Azotobacter - Organic and Natural Products

Science of Azotobacter

Uses of Azotobacter

Growth of Azotobacter

Experiments with Azotobacter

Azotobacter Vinelandii : is a free-living bacterium that can fix atmosferic nitrogen into the soil, being a great source to obtain a natural biofertilizer that can be used in the cultivation of most crops.

Azotobacter is a great source of nitrogen to meet the needs of crops because also has the capabilities to cause a rejuvenation of soil microbiology to tap out the biological fixation of nitrogen.

Azotobacter Benefits: It improves seed germination and plant growth
Azotobacter are tolerant to high salts.
It can benefit crops by Nitrogen fixation, growth promoting substances, fungi static substances.
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 thrives even in alkaline soils.
Azotobacter is less effective in soils with poor organic matter content.
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.

Azotobacter Characteristics -

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.

Azotobacter species - 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 - Soils are habited 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. A normal population of Azotobacter could be about than 10 thousand to 1 lakh/g of soil and it is mostly influenced by other micro-organisms present in soil. There are some micro-organism which stimulate the Azotobacter population 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 Functions : 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.
Azotobacter also produces fixation of 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.

Azotobacter strains - : - Selection of 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.

Azotobacter Science - Azotobacter - Azotobacter vinelandii - 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. Azotobacter is an aerobic soil-dwelling organism with a wide variety of metabolic capabilities that fixes the nitrogen from the atmosphere and does not enter into symbioses with plants.

Azotobacter has evolved a number of physiological mechanisms to allow it to fix nitrogen aerobically despite the inherent oxygen-sensitivity of nitrogenase. It has uniquely high rates of respiration coupled with specific cytochromes to ensure that nitrogenase experiences an essentially anoxic environment despite the fact that energy is being derived from aerobic metabolism.
It can also synthesise a protective 2Fe-2S protein which can bind to nitrogenase in conditions of oxygen stress to form an oxygen-stable complex that is inactive but protected from damage.

Azotobacter vinelandii is a micro organism that can fix atmospheric nitrogen into the soil by converting it to ammonia. Micro biology of Azotobacter in the nitrogen fixation process.
It is capable of synthesising the molybdenum-containing nitrogenase enzyme that typifies most diazotrophs (Like Klebsiella pneumoniae and Rhizobium leguminosarum ). Also process two additional alternative nitrogenases; one in which vanadium replaces molybdenum and a second which contains neither transition metal but only iron. This ability to carry out the chemistry of nitrogen reduction at sites that do not contain molybdenum is of particular importance to chemists and biochemists investigating the mechanism of biological nitrogen fixation. The alternative nitrogenases are encoded by distinct structural genes, vnfHDGK and anfHDGK : the vnfG and anfG genes encoding an extra small subunit not found in molybdenum nitrogenase. However many of the same ancillary genes e.g. nifUSVWZ and nifM are used in biosynthesis of all three enzymes. Synthesis of the alternative nitrogenases is regulated by availability of the appropriate metals i.e. molybdenum or vanadium, and expression of each set of genes is controlled by a specific regulatory protein, the products of the nifA , vnfA and anfA genes. Interest in this regulation has focussed research on the mechanisms whereby Azotobacter transports molybdate into the cell and distinguishes it from similar molecules such as sulphate. This has led to the dissection of the molybdate transport genes, modEABC and modG of Azotobacter that have homologues in many other bacteria.

Azotobacter Uses - Azotobacter vinelandii - Uses and Application It is a great source of nitrogen to meet the needs of crops because also has the capabilities to cause a rejuvenation of soil microbiology to tap out the biological fixation of nitrogen. - Azotobacter - Are free living bacteria which utilize atmospheric nitrogen gas for synthesis and then mineralised in the soil after the death of Azotobacter thereby contributing towards the nitrogen availability of the crop plants. Azotobacter is a free-living nitrogen-fixing bacterium, which is used as a biofertilizer in the cultivation of most crops.
All 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%.

Bacteria Features: -
Azotobacter are tolerant to high salts.
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, growth promoting substances, fungi static substances.
Azotobacter is less effective in soils with poor organic matter content.
It improves seed germination and plant growth
It thrives even in alkaline soils.
Use as Fertilizer It is recommended the application of the Azotobacter biofertilizer at the rate of 20 kg per hectare per year and this assures a substantial saving of nitrogen fertilizer (about 50 % reduction in N requirement). This indirectly reduces the costs involved in the silkworm rearing aspects as well as the soil health is improved by increased microbial activity. The process of mass multiplication of the microbial culture and packing in polythene bags along with a carrier (preferably lignite or coal) has been standardized.
The process ensures a minimum of 108 viable cells of Azotobacter per gram of carrier on dry weight basis within 15 days of preparation and 107 viable cells before the expiry date (As per the BIS norms). The shelf life of the biofertilizer is six months from date of manufacture. The Process requires locally available plant, machinery and raw materials for manufacture. The mass culture can be done by shake-flask or by using a fermentor depending on the investment capability. The culture is then mixed with the carrier material under hygienic conditions and packed immediately. The batch number and expiry date are immediately affixed on each bag. Normally, packets are available in 250 g, 500 g, 1.0 kg and 5.0 kg deno minations.

How to apply bio-fertilizer ?

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 a 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.

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 inoculum. These seedlings are then transplanted e.g. Tomato, Rice, Onion, Cole, Crops, flowers.

Self inoculation or tubez 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.

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

Use 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, 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.

Bio Fertilizer Storage - To storage bio fertilizer periods for biofertilizer must check the conditions, such temperature and humidity.