specific to form nodule in legumes, referred as cross
inoculation group.
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.
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| Azotobacter |
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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.
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| Azospirillum |
|
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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.
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| Cyanobacteria |
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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.
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.
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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.
Phosphate solubilizing microorganisms(PSM)
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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.
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AM fungi
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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.
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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.
Plant Growth Promoting Rhizobacteria (PGPR)
The group of bacteria that
colonize roots or rhizosphere soil and beneficial to crops are referred
to as plant growth promoting rhizobacteria (PGPR).
The PGPR inoculants currently
commercialized that seem to promote growth through at least one
mechanism; suppression of plant disease (termed Bioprotectants),
improved nutrient acquisition (termed Biofertilizers), or phytohormone
production (termed Biostimulants). Species of Pseudomonas and Bacillus
can produce as yet not well characterized phytohormones or growth
regulators that cause crops to have greater amounts of fine roots which
have the effect of increasing the absorptive surface of plant roots
for uptake of water and nutrients. These PGPR are referred to as
Biostimulants and the phytohormones they produce include indole-acetic
acid, cytokinins, gibberellins and inhibitors of ethylene production.
Recent advances in molecular
techniques also are encouraging in that tools are becoming available to
determine the mechanism by which crop performance is improved using
PGPR and track survival and activity of PGPR organisms in soil and
roots. The science of PGPR is at the stage where genetically modified
PGPR can be produced. PGPR with antibiotic, phytohormone and
siderophore production can be made.
Despite of promising results,
biofertilizers has not got widespread application in agriculture mainly
because of the variable response of plant species or genotypes to
inoculation depending on the bacterial strain used. Differential
rhizosphere effect of crops in harbouring a target strain or even the
modulation of the bacterial nitrogen fixing and phosphate solubilizing
capacity by specific root exudates may account for the observed
differences. On the other hand, good competitive ability and high
saprophytic competence are the major factors determining the success of
a bacterial strain as an inoculant.
Studies to know the synergistic
activities and persistence of specific microbial populations in complex
environments, such as the rhizosphere, should be addressed in order to
obtain efficient inoculants. In this regards, research efforts are
made at Agricultural College and Research Institute, Madurai to obtain
appropriate formulations of microbial inoculants incorporating nitrogen
fixing, phosphate- and silicate- solubilizing bacteria and plant
growth promoting rhizobacteria which will help in promoting the use of
such beneficial bacteria in sustainable agriculture.
Liquid Biofertilizers
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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.
Biofertilizers is one of the prime
input 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.
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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 through out 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 |
| Alfafa grp.Group |
R.mellilotiMedicago Trigonella |
Melilotus |
100- 150 |
| Beans group |
R. phaseoli |
Phaseoli |
80- 110 |
| Clover group |
R. trifoli |
Trifolium |
130 |
| Cowpea group |
R. species |
Moong, Redgram, Cowpea, Groundnut |
57- 105 |
| 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
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The colour of the liquid may be blue or dull white.
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Bad odours confirms improper liquid formulation and may be concluded as mere broth.
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Production of yellow gummy colour materials comfirms the quality product.
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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 bicolour (Sorghum) |
20 |
| 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 giberllins. All these
hormones/chemicals helps the plants in better germination, early
emergence, better root development.
Role of Liquid Azospirillum under field conditions
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Stimulates growth and imparts green colour which is a characteristic of a healthy plant.
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Aids utilization of potash, phosphorous and other nutrients.
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Encourage plumpness and succulence of fruits and increase protein percentage.
Sign of non functioning of Azospirillum in the field
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No growth promotion activity
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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
rhizosplane but are abundant in the rhizosphere region. The lack of
organic matter in the soil is a limiting factor for the proliferation of
Azotobaceter 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-
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A. chroococcum: Produces brown-black pigmentation in liquid inoculum.
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A. beijerinchii: Produces yellow- light brown pigementation in liquid inoculum
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A. vinelandii: Produces green fluorescent pigmentation in liquid inoculum.
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A. paspali: Produces green fluorescent pigmentation in liquid inoculum.
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A. macrocytogenes: Produces, pink pigmentation in liquid inoculum.
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A. insignis: Produces less, gum less, grayish-blue pigmentation in liquid inoculum.
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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:
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The performance of Azotobacter liquid inoculant was c
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omparatively better than all the treatments in 10 % MS medium followed Azospirillum.
