a 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.

b. Production:

1. 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 litre 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.

2. 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 is prepared. Depending upon the demand and supply suitable fermenter is selected.

Other production method is the 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 flasks 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.