1) The document discusses various types of biofertilizers including nitrogen, phosphorus, and compost biofertilizers. It describes the microorganisms used in biofertilizers and their benefits. 2) Key biofertilizers discussed are Rhizobium, Azotobacter, and Azospirillum. The summary describes the isolation, identification, mass production, and application of these three biofertilizers. 3) Rhizobium, Azotobacter, and Azospirillum are soil bacteria that fix atmospheric nitrogen, thereby reducing the need for nitrogen fertilizers and promoting plant growth.

2. INTRODUCTION:
Plants place a very important role in the living world. Plants are the
source of food for all most all living creations. For their growth plants
require nutrients. Plants require there nutrients in micro and macro forms.
Fertilizer is any material of natural or synthetic origin that is applied to
soil or to tissues of the plants for the supply of one or more nutrients
which helps the growth of the plant.
Management of soil fertility is followed by the human being from the
thousands of years. In the olden days the biologically fertilizers were
prepared , there after the chemical fertilizers were extremely used in the
last 60-70 years. there are many disadvantages by the use of chemical
fertilizer.
A biofertilizers is a substance which contains the living microorganisms
which when applied to soil, plants surfaces, or seeds colonizes the
rhizophore of the plant and increases the plant growth by providing the
primary nutrients to the plant

3. BIOFERTILIZERS AND TYPES OF BIOFERTILIZERS:
Biofertilizers are defined as biologically active products or
microbial inoculants of bacteria, algae and fungi (separately or in
combination), which help biological nitrogen fixation for the
benefit of plants.
1) Nitrogen Biofertilizers: Nitrogen biofertilizers help to correct the
nitrogen levels of the soil. Nitrogen is a limiting factor for plant growth
because plants need a certain amount of nitrogen in the soil to grow.
2) Phosphorus Biofertilizers: Phosphorus biofertilizers help the soil
to reach its optimum level of phosphorus and correct the phosphorus levels of
the soil.
3) Compost Biofertilizers: We can also use biofertilizers to enrich the
compost and the bacterial processes breaking down the compost waste.
Suitable biofertilizers for compost use are Cellulolytic fungal culture and
Phosphotika and Azotobacter cultures.

4. GENERAL METHOD OF PREPARATION:
The method of preparation of biofertilizers involves some of the general
steps they can be listed as follows
•Isolation of the micro organism from the leguminous plants or from the
other sources.
•Identification of the micro organism by different methods.
•Cultivation of identified micro organism by different methods.
•Mass production of the micro organism.
•Storage of the microorganism with the suitable carrier material.

5. RHIZOBIUM AS A BIOFERTILIZER:
Rhizobium is a gram negative, aerobic rod-shaped bacterium. It
contains a refractive granule. It is a soil bacterium present in large
numbers in rhizosphere of legume roots.
Different species of Rhizobium can fix 50-200 kg nitrogen/ha/year in
leguminous crops. Therefore, they have been recommended as nitrogen
biofertilizers in agriculture.
Production of Rhizobium Inoculants:
Production of Rhizobium inoculants involves following
steps 1. Isolation of Rhizobium
2. Identification of Rhizobium
3. Establishment of starter culture
4. Mass culture
5. Mixing with carrier
6. Packaging and storage

6. 1.Isolation of Rhizobium
Rhizobium occurs in the soil as well as in the root nodules of
selective legumes which is Pink-colored.
The root nodules are kept immersed in 0.1% potassium chloride
solution or in 0.1% acidified mercuric chloride The sterilized root nodules are
then washed 5 or 6 times with distilled water. They are once again sterilized
by immersing them in 90% ethyl alcohol for 10 seconds and washed
repeatedly with distilled water. They were crushed to make suspension
2. Identification of Rhizobium Test identification of rhizobium can be
done by the following tests
i) CRYEMA Test iii) Glucose Peptone Agar Test(GPA Test)
ii) Microscopic observation iv) Nodulation test
3.Establishing the Starter Culture
Pure rhizobial colony is transferred to a flask containing YEMA medium.
The flask is kept on a rotary shaker system in a constant temperature room at
28±2°C. Pure culture of rhizobium appears within a week
PRODUCTION OF RHIZOBIUM:

7. STRUCTURE RHIZOBIUM BACTREA

8. 4.Mass culture of Rhizobium
Rhizobium is mass cultured in large bioreactors (fermenter) to prepare
inoculant. YEM medium or sucrose-mannitol medium is used for this
purpose. A suitable medium is formulated and filled into the bioreactor after
proper sterilization. One liter of starter culture for 100 liter of medium is
inoculated into the bioreactor.
5.Making the Carrier-based Inoculants
A carrier is a neutral medium used to mix with a cultured broth for
handling it easily. Charcoal peat, lignite, vermiculite, paddy husk, farm
yard manure, etc. are used as carriers. The carrier holds the broth and
allows the slow growth of microbes.
6. Packing and Storage
The carrier based inoculant is packed in low density polytene bags for
storage and marketing. Usually, the biofertilizer pockets are kept in a
constant temperature room for about a week before storage. During this
time, Rhizobia grow by consuming the little amount of medium present in
the carrier.
PRODUCTION OF RHIZOBIUM(continued)

9. FIELD APPLICATIONS AND CROP RESPONSE
Field Application of Rhizobium Inoculant Rhizobium inoculants are
recommended for various leguminous crops by seed treatment:-
A 50 g of cane sugar is dissolved in 500 ml of water and boiled for 15
minutes.
200 g of gum arabic is added to the boiling sugar solution and stirred well to
dissolve it. are added into the slurry mixed well by hand. Consequently, about
105-106 Rhizobial cells get adsorbed on each and every seed.
The seeds are then allowed to
The sticker solution so formed is cooled own.
200 of Rhizobium inoculant is added into then a g the sticker solution and
mixed well to get a biofertilizer slurry.
The seeds of a legume dry by spreading them on a polytene sheet in shade. The
seeds are sown in the main field.
Crop Response
Seed inoculation of a proper Rhizobium strain increases growth and yield of
many legumes. Rhizobium usually increases the yield of legumes upto 10-35%.
The crop rotation of rhizobium inoculated legumes with cereals increases the
yield of the subsequent cereal crop.

10. AZOTOBACTER AS A BIOFERTILIZER:
Azotobacter is a gram negative, non-symbiotic, nitrogen fixing
bacterium. It is an aerobic bacterium present in large numbers in
rhizosphere soils.
Azotobacter fixes about 20-40Kg N/ha/year. Further, it produces plant growth
promoting substances like IAA, gibberellic acid and vitamins to favor the plant
growth. Hence it been recommended as a biofertilizer for rice, wheat, millets,
cotton, vegetables, mustard, sunflower, etc.
ISOLATION AND PRODUCTION:
1 A 10g rhizosphere soil is mixed well with 100ml of
distilled water and left undisturbed for some time to have a clear
suspension. The suspension is serially diluted and inoculated
into pertri dishes containing Jensen's medium
2 Azotobacter alone grows into soft, milky, mucoid
colonies on its surface. It transferred to culture flasks containing
liquid Jensen's medium Which kept on a rotary shaker for few
days . That gives a starter culture for mass production.

11. 3 For mass culture, l liter of
starter culture for 100 liters of medium is
transferred to sterilized Jensen's medium
or Ashby's medium or Burk's medium in a
bioreactor. Azotobacter attained the
concentration of 108 cells/ml, the
inoculant(broth) is harvested to make
carrier- based inoculant.A suitable carrier
is ground into fine powder Then the
inoculant is allowed for curing for a week.
Then packed in polytene bags for sales.
BENEFICIAL ROLES OF AZOTOBACTER
•Azotobacter fixes the atmospheric nitrogen in the soil and there by saves nitrogen
fertilizers in agriculture.
• Azotobacter together with 75% of recommended dosage of nitrogen fertilizers
boosts yield in many crop plants.
•Azotobacter treatment reduces crop diseases caused by these soil-borne pathogens.

12. FIELD APPLICATIONS
1.Seed Treatment
Azotobacter inoculant is mixed with a small quantity of water to make a
slurry and the seeds are kept dipped in the slurry for one night. In the early morning,
the seeds are taken out and sown in the main field.
2. Seedling Treatment
The roots of seedlings to be transplanted are kept dipped in the slurry for 10-
15 minutes. Then the seedlings are transplanted in the main field.
3. Pouring of Slurry
For crops such as sugarcane, Azotobacter inoculant is diluted properly with
water and a small volume of the slumy is poured near the root zone of the crop.
4. Top Dressing
For cereal crops like rice and wheat, Azotobacter inoculant(2Kg/ha) is
mixed with farmyard manure(20-25 Kg/ha) and broadcasted by top dressing after
transplantation.

