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AZOTOBACTER
AS
BIOFERILIZER
Presented by – Chandrakant
CONTENT
• Biofertilizer
• Role of biofertilize
• Types of biofertilizer
• Azotobacter
• Characteristics
• Nitrogen fixation
• Mode of action of Azotobacter
• Isolation of Azotobacter
• Mass production of Azotobacter
• Mixing and packaging
• Ideal characteristics of carrier material
• Preparation of inoculum packet
• Application
• Advantage and disadvantage
• Reference
BIOFERTILIZER
• A biofertilizer is a substance which contains living
micro-organisms which, when applied to seeds, plant
surfaces, or soil, colonize the rhizosphere or the
interior of the plant and promotes growth by
increasing the supply or availability of primary
nutrients to the host plant.
• Biofertilizers add nutrients through the natural
processes of nitrogen fixation, solubilizing
phosphorus, and stimulating plant growth through
the synthesis of growth-promoting substances.
Why Microbes used as Biofertilizer?
• Microbes are effective in inducing plant growth as they secrets plant growth
promoters (auxins, abscisic acid, gibberellic acid, cytokinis’, ethylene) and
enhance seed germination and root growth.
• They also play a considerable role in decomposition of organic materials and
enrichment of compost.
Role of biofertilizers:-
• Better germination.
• Makes nutrients available.
• Growth Promoting Substances are produced.
• Improve quality and quantity of produce.
• More biotic and abiotic stress tolerance.
• Improve soil health.
• Increase the crop yields
TYPES OF BIOFERTILIZER
AZOTOBACTER
 The Azotobacter genus was discovered in 1901 by Dutch microbiologist and botanist
martinus Beijerinck ,who was one of the founders of environmental microbiology .He
selected and described the species Azotobacter chroococcum.
 Azotobacter is a free living aerobic nitrogen fixing bacterium .
 Free living soil microbes which play an important role in the nitrogen cycle in nature
binding atmospheric nitrogen ,which is inaccessible to plants ,and releasing it in the
form of ammonium ions into the soil ( Nitrogen fixation ).
 It also improves seed germinations , root proliferation and reduces the damage to
crop by plant diseases .
CHARACTERISTICS OF AZOTOBACTER
 Gram positive , Rod shape , aerobic bacterium.
 Pleomorphic , ranging from rod to coccoid cells.
 Grow well at 25 -30 C high humidity aeration ,PH
7.2 -7.6 high salt concentration .
 Forms cyst which resist unfavorable condition ( UV desiccation).
 Isolation can be done from soil using N free media like Ashby’s
manitol media , jensen’s media.
 Colonies are flat , soft , milky and mucoid.
 The motility of the bacteria is with the help of peritrichous
flagella.
At present six species of Azotobacter are know
 A. Chroococcum
 A. Vinelandii
 A. Nigricans
 A. Paspali
 A. Armenicus
 A. Salinestris
 A. Chroococcum is the most commonly found species in the soil.
NITROGEN FIXATION
 Azotobacter species are free living nitrogen fixation bacteria in
construct to Rhizobium species they normally fix molecular nitrogen
from the atmosphere without symbiotic relation with plant althought
some Azotobacter species are associated with plants .
 Nitrogen fixation is inhibited in the presence of available sources
such as ammonium ions and nitrates .
 Azotobacter species have a full range of enzymes needed to
performs the nitrogen fixation ferredoxin , hydrogenase and an
important enzymes nitrogenase . This process of nitrogen fixation
requires an influx of energy in the form of adenosine triphosphate .
MODE OF ACTION
 Direct mechanism of plant growth improvement
 Biological nitrogen fixation under free-living conditions
Productions of phytohormones like indole 3-acetic acid, gibberrillin-like
substances and cytokinins
 Indirect mechanism
 plant growth improvement by biocontrol activity
 Production of antifungal compound.
ISOLATION OF AZOTOBACTER
The species of Azotobacter are isolated by soil
dilution plate technique on the nitrogen free
medium. Ex- Jensen’s media and Ashby manitol
agar media.
