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Azotobacter-Soil health - Use of Azotobacter as Biofertilizer

Nitrogen fixation is mainly responsible for improvement of crop yield. In this regard, diazotrophs like Rhizobium, Azotobacter and Azospirillum are important as they enrich nitrogen nutrition in N-deficient soils. Of these, Azotobacter promotes plant growth as well as nitrogen fixation. Thus technology has been developed for making use of Azotobacter biofertilizer for nitrogen and non-nitrogen fixing plants and popularized by educating about their benefits in agriculture to users for practicing integrated nitrogen management.

In agriculture, one of the limiting factors is providing plant nutrients, particularly nitrogen (N) and phosphorous (P), to crops. So the improvement of crop yield by inoculation with diazotrophs like Azotobacter, Rhizobiumand Azo-spirillumhas been suggested as an ecofriendly technology. These microorganisms colonize the rhizosphere of plants and remain in close asso-ciation with roots and influence their growth. Of these, Azotobacter is one of the most extensively studied plant growth-promoting microorganisms because its inoculation benefits a wide variety of crops. These are polymorphic, possess peritrichous flagella and produce polysaccharides; they are sensitive to acidic pH, high salts and temperature above 35°C and can grow on a N-free medium thus utilize atmospheric nitrogen (N2) for cell protein synthesis. Cell proteins are mineralized in soil after death of Azotobacter and contribute to N availability to crop plants.

Growth of Azotobacter:

Usually Azotobacter is grown on a solid medium free of nitrogen. After some times (6 months) old growth of Azotobacter is transferred to a fresh solid medium to renew the growth. This procedure is repeated periodically so that the culture can be maintained in good condition.

Production of azotobacter:

i. Mother culture: A pure growth of any organism on a small scale is called as a mother culture. Mother culture is always prepared in a conical flask of 500 or 1000 ml. Capacity and then this mother culture is used for further production.

For this purpose, one liter conical flasks are taken to which 500 ml of broth of nitrogen free medium is added and these flasks are then plugged with non-absorbent cotton, sterilized in an auto slave for 15-20 minutes at 75 lbs pressure for 15 minutes. Flasks are then inoculated with mother culture with the help of inoculating needle aseptically. The flasks are transferred to shaker and shaking is done for 72-90 hours so as to get optimum growth of bacteria in broth. Bacteria are multiplied by binary method i.e. cell division. After about 90 days, the number of per milliliters comes to about 100 crores. Total growth of bacteria in this broth means starter culture or mother culture, which should carefully be done, since further purity of biofertilizer or quality of biofertilizer depends upon how mother culture is prepared.

ii. Production on a large scale: Azotobacter is multiplied on a large scale by two ways viz. Fermenter and Shaker. The fermenter is most automatic and accurate method of multiplication of any micro-organism. In this method, the medium is taken in a fermenter and then sterilized. After this pH of the medium is adjusted and 1% mother culture is added. In order to get an optimum growth of the Azotobacter required temperature and oxygen supply is adjusted so that concentrated broth is made. This concentrated broth of the culture is then mixed with a carrier previously sterilized and bio-fertilizers are prepared. Depending upon the demand and supply suitable fermenter is selected.

In the 2nd method i.e. shake method, a suitable medium is prepared transferred to conical flask of suitable capacity. These flasks are then sterilized in an autoclave at 15 lbs pressure for 15 minutes. Each flask is inoclulated with 10 ml mother culture and they are transferred to shaker for multiplication where they are kept for 72-90 hours. This broth is mixed with a suitable carrier previously sterilized. Thus biofertilizer is prepared, filled in plastic bags and stored in cool place.

Selection of carrier:

A carrier is nothing but a substance which has high organic matter, higher water holding capacity and supports the growth of organism. In order to transport the biofertilizer and becomes easy to use the suitable carrier is selected. Generally Lignite cool, compost and peat soil are suitable carriers for Azotobacter. Out of these carriers lignite is most suitable for this organism, since it is cheaper, keeps organism living for longer period and does not lower the quality of bio-fertilizers.

The lignite comes in clouds and hence it is ground in fine powder by grinding machine. Its finesses should be 250-300 mesh. The pH of the carrier is adjusted to neutral by adding CaCO3. The lignite naturally has a variety of micro-organism and hence it is sterilized in autoclave at 30 lbs. Pressure for 30 minutes. After this the broth is mixed with lignite 1:2 proportion by following method.