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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.
Acetobaceter
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
bacteria Acetobacter diazotrophicus which is present in the
sugarcane stem, leaves, soils have a capacity to fix up to 300 kgs of
nitrogen. This bacteria 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 tones per ha. This leads to the assumption that
active nitrogen fixing bacteria has associated within the plant.
Do’s and Don’t for Entrepreneurs, Dealers and farmers
Do
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Don’t
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| Keep Bio-fertilizers bottles away from direct heat and sunlight. Store it in cool and dry place. |
Don’t store Bio-fertilizers bottles under heat and sunlight |
| Sell
only Bio-fertilizers bottles which contain batch number, the name of
the crop on which it has to be used, the date of manufacture and
expiry period. |
Don’t sell Bio-fertilizers bottles after their expiry period is over. |
| If the expiry period is over, then discard it as it is not effective. |
Don’t prick holes into the bottles or puncture them to pour the content |
| Keep Bio-fertilizers bottles away from fertilizer or pesticide containers and they should not be mixed directly. |
Do not mix the Bio-fertilizers with fungicides, insecticides, herbicides, herbicides and chemical fertilizers. |
Liquid Bio-fertlizer application methodology
There are three ways of using Liquid Bio-fertilizers
- Seed treatment
- Root dipping
- 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 done 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 seeds 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 over night 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:
| Crop |
Recommended Bio-fertilizer
|
Application method
|
Quantity to be used
|
Field crops
Pulses
Chickpea, pea, Groundnut, soybean, beans, Lentil,
lucern, Berseem, Green gram, Black gram, Cowpea and pigeon pea |
Rhizobium |
Seed treatment |
200ml/acre |
Cereals
Wheat, oat, barley |
Azotobacter/Azospirillum |
Seed treatment |
200ml/acre |
| Rice |
Azospirillum |
Seed treatment |
200ml/acre |
Oil seeds
Mustard, seasum, Linseeds, Sunflower, castor |
Azotobacter |
Seed treatment |
200ml/acre |
Millets
Pearl millets, Finger millets, kodo millet |
Azotobacter |
Seed treatment |
200ml/acre |
| Maize and Sorghum |
Azospirillum |
Seed treatment |
200ml/acre |
Forage crops and Grasses
Bermuda grass, Sudan grass, Napier Grass , ParaGrass, StarGrass etc. |
Azotobacter |
Seed treatment |
200ml/acre |
Other Misc. Plantation Crops
Tobacco |
Azotobacter |
Seedling treatment |
500ml/acre |
| 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 wood fodder,
fruits,gum,spice,leaves,flowers,nuts and seeds puppose |
Azotobacter |
Soil treatment |
2-3 ml/plant at nursery |
| Leguminous plants/ trees |
Rhizobium |
Soil treatment |
1-2 ml/plant |
Note:
Doses recommended when count of
inoculum is 1 x 108 cells/ml then doses will be ten times more besides
above said Nitrogen fixers, Phosphate solubilizers and potash
mobilizers at the rate of 200 ml/ acre could be applied for all crops.
Equipments required for Biofertilizer production
In biofertilizer production industry,
equipments are the major infrastructure, which involves 70 percent of
capital investment. Any compromise on the usage of the following
mentioned equipments may finally decline in the quality of
biofertilizer.After studying the principle behind the usage of all
instruments, some of the instruments can be replaced with a culture
room fitted with a U.V.Lamp. Autoclaves, Hot Air Oven, Incubators and
sealing machines are indigenously made with proper technical
specifications. The correct use of equipments will give uninterrupted
introduction with quality inoculum.
Essential equipments
Autoclave
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It
is an apparatus in which materials are sterilized by air free
saturated steam (under pressure) at a temperature above 100OC. If the
steam pressure inside the autoclave is increased to 15 psi, the
temperature will rise to 121°C. this is sufficient to destroy all
vegetative cells. Normally all growth medium are sterilized in the
autoclave.
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Laminar air flow chamber
Laminar air flow chamber provides a
uniform flow of filtered air. This continuous flow of air will prevent
settling of particles in the work area.Air borne contamination is
avoided in this chamber. Culture transfers and inoculation can be done
here.
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BOD incubators
Incubators providing controlled
conditions (light, temperature, humidity, etc.) required for the growth
and development of microorganisms. Multiplication of starter culture
can be done in this instrument.
