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
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.
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.
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.
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
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
produces some substances which check the plant pathogens such as Alternaria, Fusarium and
Helminthosporium. Hence Azotobacter also acts as a biological control agent.
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.
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
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;
- 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
- 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.
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.
- Field Test: Strains found
efficient under glass house conditions are required to undergo field test which is most
important test from the farmers 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.
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.