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PRODUCTION 
OF 
BIOFERTILIZERS
Soil is a natural habitat of variety of agriculturally 
benef icial microorganisms. Certain soil 
microorganisms have an ability to absorb and 
convert atmospheric nitrogen to the readily 
available form to the plants. Whereas certain soil 
microorganisms solubilize part of the bound 
phosphates of the soil and thereby make them 
available to the plants. Both these attribute make 
them important to be used as Biofertilizers 
.
Production of Biofertilizers
Nitrogen fixing Bacteria for Leguminous Crops 
Crops Rhizobium fertilizer contains Rhizobium micro-organisms. 
These micro-organisms live in association with 
the roots of leguminous plants in the form of root nodules. 
These micro-organisms fix the atmospheric Nitrogen in the 
roots & make it available to plants, which plants cant fix 
directly and makes the soil fertile & also increases the plant 
health.
Phosphate solubilizing micro-organisms for all Crops 
There are many types of micro-organisms present in soil out of which 
some microbes & fungi can solubilise the complex insoluble form of 
phosphorus into simple soluble forms that can be taken up by plants. 
Generally these micro-organisms are very few in soil. 
PSB culture contains millions of soil phosphate solubilising micro-organism 
per gram. These soil phosphate solubilising bacteria stay near 
the roots & make the phosphorus available to plants from soil as well as 
fertilizers & increase the production drastically.
Nitrogen fixing Bacteria for all Non –Leguminous 
Crops 
This microorganism fixes the atmospheric nitrogen and 
makes it available to plants in asymbiotic manner. 
Azotobacter is mainly useful for monocot vegetables. 
Azotobacter also secretes some fungicides, enzymes but in 
minute amount. Use of Azotobacter increases the crop 
production in large scale.
Nitrogen fixing Bacteria –azospirillum 
for all Non –Leguminous Crops 
This microorganism fixes the atmospheric nitrogen and 
makes it available to plants in asymbiotic 
manner.Azospirillum is mainly useful for monocot 
vegetables.Azospirillum biofertilizer also secretes some 
fungicides, enzymes but in minute amount. Use of 
Azospirillum biofertilizer increases the crop production in 
large scale.
ISOLATION OF RHIZOBIUM 
RHIZOBIUM IS ISOLATED ON CONGO RED MEDIUM 
The Rhizobium from the freshly crushed root nodule 
was Gram stained onto a slide and examined at 100X 
power with oil immersion. The bacteria cells were rods 
in chains, and clumps that stained Gram negative as 
indicated by the faint pink red colour of the rod 
membrane walls.
Samples were collected from different stations viz. Station-1 
(Thondi open sea-I), Station-2 (Under the Jetty), Station-3 
(Thondi open sea-II), Station-4 (Beach). Sediment samples were 
collected by sediment sampler (Peterson crab), it was sterilized 
with alcohol before sampling at each station. The central portion 
of the top 2 cm sediment samples was taken out with the help of 
a sterile spatula. The samples were then transferred to a sterile 
polythene bag and transported 
The serially diluted samples were plated on Pikovskaya’s agar 
media to isolate the phosphate solubilizing bacteria. The plates 
were incubated at 28±2 ºC. After 3 days,
Soil samples were collected during Spring and Autumn 
2000 in different areas of central Italy both from cultivated 
and uncultivated soils. Samples were withdrawn at a depth 
of 10 – 15 cm below the surface, collected into sterile vials. AND 
THEN 
AZOTOBACTER IS ISOLATED ON JENSEN’S MEDIUM
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 forRhizobium; 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.
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.
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).
Use for the non-leguminous crops before the mentioned expiry date. 
Mix the inoculants uniformly with the seeds gently with the minimum 
amount of water taking care to avoid damage to seed coat. Dry the 
inoculated seeds under shade over clean paper or gunny bag and sow 
immediately. 
For transplanted crops: Mix the inoculants in bucket of water stir the 
mixture vigorously. Dip the roots of seedlings in this mixture before 
transplanting. Transplant as usual. 
It can be mixed with pit mixture before planting of vegetables / fruit 
crop. 
If the seed is to be treated with pesticides; first follow the pesticide 
treatments and finally treat seeds with Azotobacter inoculant.
Use for the non-leguminous crops before the mentioned expiry date. 
Mix the inoculants uniformly with the seeds gently with the minimum amount 
of water taking care to avoid damage to seed coat. Dry the inoculated seeds 
under shade over clean paper or gunny bag and sow immediately. 
For transplanted crops: Mix the inoculants in bucket of water stir the mixture 
vigorously. Dip the roots of seedlings in this mixture before transplanting. 
Transplant as usual. 
It can be mixed with pit mixture before planting of vegetables / fruit crop. 
If the seed is to be treated with pesticides; first follow the pesticide treatments 
and finally treat seeds with Azospirillum inoculant. 
It reduces dose of nitrogenous fertilizers like Urea and Increases the protein 
content of seeds. 
It provides nitrogen to the plant and increases the yield.
Use for the crops before the mentioned expiry date. 
Mix the inoculants uniformly with the seeds gently with the minimum 
amount of water taking care to avoid damage to seed coat. Dry the 
inoculated seeds under shade over clean paper or gunny bag and sow 
immediately. 
For transplanted crops: Mix the inoculants in bucket of water stir the 
mixture vigorously. Dip the roots of seedlings in this mixture before 
transplanting. Transplant as usual. 
It can be mixed with pit mixture before planting of vegetables / fruit 
crop. 