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Rotary shaker
It is used for agitating culture flasks
by circular motion under variable speed control. Shaking provides
aeration for growth of cultures. Shakers holding upto 20-50 flasks are
generally used. The capacity of the shaker may be increased if it is a
double- decker type.
Hot air oven
Hot air oven is meant for
sterilizing all glassware materials. Dry heat is used in this apparatus
to sterilize the materials. Normally 180OC is used for two hours for
sterilizing glasswares.
pH meter
An instrument for measuring pH of
the solution using a 0-14 scale in which seven represents neutral
points, less than seven is acidity (excess of H‘ over OH-) and more
than seven is alkality (excess of OH- over H‘ ) useful in adjusting
the pH of the growth medium.
Refrigerator
This equipment is used preserving
all mother cultures used for biofertilizer production. The mother
culture is periodically sub-cultured and stored in the refrigerator for
long- term usage.
Fermentor
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A fermentor is the equipment, which provides the proper environment for the growth of a desired organism.
It is generally a large vessel in
which, the organism may be kept at the required temperature, pH ,
dissolved oxygen concentration and substrate concentration. Different
models of fermentors are available depending upon the necessity.
A simple version model contains steam generator, sterilization process devices and agitator.
A sophisticated fermentor contains pH regulator, oxygen level regulator, anti-foam device, temperature controller, etc.
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3. Mass production of Bacterial Biofertilizer
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| 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 more 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 orthophosphate |
- |
0.5g |
| Magnesium sulphate |
- |
0.2g |
| Sodium chloride |
- |
0.1g |
| Calcium chloride |
- |
0.2g |
| Fe-EDTA (1.64% w/v aqueous) |
- |
4.0 ml |
| Trace element solution |
- |
2.0 ml |
| BTB (0.5% alcoholic solution) |
- |
2.0 ml |
| 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 micron 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 biofertilizerpacket
- 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 multiplication of AM |
2. Sprinkling of water in tank with vermiculite |
3. Making of furrows to sow maize seeds |
 |
 |
 |
| 4. Sowing the seeds in furrows |
5. View of the maize sown AM pit |
6. Vermiculite contained raised AM infected maize 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
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)
| Crop |
Seed |
Nursery |
Seedling dip |
Main field |
Total requirement 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 |
| Sugarcane |
10 |
- |
- |
36
(3 split) |
46 |
| Turmeric |
- |
- |
- |
24
(2 split) |
24 |
| Tobacco |
1 |
3 |
- |
10 g/pit |
14 |
| Papaya |
2 |
- |
- |
10 |
- |
Mandarin
Orange |
2 |
- |
- |
10 g/pit |
- |
| Tomato |
1 |
- |
- |
10 |
14 |
| Banana |
- |
- |
5 |
10 g/pit |
- |
Rhizobium (only seed application is recommended)
| Crop |
Total requirement of packets 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 portion) |
3.9 |
3.4 |
14.70 |
| Globulin (g /100 g of edible portion) |
10.1 |
9.5 |
6.31 |
| Total protein (g/ 100 g of edible portion) |
14.0 |
12.9 |
8.52 |
| Carotenes (µg / 100 g of edible portion) |
440 |
405 |
8.64 |
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) were 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 |
6. List of Biofertilizer production units in Tamil Nadu
Department of Agricultural Microbiology, Agriculture College and Research Institute,
Tamil Nadu Agricultural University
Dr. S. Anthoniraj
MADURAI-625 104
( 0452-422956 fax: 422785
e-mail: s_anthoniraj@yahoo.com |
Biofertilizer Production Unit, Department of Agriculture, Govt. of Tamil Nadu
Gundusalai Road, Sommandalam,
CUDDALORE-607 001 (TN) |
Biofertilizer Production Unit, Department of Agriculture, Govt. of Tamil Nadu
Agricultural Chemist
Sakkottai,
THANTAVUR-612 401 (TN) |
Biofertilizer Production Unit, Department of Agriculture, Govt. of Tamil Nadu
Jamal Mohd. College Post, Khajamalai,
TRICHY-620 020 (TN) |
KRIBHCO
Sidco Garment Complex, Thiruvika Industrial Estate, Guidy,
CHENNAI-32 |
Regional Research Station
Tamil Nadu Agricultural University,
PIYUR-635 112
Via-Kaveripattinam
Dharmapuri District
( 04343-50043 |
Monarch Bio-Fertilisers and Research Centre
12, SIDCO Industrial Estate, Thirumazhisai,
CHENNAI-602 107 (TN)
( 6272780 |
Lakshmi Bio-Tech
Mr. V. Sithanandham
Nellikuppam Road, Thottapattu,
CUDDALORE-607 109 (TN)
( 04142-210136 |
Marygreen Afrotech (P) Ltd.