13. AZOSPIRULLUM AS BIOFERTILIZER:
Azospirillum is a gram negative, symbiotic, vibrioid soil bacterium. It
occurs in large numbers in association with roots of cereals, grasses and
tuber crops
Azospirillum fixes about 20-40 Kg of the atmospheric nitrogen under micro
aerobic conditions. And it increases the vegetative growth and crop yield in many
plants
1.Isolation of Azospirillum
Azospirillum is present both inside and outside the plant roots. A proper host
plant is uprooted from the soil and washed then cut into small pieces and sterilized
in 0.1% mercuric chloride for one minute. Then washed with phosphate buffer.
Semi-solid malic acid medium is formulated suspension is inoculated and
incubated at room temperature for 3-5 days. A white pellicle like layer of
Azospirillum appears.
PRODUCTION OF AZOSPIRILLUM :
2.Confirmation of Azospirillum
Development of blue color in nitrogen free malic acid medium is the
confirmative test for Azospirillum

15. 3. Making the Starter Culture
A loopful of Azospirillum colony in the malic acid medium is transferred to
okan's medium in culture flasks. The culture flasks are maintain-ed at room
temperature for 3-5days.The bacterial cells grow into a starter culture.
4. Mass Culture
Okan's medium containing a sufficient amount of ammonium chloride is filled in a
fermenter. The starter culture is inoculated into the fermenter at the rate of 1% to
the total volume of the medium. The temperature is maintained at 35ºC for a few
days. Sterile air is pumped into the broth to supply oxygen. The broth is tested at
specific intervals for its purity and cell number. When the culture attained 109
cells/ml, the inoculum is harvested to make carrier-based inoculant.
5.Carrier-Based Inoculant
Powdered farmyard manure (FYM) and soil or FYM alone or FYM plus
charcoal is used as a carrier to make carrier-based Azospirillum inoculant. The
harvested inoculum is mixed with the carrier till it attains 40% moisture.
The inoculant is then packed in polytene bags and stored at 4ºC for a
week. The Azospirillum bags can be stored for about 3l weeks without any loss in
the viability.
PRODUCTION OF AZOSPIRILLUM(continued)

16. 1.Seed Treatment The seeds are soaked in the slurry overnight before
sowing. About 2 kg inoculant is required for a hectare.
2.Seedling Treatment One kilogram of Azospirillum inoculant is mixed with
40 liters of water to make a slurry. The roots of seedlings to be transplanted
are kept dipped in the slurry
3. Top Dressing In this method, 3 kg of Azospirillum inoculant is mixed
well with 25 kg of FYM and 25 kg of soil. This mixture is broadcasted
throughout the field by top dressing.
*Azospirillum fixes the atmospheric nitrogen and releases plant growth
promoting substances. So the crops grow well and give higher yield
*Azospirillum increases the straw yield as well as grain yield in rice, wheat,
barley, sorghum, bajra and fodder oats.
* Azospirillum treatment increases phenolic contents in sorghum. Hence the
crop becomes resistant to sorghum shoony and Antherigona soccata.
CROP RESPONSE :
FIELD USE OF AZOSPIRILLUM:

17. BLUE GREEN ALGAE (BGA):
Blue-green algae are photoautotrophic, prokaryotic algae, They are
mostly free living. They are otherwise called Cyanobacteria. Many species
of BGA fix the atmospheric nitrogen in moist soils.
PRODUCTION OF BGA INOCULANT
1. Isolation of BGA
About 5 gm of top soil is taken from a paddy field and added into a 100 ml of
Fogg's medium in a flask and shaken well and incubated at 1500lux illumination to
facilitate algal growth. Resulting algal culture is transferred to 10 ml of water.
shaken to separate algal filaments. Content is serially diluted.
A drop from each dilution is inoculated into Fogg's medium A drop from each
culture is examined under the microscope.
2. Starter Culture
The isolated pure cultures of BGA are grown separately in Fogg's
medium in culture flasks. Enough light is provided to the flask to favor the
algal growth. The algal cultures are used a starter cultures to initiate mass
culture of BGA.

18. 3.Mass culture of BGA
BGA can be mass cultured in four ways. They are-
Trough method
Pit method
Field method
Nursery method
4.Storage
Harvested flakes of BGA are well dried in the sunlight and packed in
gunny bags. The bags are stored in a cool dry place. The BGA inoculants can be
p for the maximum period of 3 years without any loss in the viability

19. A 10 kg of BGA inoculant is
recommended for one hectare of flooded
rice. The BGA flakes are dispersed and
broadcasted over the field 10 days after
transplantation. The application of BGA
to the crops is called algalization.
FIELD USE OF BGA:
Inoculation of BGA in rice
fields increases the yield upto 34 per
cent (ie.586 kh/ha). It saves about 30%
of the recommended dose of nitrogen
fertilizers for flooded rice.
CROP RESPONSE:
STRUCTURE OF BLUE GREEN ALGAE:

20. PHOSPHATE BIOFERTILIZERS:
Microbes which solubilise the bound phosphates and rock phosphates into
simple soluble phosphates are called phosphate solubiliers, phosphate
solubilizing bacteria or phosphobacteria.
The phosphate biofertilizers can save 30-50 kg of super phosphate per
hectare and increase the yield up to 200-500 kg/ha
PRODUCTION OF PHOSPHATE SOLUBILIZERS:
Isolation of Phosphate
Pikovskaias's medium is a specific medium for the phosphate solubilizing microbes.
The formulated Pikovskaia's medium is treated with 0.5% gum arabic solution and distributed
into petri dishes.
One gram of rhizosphere soil is suspended in 10ml of distilled water and the tube is
shaken well. And Serial diluted then inoculated into medium and incubated at 28±2ºC for two
weeks .The microbial colonies formed acts as a starter culture
Mass Production
Phosphobacteria are mass-cultured in Pikovskaia's liquid medium or Burnt and
Rovira medium in large bioreactors. About 1 liter culture for 100 liters of medium is
inoculated into the bioreactor and the temperature is maintained at 28±2ºC for 7-17days.
Wood charcoal, peat or mixture of wood charcoal and oil are used as carriers for inoculum
production

21. The phosphate biofertilizer is diluted
with water and then treated with gum
arabic and calcium carbonate to make a
slurry. Seeds to be sown are soaked in the
slurry for some time and dried in shade.
By this process, the inoculant is made to
form a pellet on the seed. The seeds are
then sown in the main field.
FIELD APPLICATION:
Phosphobacteria solubilize bound phosphates and contribute 20-30% of
phosphate required for the crops. Phosphate biofertilizer is suited for all types of
crops.
Co-inoculation of B.megaterium and Pseudomonas striata with
Azotobacter chroococcum increases the yield in sorghum and cotton. rice,
Sorghum and cotton.
USES:

22. VAM FUNGI:
The vesicular arbuscular mycorrhizal fungi(VAM Fungi) are a group
of symbiotic, endotrophic mycorrhizal fungi found in roots of higher plants.
They are included in the family Endogonaceae of Zygomycetes
VAM has three distinct regions-
• External hyphae
• Arbuscles
• Vesicles
VAM spores are isolated from the soil
in two Ways they are
1) Sieving method
2) Floatation method
ISOLATION OF VAM SPORES
The VAM spores are immersed in a solution containing chloramin-T
and streptomycin(200ppm) for 15 minutes and then washed with distilled water
The spores are mixed with sterilized soil. The soil is filled in pots and seedlings
of a host plant is transplanted in the pots. The pots are kept in a green house for
3-4 months. Finally, the soil in the pots along with roots of host plants is
macerated and dried till it attains roots 5% moisture. The dried soil-inoculant
mixture is used for field application.
MASS PRODUCTION OF VAM

23. VAM fungal inoculant is diluted with water and mixed with seeds to make
a pellet of inoculant on them. The seeds are then sown in the main field as usual.
In another method, the inoculant is spread uniformely all over the field before
ploughing and then crops are transplanted or seeds are sown in that field as usual.
• VAM fungi play the following important roles in agriculture
• VAM fungi help the plants to intake more Zn, S, Cu, P, Ca, K, Fe, Mn and
Br from the soil.
• VAM fungal infection increases the concentration of cytokinins and
chlorophylls in the plants.
• It reduces sensitivity of crops towards high level of salts and heavy metals
in the soil.
• VAM provides resistance to plants against various soil bore plant pathogens
causing root diseases.
• In fumigated soils plants show stunted growth. VAM fungal infection
reduces the stunting of the plants in such soils.
IMPORTANCE OF VAM FUNGI
FIELD APPLICATION

24. AZOLLAAS A (A GREEN MANURE CUM BIOFERTILIZER):
Azolla is a free-floating freshwater fern. The plant body consists of crowded
mass-like leaves on a branched fragile stem and submerged root. The ventral
surface of the aerial lobe bears mucilage cavities harboring the blue-green alga
A simple method of Azolla production is given here:
1) The land is ploughed well and levelled properly. It is divided into small plots of
20 2m size with bunds and irrigation channels.
2) The plots are irrigated through the channels to a height of 10 cms
3) The soil pH is adjusted to 8 by adding lime or sulphuric acid. A 10 kg of fresh
cattle dung is mixed with 20 liter of water to make a slurry and the slurry is
sprinkled uniformly all over the plot The 8 kg of Azolla inoculum is spread in
each plot.
4) 100 gm of super phosphate is added to each plot by dressing. top
5) 100 of furadon-G is top dressed over the plot 7 gm days after spreading the
inoculum to control pests.
6) Azolla forms a dense mat within 15 days after inoculation. After 15 days, Azolla
is harvested and used as a source of primary inoculum for main fields. About 40-
50 kg fresh Azolla can be harvested from a plot.
MASS CULTIVATION OF AZOLLA