After 48 hour or 3 days to 30 C incubation, flat soft,
milky and mucoid colonies of Azotobacter grow on
agar surface of the plates.
Besides, it can also be isolated directly from soil by
spreading a lump of soil on nitrogen free nutrient
medium.
After incubation for 3 days colonies of Azotobacter
grow on the surface of agar medium.
MASS PRODUCTION OF AZOTOBACTER
Culture mass multiplied in two levels
 at primary level in flasks using shaker
 Secondary stage multiplication in fermenters
The steps involved are
 Culture selection and maintenance
 Mass multiplication
 Mixing of the culture with carrier material and packing
After isolation, the pure culture of Azotobacter is transferred to a flask containing
sterile Jensen’s medium.
The flask is incubated on a rotary shaker or batch fermenters for a few days at 30°C.
The pure cells of Azotobacter developed in broth acts as starter culture which may be
used for mass production of inoculants.
For 1 litre of starter culture, 100 litres of medium is transferred to sterilized Jensen’s
medium in a bioreactor with proper maintenance of temperature of 30°C
and continuous agitation or aeration.
Azotobacter multiplies in mediumWhen inoculum density has reached to 10-10°
cell/ml broth, it should be harvested so that carrier-based inoculants should be
prepared.
MIXING AND PACKAGING
Suitable carrier is dried and powered passing through a sieve.
 Calcium carbonate powder is added to neutralize the carrier followed by
sterilization by autoclaving.
The harvested broth is poured over the carrier in such a way that 40%
moisture is maintained.
The inoculants is mixed and curing is done for a week.
 Then carrier based inoculants is packed in polythene bags so that it can
be stored and sent in market for sale.
IDEAL CHARACTERICTICS CARRIER MATERIAL
Carrier material- the use of ideal carrier material is necessary for the
production of good quality of biofertilizer
 Peat soil, lignite,, charcoal, press mud, farmyard manure and soil mixture
are used as a carrier
Ideal carrier material should be Cheaper in cost
 Locally available
 High organic matter content
No toxic chemical Water holding capacity of more than 50% Easy to process
PREPARATION INOCULUM PACKET
Neutralized and sterilized carrier material is spread in a clean, dry, sterile metallic
or plastic
Bacterial culture drawn from the fermenter is added to the sterilized carrier and
mixed well by manual or mechanical mixer
Inoculants are packed in a polythene bags sealed with electric sealer
APPLICATION
Application of Azotobacter inoculants in field
Azotobacter inoculants can be applied in different ways for different types of
plants in varying conditions.
FOLIAR APPLICATION:
▸ Foliar application of Azotobacter (108-109 cells/ml broth diluted to 1: 10 Ratio
with water) biofertilizer individually or in combination with half of normal dose of
chemical nitrogen fertilizer which increases leaf production in mulberry.
▸ In this case foliar application is better than soil application.
SEEDLING TREATMENT
This method is applicable for transplantation crop.
Ex-tomato, rice, onion, flower etc. Where seedling
are used for transplantation.
2kg of Azotobacter biofertilizer Is Add in buckets
which is full of water and mixed properly
And the and the root of seedling is dipping in this
mixture
And ready to transplant in the agricultural field
SOIL APPLICATION –
Azotobacter biofertilizer is applied to the soil in the case of fruits, crops,
sugarcane
It is recommended that 20g of biofertilizer mixed with compost is applied to each
sampling
Ex- in the case of sugarcane 5-6 kg of biofertilizer mixed with compost is applied
per arch
After two to three month of planting.
POURING OF SLURRY -
Carries based inoculants is diluted in water small amount of slurry is poured the
root zone
Slurry may be mixed with farmyard manure and administered near root zone .
ADVANTAGE
Azotobacter is heaviest breathing organism and requires a large amount of
organic carbon for its growth.
 It is poor competitor for nutrients in soil and hence its growth promoting
substances, fungistatic substances.
It can benefit crops by Nitrogen fixation.