Dr. Y. Joe
5/302, Srisaibaba St., Santosh Nagar, Kandanchavadi, Perungudi Post,
CHENNAI-600 096 (TN)
4964202, 4745957
e-mail: marygreen45@hotmail.com |
Tamil Nadu Agricultural University
Prof. & Head
Deptt. of Agricultural Microbiology,
COIMBATORE-3 (TN)
( 431222 ext. 294 Fax: 0422-431672
e-mail: vctnau@vsnl.com |
T Stanes & Company Limited
Dr. S. Ramarethinam
8/23-24, Race Course Road,
COIMBATORE-641 018 (TN)
( 0422-211514, 213515 Fax: 217432
e-mail: tstanes@vsnl.com |
Esvin Advanced Technologies Limited
Mr. T. S. Venkataraman
“Esvin House” Perungudi,
CHENNAI-600 096 (TN)
( 4961056,4960690 Fax: 4961002
e-mail: tsv@vsnl.com |
Southern Petrochemical Industries Corporation Limited,
Mr. K. Raju
SPIC Ltd. Biotechnology Division, Chettiar Agaram Road, Gandhi Nagar, Porur,
CHENNAI-600 116 (TN)
( 044-4768064 Tele-Fax: 044-4767347
e-mail: biotech.por@spic.co.in |
Biofertiliser Unit-Manali, Madras Fertilizers Limited
Mr. P. Mallikarjuna Reddy
Chief Manager –Bioproducts
Commercial Group, Madras Fertilizers Ltd., Manali,
CHENNAI-600 068 (TN)
( 044-5941001 ext. 2750 Fax: 5941010
e-mail: edcomm@mfl.tn.nic.in |
Biofertilizer Production Unit
Mr. S. Murugan
Agricultural Chemist, Biofertilizer Production Unit, Seelanaickenpatty,
SALEM-636 201 (TN) |
Biofertilizer Production Unit,
Mr.Thiru P. Raman
Agricultural Chemist, Biofertilizer Production Unit,
KUDUMIAMALAI-622 104
Distt. Pudukkottai |
Main Biocontrol Research Laboratory
(Unit of Tamilnadu Cooperative Sugar Federation)
2E/1, Rajeshwari Vedhachalam Street,
CHENGALPATTU-603 001 (TN)
( 04114-431393 |
The SIMA Cotton Development and Research Association
Dr. M.A. Shanmugham
“Shanmukha Manram”, Post Box No. 3871, Race Course,
COIMBATORE-641 018 (TN)
( 0422-211391 Tele-Fax: 0422-216798 |
7. Constraints in Biofertilizer Technology
Though the biofertilizer technology is a low
cost, ecofriendly technology, several constraints limit the application
or implementation of the technology the constraints may be
environmental, technological, infrastructural, financial, human
resources, unawareness, quality, marketing, etc. The different
constraints in one way or other affecting the technique at production,
or marketing or usage.
Technological constraints
- Use of improper, less efficient strains for production.
- Lack of qualified technical personnel in production units.
- Unavailability of good quality carrier material or use of
different carrier materials by different producers without knowing the
quality of the materials.
- Production of poor quality inoculants without understanding the basic microbiological techniques
- Short shelf life of inoculants.
Infrastructural constraints
- Non-availability of suitable facilities for production
- Lack of essential equipments, power supply, etc.
- Space availability for laboratory, production, storage, etc.
- Lack of facility for cold storage of inoculant packets
Financial constraints
- Non-availability of sufficient funds and problems in getting bank loans
- Less return by sale of products in smaller production units.
Environmental constraints
- Seasonal demand for biofertilizers
- Simultaneous cropping operations and short span of sowing/planting in a particular locality
- Soil characteristics like salinity, acidity, drought, water logging, etc.
Human resources and quality constraints
- Lack of technically qualified staff in the production units.