Galvanized trays are sterilized and used. To these trays, previously sterilized lignite is transferred and broth is then added (lignite2: broth 1) and mixed properly. Trays are then kept one above the other for 10-12 hours for allowing the organism to multiply in the carrier. This mixture is then filled in plastic bags of 250 g or 500 g capacity. Plastic bags are properly. Trays are then kept one above the other for 10-12 hours for allowing the organism to multiply in the carrier. This mixture is then filled in plastic bags of 250 g or 500 g capacity. Plastic bags are properly sealed. All the required information such as name of biofertilizer, method of use expiry date, etc. is printed on plastic bags. In this way biofertilizer is ready to sell or use. If biofertilizer is used immediately then bags are stored in cool place otherwise they should be stored in cold storage in order to keep biofertilizer in good quality.

As per ISI standards, one gram of biofertilizer immediately after it is prepared should have one crore cells of bacteria and 15 days before expiry date one gram of biofertilizer should have 10 lakh bacteria. If biofertilizer is stored at 15-20 0C then it will remain effective for 6 months. However, at 0 to 4 0C (cold storage) the bacteria will remain active for 2 years. The storage periods are decided after testing the biofertilizer for that particular storage conditions, such temperature and humidity.

Use of Azotobacter as Biofertilizer:

Plant needs nitrogen for its growth and Azotobacter fixes atmospheric nitrogen non-symbiotically. Therefore, all plants, trees, vegetables, get benefited. However, especially cereals, vegetables, fruits, trees, sugarcane, cotton, grapes, banana, etc. are known to get addition nitrogen requirements from Azotobacter. Azotobacter also increases germination of seeds. Seeds having less germinating percent if inoculated can increase germination by 20-30%.

How to apply Azotobacter bio-fertilizer?

a. Seed inoculation:
On the basis of efficiency of Azotobacter, other micro-organisms present in the soil, benefits obtained from biofertilizer and expenditure it has been fixed to use Azotobacter - bio-fertilizer at the rate of 250 g biofertilizer for 10-15 kg. If one knows this proportion then take a definite quantity of seed to be inoculated. The required quantity of fresh biofertilizer is secured and slurry is made by adding adequate, quantity of water. This slurry is uniformly applied to seed, seed is then dried in shed and sown. Some stickers are used in order to adher biofertilizer to seeds. Viz. Jaggery or gum arebia.

b. Seedling inoculation:
This method of inoculation is used where seedlings are used to grow the crop. In this method, seedlings required for one acre are inoculated using 4-5 packets (2-2.5 kg). For this, in a bucket adequate quantity of water is taken and biofertilizer from these packets is added to bucket and mixed properly. Roots or seedlings are then dipped in this mixture so as to enable roots to get inoculums. These seedlings are then transplanted e.g. Tomato, Rice, Onion, Cole, Crops, flowers.

c. Self inoculation or tube inoculation:
In this method 50 litres of water is taken in a drum and 4-5 kg of Azotobacter biofertilizer is added and mixed properly. Sets are required for one acre of land are dipped in this mixture. Potato tubers are dipped in the mixture of biofertilizer and planting is done.

d. Soil application:
This method is mostly used for fruit crops, sugarcane, and trees. At the time of planting fruit tree 20 g of biofertilizer mixed with compost is to be added per sappling, when trees became matured the same quantity of biofertilizer is applied.

In sugarcane after two to three months of planting i.e. before earthing up 5-6 kg of biofertilizer per acre is applied by mixing with compost or soil. Although, Azotobacter fixes nitrogen non-symbiotically, it also fixes atmospheric nitrogen in the rhizospere region i.e. soil around the seedlings or trees. Biofertilizer applied to seed or seedlings bacteria remain around seeds or seedlings and use organic carbon for their metabolism. When seeds are germinated or seedlings set in soil they leave or exude root exudates which become food of these bacteria. They grow on these substances which include sugars, organic acids, and amino acids and fix atmospheric nitrogen most efficiently. Nitrogen so fixed by these bacteria becomes available to plants after dead and degradation of bacterial cells.

Azotobacter and Nitrogen Fixation:

Azotobacter belongs to the Azotobacteriaceae family. These are Gram-negative, non-symbiotic, aerobic diazotrophs. The young rod-shaped cells vary from 2.0-7.0 to 1.0-2.5 μm and occasionally an adult cell may increase up to 10-12 μm, and be oval, spherical or rod-shaped cells. Azotobacter can grow well on simple N-free nutrient medium containing phosphate, magnesium, calcium, molybdenum, iron and carbon sources. Its catabolic versatility in utilizing several aromatic compounds such as protocatechuic acid, 2-4-D (2,4-dichlorophenoxyacetic acid), 2-chlorophenol, 4-chloro-phenol, 2,4,6-chlorotriphenol, aniline, lindane, toluene, p-hydroxy benzoate, benzoate and benzene is well docu-mented. Azotobacter contributes significant amounts of fixed N2 in, on, or near a plant. The energy requirement for the process of N2fixation is met by a very high rate of aerobic metabolism which contributes to high oxygen demand for the maintenance of minimal intracel-lular oxygen tension, a requirement of the oxygen-sensitive nitrogenase to accomplish N2fixation. Diazotrophic bacteria in the rhizosphere of plants utilize the products of N2fixation for their own growth and release little while they are alive. When bacteria die, only a small quantity of fixed N2 is assimilated by the plant. N2fixation by heterotrophic bacteria in the rice rhizosphere develops in response to a deficiency in the availability of the combined N2. When fixed N2 is not readily available for plant growth, the plants become N2deficient where rhizobcteria contribute significantly. The highest rates of root-associated Nitro-genase activity were measured in N-deficient plants. In native bacteria the process of N2fixation is inhibited by combined N2in the environment. The N2fixation process in root-associated bacteria can fix N2gas in the presence of repressive levels of combined N2and export a major portion of the Nitrogenase-produced ammonia or organic N 2 by-product from their cells into the rhizosphere and/or roots. Thus, plants which form associations with desired bacteria have an additional source of combined N2available for growth. This microbe possesses three genetically distinct Nitrogenase complexes and the expression of these Nitro-genase varies with vanadium, molybdenum and ammonium in the culture medium. Nitrogenase-I is expressed only when molybdenum is present in the medium, Nitrogenase-II is expressed only when vanadium is present while Nitrogenase-III is expressed when both molybdenum and vanadium are absent.

Advantages of Azotobacter:

1. Azotobacter contributes moderate benefits

2. Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its growth.

3. It is poor competitor for nutrients in soil and hence its growth promoting substances, fungistatic substances.

4. It can benefit crops by Nitrogen fixation, growth promoting substances, fungi static substances.

5. Azotobacter is less effective in soils with poor organic matter content.

6. It improves seed germination and plant growth

7. It thrives even in alkaline soils.

8. Azotobacter is tolerant to high salts.


Azotobacter is a broad spectrum biofertilizer and can be used as inoculant for most agricultural crops. Earlier, its utility as a biofertilizer was not a priority due to its relatively low population in the plant rhizosphere. However, seeding treatment with Azotobacter of several crops brought about an increase in yield. Besides, because of its well known N2 nutritional function, it is now recognized to play a multiple role in helping crop plants to improve their growth potential, yield and maintenance of soil health for sustainable agriculture. Hence there is renewed interest in this rhizobacterium. However, quantitative understanding of the ecological factors that control the performance of biological N2fixation systems of the bacterium in crop fields is essential for promotion and successful adoption of the bio-fertilizer production technology.

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The worldwide spread of inflation, initiated by several fold rise in Petroleum price thereby depicting its striking influence on the prices of chemical nitrogenous fertilizers, the prices of nitrogenous fertilizers have nearly doubled during the last 3-4 years. This has necessiated to search for cheaper source of nitrogen to meet the needs of crops. This has rejuvenation of soil microbiology to tap out the biological fixation of nitrogen.

Azotobacter spp:

These are free living bacteria which grow well on a nitrogen free medium. These bacteria utilize atmospheric nitrogen gas for their cell protein synthesis. This cell protein is then mineralised in soil after the death of Azotobacter cells thereby contributing towards the nitrogen availability of the crop plants.

Characteristics of Azotobacter :

Azotobacter is Gram negative bacteria, polymorphic i.e. they are of different sizes and shapes. Their size ranges from 2-10x1-2.5 m ., young cell possess peritrichous flegella and are used as locomotive organs. Old population of bacteria includes encapsulated forms and have enhanced resistant to heat, desication and adverse conditions. The cyst germinates under favourable conditions to give vegetative cells. They also produce polysachharides. Azotobacter spp.,are sensitive to acidic pH, high salts, and temperature above 350C.

There are four important species of Azotobacter viz. A.Chroococcum, A.agilis, A.paspali and A.vinelandii of which A.chroococcum is most commonly found in our soils.

Nitrogen fixation by Azotobacter:

The species of Azotobacter are known to fix on an average 10 mg.of N/g of sugar in pure culture on a nitrogen free medium. A maximum of 30 mg. N fixed per gram of sugar was reported by lopatina. However, Azotobacter is a poor competitor for nutrients in soil. Most efficient strains of Azotobacter would need to oxidise about 1000 kg of organic matter for fixing 30 kg of N/ha. This does not sound realistic for our soils which have very low active carbon status. Besides, soil is inhabitated by a large variety of otherr microbes, all of which compete for the active carbon.