 It improves seed germination and plant growth.
 It thrives even in alkaline soils.
DISADVANTAGE
Limited shelf life
Do not replace chemical fertilizers
REFERENCE
 A text book of Biotechnology by R.C. Dubey.
 https://www.slideshare.net/slideshow/azotobacter-pptx/265597432

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AZOTOBACTER AS BIOFERILIZER.PPTX

  • 2. CONTENT • Biofertilizer • Role of biofertilize • Types of biofertilizer • Azotobacter • Characteristics • Nitrogen fixation • Mode of action of Azotobacter • Isolation of Azotobacter • Mass production of Azotobacter • Mixing and packaging • Ideal characteristics of carrier material • Preparation of inoculum packet • Application • Advantage and disadvantage • Reference
  • 3. BIOFERTILIZER • A biofertilizer is a substance which contains living micro-organisms which, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant. • Biofertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances.
  • 4. Why Microbes used as Biofertilizer? • Microbes are effective in inducing plant growth as they secrets plant growth promoters (auxins, abscisic acid, gibberellic acid, cytokinis’, ethylene) and enhance seed germination and root growth. • They also play a considerable role in decomposition of organic materials and enrichment of compost. Role of biofertilizers:- • Better germination. • Makes nutrients available. • Growth Promoting Substances are produced. • Improve quality and quantity of produce. • More biotic and abiotic stress tolerance. • Improve soil health. • Increase the crop yields
  • 6.
  • 7. AZOTOBACTER  The Azotobacter genus was discovered in 1901 by Dutch microbiologist and botanist martinus Beijerinck ,who was one of the founders of environmental microbiology .He selected and described the species Azotobacter chroococcum.  Azotobacter is a free living aerobic nitrogen fixing bacterium .  Free living soil microbes which play an important role in the nitrogen cycle in nature binding atmospheric nitrogen ,which is inaccessible to plants ,and releasing it in the form of ammonium ions into the soil ( Nitrogen fixation ).  It also improves seed germinations , root proliferation and reduces the damage to crop by plant diseases .
  • 8. CHARACTERISTICS OF AZOTOBACTER  Gram positive , Rod shape , aerobic bacterium.  Pleomorphic , ranging from rod to coccoid cells.  Grow well at 25 -30 C high humidity aeration ,PH 7.2 -7.6 high salt concentration .  Forms cyst which resist unfavorable condition ( UV desiccation).  Isolation can be done from soil using N free media like Ashby’s manitol media , jensen’s media.  Colonies are flat , soft , milky and mucoid.  The motility of the bacteria is with the help of peritrichous flagella.
  • 9. At present six species of Azotobacter are know  A. Chroococcum  A. Vinelandii  A. Nigricans  A. Paspali  A. Armenicus  A. Salinestris  A. Chroococcum is the most commonly found species in the soil.
  • 10. NITROGEN FIXATION  Azotobacter species are free living nitrogen fixation bacteria in construct to Rhizobium species they normally fix molecular nitrogen from the atmosphere without symbiotic relation with plant althought some Azotobacter species are associated with plants .  Nitrogen fixation is inhibited in the presence of available sources such as ammonium ions and nitrates .  Azotobacter species have a full range of enzymes needed to performs the nitrogen fixation ferredoxin , hydrogenase and an important enzymes nitrogenase . This process of nitrogen fixation requires an influx of energy in the form of adenosine triphosphate .
  • 11. MODE OF ACTION  Direct mechanism of plant growth improvement  Biological nitrogen fixation under free-living conditions Productions of phytohormones like indole 3-acetic acid, gibberrillin-like substances and cytokinins  Indirect mechanism  plant growth improvement by biocontrol activity  Production of antifungal compound.
  • 12. ISOLATION OF AZOTOBACTER The species of Azotobacter are isolated by soil dilution plate technique on the nitrogen free medium. Ex- Jensen’s media and Ashby manitol agar media. After 48 hour or 3 days to 30 C incubation, flat soft, milky and mucoid colonies of Azotobacter grow on agar surface of the plates. Besides, it can also be isolated directly from soil by spreading a lump of soil on nitrogen free nutrient medium. After incubation for 3 days colonies of Azotobacter grow on the surface of agar medium.