- Lack of suitable training on the production techniques.
- Ignorance on the quality of the product by the manufacturer
- Non-availability of quality specifications and quick quality control methods
- No regulation or act on the quality of the products
- Awareness on the technology
- Unawareness on the benefits of the technology
- Problem in the adoption of the technology by the farmers due to different methods of inoculation.
- No visual difference in the crop growth immediately as that of inorganic fertilizers.
Awareness on the technology
- Unawareness on the benefits of the technology.
- Problem in the adoption of the technology by the farmers due to different methods of inoculation.
- No visual difference in the crop growth immediately as that of inorganic fertilizers.
- Unawareness on the damages caused on the ecosystem by continuous application of inorganic fertilizer.
Marketing constraints
- Non availability of right inoculant at the right place in right time.
- Lack of retain outlets or the market network for the producers.
8. Biofertilizer strains developed from TNAU
| Azospirillum |
Strains |
| Normal soils |
Az. 204 |
| Acid soils |
Az Y2 |
| Dry lands |
Azt. 11 |
| Rhizobium |
|
| Groundnut |
TNAU 14 |
| Soybean |
Cos 1 |
| Cowpea |
Coc 10 |
| Redgram |
Cc 1 |
Greengram &
Blackgram |
COG 15 |
| GMBS 1 |
| Bengalgram |
CoBe 13 |
| Phosphobacteria |
|
| All crops |
PB 1 |
The increasing demand for the biofertilizers and
the awareness among farmers and planters in the use of biofertilizers
have paved way for the fertilizer manufactures and new entrepreneurs to
get into biofertilizer production. A number of biofertilizer
production units have been started recently particularly in the southern
states of our country.
Nationalized banks have started their Hi-Tech
agricultural programme providing loan and motivated the entrepreneurs
to start their own production units. The Government of India is also
encouraging this low cost technology by providing a subsidy upto Rs.20
lakhs to start a production unit with the capacity of 150 metric tonnes
per annum. However, we are all aware that the success of the project
entirely depends on the economic viability. With the objective of
giving an overall economics of the biofertilizer production and sales,
an approximate estimate is prepared.
Total estimate for starting a biofertilizer production unit with the capacity of 150 metric tonnes/annum.
| S. No. |
Particulars |
Amount
(Rs.in lakhs) |
| I. |
Expenditure* |
|
| A. |
Capital Investment (Fixed cost) |
|
| i. |
Building including cost of site (App. 1200 sq. ft.) |
12.00 |
| ii. |
Equipment and apparatus |
41.00 |
| B. |
Operational cost (variable cost) |
|
| i. |
Working capital (Raw materials) |
10.00 |
| ii. |
Staff salary |
2.04 |
| iii. |
Labour |
2.50 |
| iv. |
Electricity |
0.50 |
| v. |
Travelling expenses |
0.50 |
| vi. |
Administrative expenses |
0.50 |
| vii. |
Interest on loan and depreciation |
0.70 |
| viii. |
Miscellaneous expenses |
0.26 |
|
Total (variable cost) |
17.00 |
|
Total investment |
70.00 |
|
Actual initial investment |
50.00 |
* The expenditures does not include the marketing expenses
Expenditure details (Rupees in lakhs)
| S.No. |
Equipment and apparatus |
Qty (Nos.) |
Amount (Rs.in lakhs) |
| 1. |
Fermentor (200 lit. capacity) |
4 |
26.00 |
| 2. |
Shaker |
2 |
1.50 |
| 3. |
Laminar air flow chamber |
1 |
0.60 |
| 4. |
Autoclave |
2 |
0.30 |
| 5. |
Hot air oven |
1 |
0.10 |
| 6. |
Incubator |
1 |
0.10 |