25. SYMBIOTIC ASSOSIATION OF AZOLLA LEAF AND ANABENA
Azolla is added to the main fields either as a green manure or as a dual
crop.
FIELD APPLICATION OF AZOLLA
The main field is ploughed and leveled about 15 days before transplanting
the rice seedlings. Then Azolla is raised throughout the main field. After forming a
dense growth. Azolla is mixed with the mud by gentle ploughing and leveling just
before transplantation.
AZOLLAAS A GREEN MANURE

26. Azolla is grown as an intercrop with rice 1-2 weeks after transplanting
the rice. Usually it forms a dense growth within 3-4 weeks. Thereafter, the Azolla
biomass is burried in the mud by using a weeder. Azolla may once again be
cultivated in the same field. By this method, 0.5-1 tonne of Azolla biomass can
be added to one hectare of rice field during a season.
• Azolla saves 20-30% nitrogen fertilizers required for rice.
• Azolla reduces the multiplication of weeds and their dispersal in paddy fields.
Azolla reduces about 75% of Echinocola and 98% of Marsilia and eliminates
Cyperus in paddy fields.
• In China, fishes have been cultured in paddy fields. Azolla is grown in such
paddy fields. The fishes feed on Azolla and excrete their wastes in the water.
Azolla takes up the excreta for its growth. It is noticed that Azolla increaes the
productivity of fishes and rice.
• Azolla has also been used as a nitrogenous feed for carps growing in ponds.
AZOLLAAS A DUAL CROP
USES

27. BENEFITS FROM BIOFERTILIZERS:
1. It is a low cost and easy technique, and can be used by small and marginal farmers.
2. It is free from pollution hazards and increase soil fertility
3. Using biofertilizers can increase the crop yield by 20 or 30 percent
4. No special care is required while using biofertilizers
5. The farmers themselves can grow BGA biofertilizers and Azolla biofertilizer
6. Biofertilizers can be mass produced by using renewable biological wastes.
7. The biofertilizers increase chemical properties of soils soil structure, such as
texture, water holding capacity, cation exchange capacity and pH by providing
several nutrients and sufficient organic matter
8. Biofertilizers are a natural way to get the benefits of synthetic fertilizers without
risking the quality of soil health and crop products.
9. They reduce the attack by the soil borne pathogens. They improve the quality of
soil for more productivity.

28. Biofertilizers place a very important role in the present agriculture.
Because biofertizers are ecofriendly and easily grown and cost effective and
have a high rate of action on the plants. Using biofertilizers can increase the
crop yield by 20 or 30 percent(average).
Seed inoculation of a proper Rhizobium strain increases growth
and yield of many legumes. Rhizobium usually increases the yield of
legumes upto 10-35%.
Azotobacter fixes the atmospheric nitrogen in the soil and there
by saves nitrogen fertilizers in agriculture
Azospirillum fixes the atmospheric nitrogen and releases plant
growth promoting substances.
SUMMARY:

29. Inoculation of BGA in rice fields increases the yield upto 34 per
cent. It saves about 30% of the recommended dose of nitrogen
fertilizers for flooded rice
Phosphobacteria solubilize bound phosphates and contribute 20
to 30% of phosphate required for the crops
VAM fungi help the plants to intake more Zn, S, Cu, P, Ca, K, Fe,
Mn and Br from the soil. VAM fungal infection increases the growth rate
in plants
Azolla saves 20 to30% nitrogen fertilizers required for rice. Azolla
reduces the multiplication of weeds and their dispersal in paddy fields
In this way different biofertilizers can be used for different types of the
crops. Even though biofertilizers process the some of the disadvantages
biofertilizers have major advantages in the field of agriculture.

30. REFFERENCE
1. Satyanarayana. U. (2005) A text book of Biotechnology. Arunabha books
and applied (P)Limited. India. Fifth edition (Page No.644-647)
2. Kumarasan. V. (1994) Biotechnology. Saras publication. India Revised
edition 2011. (Page No.414-442)
3. R.C. durbey. (1993) A text book of Biotechnology. S.chand and company
Limited. India. Ninth edition (Page No.217-229)
4. Arun K. Sharma. (2002).Biofertilizers for sustainable agriculture Agrobios
publication. India First edition (general reference)
5. http://en.wikipedia.org/wiki/Biofertilizer
6. http://orbitbiotech.com/bio-fertilizers

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