  • 13. MASS PRODUCTION OF AZOTOBACTER Culture mass multiplied in two levels  at primary level in flasks using shaker  Secondary stage multiplication in fermenters The steps involved are  Culture selection and maintenance  Mass multiplication  Mixing of the culture with carrier material and packing
  • 14. After isolation, the pure culture of Azotobacter is transferred to a flask containing sterile Jensen’s medium. The flask is incubated on a rotary shaker or batch fermenters for a few days at 30°C. The pure cells of Azotobacter developed in broth acts as starter culture which may be used for mass production of inoculants. For 1 litre of starter culture, 100 litres of medium is transferred to sterilized Jensen’s medium in a bioreactor with proper maintenance of temperature of 30°C and continuous agitation or aeration. Azotobacter multiplies in mediumWhen inoculum density has reached to 10-10° cell/ml broth, it should be harvested so that carrier-based inoculants should be prepared.
  • 15. MIXING AND PACKAGING Suitable carrier is dried and powered passing through a sieve.  Calcium carbonate powder is added to neutralize the carrier followed by sterilization by autoclaving. The harvested broth is poured over the carrier in such a way that 40% moisture is maintained. The inoculants is mixed and curing is done for a week.  Then carrier based inoculants is packed in polythene bags so that it can be stored and sent in market for sale.
  • 16. IDEAL CHARACTERICTICS CARRIER MATERIAL Carrier material- the use of ideal carrier material is necessary for the production of good quality of biofertilizer  Peat soil, lignite,, charcoal, press mud, farmyard manure and soil mixture are used as a carrier Ideal carrier material should be Cheaper in cost  Locally available  High organic matter content No toxic chemical Water holding capacity of more than 50% Easy to process
  • 17. PREPARATION INOCULUM PACKET Neutralized and sterilized carrier material is spread in a clean, dry, sterile metallic or plastic Bacterial culture drawn from the fermenter is added to the sterilized carrier and mixed well by manual or mechanical mixer Inoculants are packed in a polythene bags sealed with electric sealer
  • 18. APPLICATION Application of Azotobacter inoculants in field Azotobacter inoculants can be applied in different ways for different types of plants in varying conditions. FOLIAR APPLICATION: ▸ Foliar application of Azotobacter (108-109 cells/ml broth diluted to 1: 10 Ratio with water) biofertilizer individually or in combination with half of normal dose of chemical nitrogen fertilizer which increases leaf production in mulberry. ▸ In this case foliar application is better than soil application.
  • 19. SEEDLING TREATMENT This method is applicable for transplantation crop. Ex-tomato, rice, onion, flower etc. Where seedling are used for transplantation. 2kg of Azotobacter biofertilizer Is Add in buckets which is full of water and mixed properly And the and the root of seedling is dipping in this mixture And ready to transplant in the agricultural field
  • 20. SOIL APPLICATION – Azotobacter biofertilizer is applied to the soil in the case of fruits, crops, sugarcane It is recommended that 20g of biofertilizer mixed with compost is applied to each sampling Ex- in the case of sugarcane 5-6 kg of biofertilizer mixed with compost is applied per arch After two to three month of planting. POURING OF SLURRY - Carries based inoculants is diluted in water small amount of slurry is poured the root zone Slurry may be mixed with farmyard manure and administered near root zone .
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  • 23. ADVANTAGE Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its growth.  It is poor competitor for nutrients in soil and hence its growth promoting substances, fungistatic substances. It can benefit crops by Nitrogen fixation.  It improves seed germination and plant growth.  It thrives even in alkaline soils. DISADVANTAGE Limited shelf life Do not replace chemical fertilizers
  • 24. REFERENCE  A text book of Biotechnology by R.C. Dubey.  https://www.slideshare.net/slideshow/azotobacter-pptx/265597432