| 7. |
Refrigerator |
1 |
0.30 |
| 8. |
Microscope |
1 |
0.75 |
| 9. |
pH meter |
1 |
0.15 |
| 10. |
Physical balance |
1 |
0.10 |
| 11. |
Electronic balance |
1 |
0.75 |
| 12. |
Counter-poise balance |
5 |
0.25 |
| 13. |
Sealing machine |
5 |
0.25 |
| 14. |
Work benches |
4 |
0.30 |
| 15. |
Plastic trays |
50 |
0.25 |
| 16. |
Trays (Zinc/Aluminium) |
10 |
0.20 |
| 17. |
Trolley |
1 |
0.10 |
| 18. |
Automatic packing machine (optional) |
1 |
9.00 |
|
Total |
|
41.00 |
Working capital
| cost of mother culture |
:
|
0.05 |
| Glasswares |
:
|
0.70 |
| Chemicals |
:
|
2.50 |
| Polythene bags |
:
|
3.50 |
| Carrier materials |
:
|
3.00 |
| Miscellaneous items |
:
|
0.25 |
| Total |
:
|
10.00
|
Staff salary
| Technical staff (1 No.) |
: |
9000 x 12 |
1,08,000 |
| Laboratory staff (2 Nos.) |
: |
4000 x 2 x 12 |
96,000 |
Total
|
|
|
2,04,000
|
II. Production
| 60% capacity |
: |
90 MT per year |
| 75% capacity |
: |
112.5 MT per year
|
100% capacity
|
: |
150 MT per year |
III. Receipts
Cost of 1 kg of biofertilizer
(present Govt./University rate) |
:
|
Rs.25/- |
| Cost of 90 MT (60% capacity) |
:
|
22.500 lakh rupees
|
| 112.5 MT (75% capacity) |
:
|
28.125 lakh rupees |
| 135 MT (90% capacity) |
:
|
33.750 lakh rupees |
| 150 MT (100% capacity) |
|
37.500 lakh rupees
|
IV. Profitability
| Year |
Production |
Receipt
(Lakh Rs.) |
Expenditure (Lakh Rs.) |
Gain
(Lakh Rs.) |
| I |
60% |
22.500 |
50.000 |
-27.500 |
| II |
75% |
28.125 |
18.700* |
9.425 |
| III |
90% |
33.750 |
20.570* |
13.180 |
| IV |
100% |
37.500 |
22.630* |
14.870 |
|
Profit anticipated after 4 years |
9.975 |
*Every year 10% increase in the expenditure is calculated to balance the price escalation
Economics of AM biofertilizer – Mass production
| Capital cost ( for construction of pits size of 4x 3x1.5 ft including construction material sand labour cost) |
Rs.3,000/- |
| Inoculum cost ( from TNAU) 20 KG @ Rs.20/- per kg |
Rs.400/- |
| Vermiculite cost (including transport charges) 500kg@ Rs.6.50 |
Rs.3,250/- |
| Labour cost-Since it is a single pit, family members can look after |
NA |
| Seed materials and mesh for covering for pits |
Rs.100+100 |
| Quality
control charges at TNAU (This will be done after 1 year and before
selling the product & need not be carried out after each harvest) |
Rs.1,000/- |
Bag- cost of packing the materials-30 @ Rs.10 each
Labour cost of harvesting and packing |
Rs.300/-
Rs.200/- |
| Total |
Rs.8,350/- |
| Benefit expected by the sale of produced inoculum 500kg @ Rs.20/- per kg (In TNAU) Rs.35/- per kg ( In Private) |
Rs.10,000/-
Rs.17,500/- |
Net
Income ( First
harvest)
Rs.10,000-8,350( Sl.No.8 – Sl.No 1 to 7)
Rs.17,500-8,350 |
Rs.1,197/-
Rs.9,150/- |
| For the II harvest the cost will be |
Rs.4,950/- |
From the second harvest benefit will be of
Rs.10,000/ - Rs.4,950/
Rs.17,500/ - Rs.4,950/ |
Rs. 5,050/-
Rs.12,550/- |
The Net Income for one year will be
Rs.50,000/ -Rs.24,750/
Rs.87,500/ -Rs.24,750/ |
Rs.25,250/-
Rs.62,750/- |
| Name of Biofertilizers |
Cost of Biofertilizers |
Availabilty |
| Azospirillum |
Rs.40/Kg |
Professor and Head
Department of Agricultural Microbiology
Tamil Nadu Agricultural University
Coimbatore - 641 003
Phone: 91-422-6611294
Fax: 91-422-2431672
Email: microbiology@tnau.ac.in |
| Phosphobacteria |
Rs.40/Kg |
| Rhizobium |
Rs.40/Kg |
| Azotobacter |
Rs.40/Kg |
| VAM |
Rs.30/Kg |
Source
Entrepreurial Training Manual
The Professor and Head
Department of Microbiology
Tamil Nadu Agricultural University,
Coimbatore-3
|