Azotobacter in soil:

In Indian soils, the population of Azotobacter is not more than 10 thousand to 1 lakh/g of soil. The Population of Azotobacter is mostly influenced by other micro-organisms present in soil. There are some micro-organism which stimulate the Azotobacterpopulation in soil thereby increasing the nitrogen fixation by Azotobacter. On the other hand there are some micro-organisms which adversely affect the Azotobacter population and hence nitrogen fixation process is hampered. For example cephallosporium is most commonly found organisms in soil which restricts the growth of Azotobacter.

Azotobacter also produces some substances which check the plant pathogens such as Alternaria, Fusarium and Helminthosporium. Hence Azotobacter also acts as a biological control agent.

Functions of Azotobacter:

Azotobacter naturally fixes atmospheric nitrogen in the rhizosphere. There are different strains of Azotobacter each has varied chemical, biological and other characters. However, some strains have higher nitrogen fixing ability than others.. Azotobacter uses carbon for its metabolism from simple or compound substances of carbonaceous in nature. Besides carbon, Azotobacter also requires calcium for nitrogen fixation. Similarly, a medium used for growth of Azotobacter is required to have presence of organic nitrogen, micro-nutrients and salt in order to enhance the nitrogen fixing ability of Azotobacter.

Besides, nitrogen fixation, Azotobacter also produces, Thiomin, Riboflavin, Nicotin, indol acitic acid and giberalin. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter.

Selection of Azotobacter strains :

After isolation of Azotobacter from soil its purity is tested in the laboratory in a pure form. In fertile soil spp.A.Chroococcum is found, commonly. The organism is aerobic in nature, requires oxygen for its growth. In old culture Malinin chemical is formed which gives the blackish colour to the culture. The organism is prominently found in alkaline or neutral soils. Strains of Azotobacters vary in their nitrogen fixing ability which depends upon pH of soil, crop and atmosphere of soil. Therefore nitrogen fixing capacity of strains is tested frequently. In order to obtain most efficient strains of Azotobacter one has to conduct different tests or experiments in glass house, earthern pots and field under the guidance of micro-biologists, some of them are explained below;

  1. Azetylene reduction Test: Different strains of Azotobacter in Pure form are grown in the laboratory in separate conical flasks. These flasks are then kept on shaker for about 72-50 hours so as to obtain full growth of bacteria in the medium of which 10-15 ml.,of both is transferred to a bottle, to this bottle 10 ml of acetylene gas is added and bottle is closed with cork borer and allowed to stand in the shed for 2-4 hours to have reaction of enzyme nitrogenase with acetylene gas. During this period, acetylene is converted to ethylene. Percentage of both the gases is measured chromatographically. The strain which has more nitrogenase enzyme forms more ethylene gas. Naturally, this strain will be selected for further use.
  2. Pot culture experiment: After having tested different strains in the laboratory and selected efficient strains next test comes the pot culture experiment. In this test, earthern pots are cleaned properly and disinfected with some common laboratory disinfectent and filled in with uniform amount of garden soil already sterilized.

Strain found efficient in acetylene reduction test are selected and multiplied in a pure form. The broth is mixed with liquite and inoculant so prepared is used to inoculate the seeds. Seeds are then dried in shed and sown in pots. Suitable inoculated control plants are kept for comparision. Plants are watered as and when required and allowed to grow for about 45-60 days. Differences between inoculated and uninoculated plants in respect of height, nitrogen content of plant and soil, dry weight of plants are noted. Efficient strains are used for field tests.

  1. Field Test: Strains found efficient under glass house conditions are required to undergo field test which is most important test from the farmer’s point of view. Strains found efficient in glass house and acetylent tests when used in field are required to compete with native flora for their nutrition. Efficient strains of Azotobacter are applied to seeds of particular and sown. An adequate control is kept for comparision. All other factors are kept similar except seed inoculation with efficient strains of Azotobacter.

After maturity yield figures are noted and comparision is made. Such experiments are repeated for 3 to 4 years at different places. From this data an efficient strain is selected and used for the production of Azotobacter on a large scale. Such strains are stored under cold storage conditions or in refrigerator. For production of bio-fertilizer, it is always advisible to use more than one strain on safer side.

Experience of biofertilizer in Russia:

Field experiments conducted in Russia with Azotobacter as a biofertilizer for wheat, barley, oat maize, sugarcane, etc. revealed that the performance of Azotobacter was medium to poor. Experiments on the use of Azotobacter for seed inoculation of various crops have also been conducted in other countries including India. The results of these experiments indicate that benefits obtained from inoculation of seeds with biofertilizer were marginal in soil with poor organic matter content. While in rich soils results were quite encouraging.

The field soils are inhabited by a very large number of microbial species. The co-existence of the relative populations of each one of the species is determined by ecological factors prevailing in the soil. These various species survive in soil while maintaining a balance of population is between various microbial species within certain limits.