If the seed is to be treated with pesticides; first follow the pesticide 
treatments and finally treat seeds with PSB culture. 
It coverts insoluble complex form of phosphate in to simple soluble 
form to increases the yield.
TO ALL OF YOU

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Production of Biofertilizers

  • 2. Soil is a natural habitat of variety of agriculturally benef icial microorganisms. Certain soil microorganisms have an ability to absorb and convert atmospheric nitrogen to the readily available form to the plants. Whereas certain soil microorganisms solubilize part of the bound phosphates of the soil and thereby make them available to the plants. Both these attribute make them important to be used as Biofertilizers .
  • 4. Nitrogen fixing Bacteria for Leguminous Crops Crops Rhizobium fertilizer contains Rhizobium micro-organisms. These micro-organisms live in association with the roots of leguminous plants in the form of root nodules. These micro-organisms fix the atmospheric Nitrogen in the roots & make it available to plants, which plants cant fix directly and makes the soil fertile & also increases the plant health.
  • 5. Phosphate solubilizing micro-organisms for all Crops There are many types of micro-organisms present in soil out of which some microbes & fungi can solubilise the complex insoluble form of phosphorus into simple soluble forms that can be taken up by plants. Generally these micro-organisms are very few in soil. PSB culture contains millions of soil phosphate solubilising micro-organism per gram. These soil phosphate solubilising bacteria stay near the roots & make the phosphorus available to plants from soil as well as fertilizers & increase the production drastically.
  • 6. Nitrogen fixing Bacteria for all Non –Leguminous Crops This microorganism fixes the atmospheric nitrogen and makes it available to plants in asymbiotic manner. Azotobacter is mainly useful for monocot vegetables. Azotobacter also secretes some fungicides, enzymes but in minute amount. Use of Azotobacter increases the crop production in large scale.
  • 7. Nitrogen fixing Bacteria –azospirillum for all Non –Leguminous Crops This microorganism fixes the atmospheric nitrogen and makes it available to plants in asymbiotic manner.Azospirillum is mainly useful for monocot vegetables.Azospirillum biofertilizer also secretes some fungicides, enzymes but in minute amount. Use of Azospirillum biofertilizer increases the crop production in large scale.
  • 8. ISOLATION OF RHIZOBIUM RHIZOBIUM IS ISOLATED ON CONGO RED MEDIUM The Rhizobium from the freshly crushed root nodule was Gram stained onto a slide and examined at 100X power with oil immersion. The bacteria cells were rods in chains, and clumps that stained Gram negative as indicated by the faint pink red colour of the rod membrane walls.
  • 9. Samples were collected from different stations viz. Station-1 (Thondi open sea-I), Station-2 (Under the Jetty), Station-3 (Thondi open sea-II), Station-4 (Beach). Sediment samples were collected by sediment sampler (Peterson crab), it was sterilized with alcohol before sampling at each station. The central portion of the top 2 cm sediment samples was taken out with the help of a sterile spatula. The samples were then transferred to a sterile polythene bag and transported The serially diluted samples were plated on Pikovskaya’s agar media to isolate the phosphate solubilizing bacteria. The plates were incubated at 28±2 ºC. After 3 days,
  • 10. Soil samples were collected during Spring and Autumn 2000 in different areas of central Italy both from cultivated and uncultivated soils. Samples were withdrawn at a depth of 10 – 15 cm below the surface, collected into sterile vials. AND THEN AZOTOBACTER IS ISOLATED ON JENSEN’S MEDIUM
  • 11. 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 forRhizobium; 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.
  • 12. 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.
  • 13. 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).
  • 14. Use for the non-leguminous crops before the mentioned expiry date. Mix the inoculants uniformly with the seeds gently with the minimum amount of water taking care to avoid damage to seed coat. Dry the inoculated seeds under shade over clean paper or gunny bag and sow immediately. For transplanted crops: Mix the inoculants in bucket of water stir the mixture vigorously. Dip the roots of seedlings in this mixture before transplanting. Transplant as usual. It can be mixed with pit mixture before planting of vegetables / fruit crop. If the seed is to be treated with pesticides; first follow the pesticide treatments and finally treat seeds with Azotobacter inoculant.
  • 15. Use for the non-leguminous crops before the mentioned expiry date. Mix the inoculants uniformly with the seeds gently with the minimum amount of water taking care to avoid damage to seed coat. Dry the inoculated seeds under shade over clean paper or gunny bag and sow immediately. For transplanted crops: Mix the inoculants in bucket of water stir the mixture vigorously. Dip the roots of seedlings in this mixture before transplanting. Transplant as usual. It can be mixed with pit mixture before planting of vegetables / fruit crop. If the seed is to be treated with pesticides; first follow the pesticide treatments and finally treat seeds with Azospirillum inoculant. It reduces dose of nitrogenous fertilizers like Urea and Increases the protein content of seeds. It provides nitrogen to the plant and increases the yield.
  • 16. Use for the crops before the mentioned expiry date. Mix the inoculants uniformly with the seeds gently with the minimum amount of water taking care to avoid damage to seed coat. Dry the inoculated seeds under shade over clean paper or gunny bag and sow immediately. For transplanted crops: Mix the inoculants in bucket of water stir the mixture vigorously. Dip the roots of seedlings in this mixture before transplanting. Transplant as usual. It can be mixed with pit mixture before planting of vegetables / fruit crop. If the seed is to be treated with pesticides; first follow the pesticide treatments and finally treat seeds with PSB culture. It coverts insoluble complex form of phosphate in to simple soluble form to increases the yield.
  • 17. TO ALL OF YOU