2. Biofertilizers are defined as preparations containing living cells or latent cells of efficient strains of microorganisms
that help crop plants’ uptake of nutrients by their interactions in the rhizosphere when applied through seed or to soil.
Biofertilizer from N2 fixing bacteria (both free living and symbiotic)come in three forms: liquid, solid and
lyophilized.
For liquid and lyophilized ones, only solution medium is used, but for solid form, carriers such as peat, activated
charcoal and chicken dung are needed.
Biofertilizers
Examples of nitrogen-fixing bacteria
Free living
1. Aerobic (Azotobacter, Beijerinckia, Klebsiella (some), Cyanobacteria (some)* )
2. Anaerobic (Winogradsky column) (Clostridium (some), Desulfovibrio, Purple sulphur bacteria*, Purple non-
sulphur bacteria*, Green sulphur bacteria*
Symbiotic with plants
1. Legumes (Rhizobium)
2. Other plants (Azospirillum, Frankia)
* denotes a photosynthetic bacterium
3. The genus, Azotobacter chroococcum, was discovered and described in 1901 by the Dutch microbiologist and botanist
Martinus Beijerinck.
1. Azotobacter are found in neutral and alkaline soils.
2. Azotobacter is free living, gram-negative, motile, pleomorphic aerobic bacterium.
3. They are oval or spherical that form thick-walled cysts and may produce large quantities of capsular slime.
4. Azotobacters are the most intensively investigated heterotrophic group possessing the highest respiratory rates.
5. Members of these genera are mesophilic, which require optimum temperature of about 30ºc.
6. There are some microorganism which establish symbiotic relationships with different parts of plants and may
develop special structures as the site of nitrogen fixation.
7. Non nodule forming diazotrophs for example , Azotobacter, Beijerinckia 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.
4. 8. Apart from being a model organism, it respire aerobically which uses the organic matter present in soil to fix
nitrogen asymbiotically and receiving energy from redox reactions, using organic compounds as electron donors.
9. Azotobacter can use a variety of carbohydrates, alcohols and salts of organic acids as sources of carbon and can fix
at least 10 ug of nitrogen per gram of glucose consumed so used by humans for the production of biofertilizers,
food additives and some biopolymers.
10. Azotobacter, a free living microbe, acts as plant growth promoting rhizobacteria (PGPR) in the rhizosphere of
almost all crops.
11. PGPR is a group of beneficial plant bacteria, as potentially useful for stimulating plant growth and increasing crop
yields has evolved over the past several years to where today researchers are able to repeatedly use them
successfully in field experiments.
12. Such PGPRs also fix nitrogen for non-legume crops like wheat, rice, sunflower, sugarcane, cauliflower, cotton,
maize and sorghum which helps in saving 20-40kg chemical nitrogen i.e.45-90 kg urea per hectare.
13. Yield of several non-legume was increased by PGPRs symbionts through plant growth promoting substances, it
helps in root expansion, improve uptake of plant nutrients.
5. Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Pseudomonadales
Family: Pseudomonadaceae/Azoto
bacteraceae
(unranked): Azotobacter group
Genus: Azotobacter
1. Azotobacter species are ubiquitous in neutral and
weakly basic soils, but not acidic soils.
2. They are also found in the Arctic and Antarctic soils, despite
the cold climate, short growing season, and relatively low
pH values of these soils. In dry soils, Azotobacter can
survive in the form of cysts for up to 24 years.
3. Representatives of the genus Azotobacter are also found in
aquatic habitats, including fresh water and brackish
marshes.
4. Several members are associated with plants and are found in
the rhizosphere, having certain relationships with the
plants. Some strains are also found in the cocoons of the
earthworm Eisenia fetida.[37]
Distribution
6. 1. The genus Azotobacter are relatively large
for bacteria (2–4 μm in diameter). They are
usually oval, but may take various forms
from rods to spheres.
2. In microscopic preparations, the cells can be
dispersed or form irregular clusters or
occasionally chains of varying lengths.
3. The cells are mobile due to the
numerous flagella. Later, the cells lose their
mobility, become almost spherical, and
produce a thick layer of mucus, forming the
cell capsule. The shape of the cell is affected
by the amino acid glycine, which is present in
the nutrient medium peptone.
4. Under magnification, the cells show
inclusions, some of which are colored.The
colored grains are composed of volutin,
whereas the colorless inclusions are drops of
fat, which act as energy reserves.
Azotobacter species cells
7. 1. Azotobacter is aerobic, receiving energy from organic compounds as electron donors, and can use a variety of
carbohydrates, alcohols, and salts of organic acids as sources of carbon.
2. Azotobacter can fix at least 10 μg of N2/ gram of glucose consumed.
3. Nitrogen fixation requires molybdenum ions, but they can be partially or completely replaced by vanadium ions. If
atmospheric nitrogen is not fixed, the source of nitrogen can alternatively be nitrates, ammonium ions, or amino
acids.
4. The optimal pH for the growth and nitrogen fixation is 7.0–7.5.
5. Azotobacter can also grow mixotrophically, in a nitrogen-free medium containing mannose; this growth mode is
hydrogen-dependent. Hydrogen is available in the soil, thus this growth mode may occur in nature.
6. Azotobacter produces flat, slimy, paste-like colonies with a diameter of 5–10 mm, which may form films in liquid
nutrient media.
7. The colonies can be dark-brown, green, or other colors, or may be colorless, depending on the species. The growth
is favored at a temperature of 20–30°C.
8. Bacteria of the genus Azotobacter are also known to form intracellular inclusions of polyhydroxyalkanoates under
certain environmental conditions (e.g. lack of elements such as phosphorus, nitrogen, or oxygen combined with an
excessive supply of carbon sources).
9. Pigments
Azotobacter produces pigments. For example, Azotobacter chroococcum forms a dark-brown water-soluble
pigment melanin. This process occurs at high levels of metabolism during the fixation of nitrogen, and is thought to
protect the nitrogenase system from oxygen. Other Azotobacter species produce pigments from yellow-green to purple
colors, including a green pigment which fluoresces with a yellow-green light and a pigment with blue-white
fluorescence.
10. Media is used
Ashby’s Media/Waksman Media/ N2 free mannitol Media/ N2 free glucose Media.
Sample
Soil from neutral/alkaline field.
Isolation Procedure
Dilution streak method/Sprade plate method/ Enrichment culture technique
Dilution streak method/Sprade plate method
1. A 10 g largest sphere soil is mixed well with hundred ml of distilled water and left undisturbed for some
time to have a clear suspension.
2. the suspension is serially diluted and inoculated into Prati dishes containing above said media.
3. After incubation at optimum temperature 30 degree centigrade a soft milky mucoid colonies were grown on
the surface of plate.
4. The pure culture of Azotobacter was subcultured in a culture tube with above state media.
Procedure for isolation of Azotobacter
11.
12. 1. Nitrogen free glucose/mannitol medium (50
ml) was inoculate with a sample of garden soil
(0.5g).
2. Rich garden soil was neutral or alkaline. with a
small spatula put about 1 gm of soil into the
bottle of medium. The container was kept in
continuous shake and incubate in optimum
temp 30ºC for 4 to 7 days.
3. Since the organisms are strict aerobes, it is best
to incubate the bottle horizontally to provide
maximum surface exposure to air.
4. After incubation broth was taken for further
culture for pure culture.
5. The pure culture of Azotobacter was
subcultured in a culture tube with above state
media.
Enrichment culture technique
13. Mass culture of Azotobacter
1. For mass culture, 1 liter of starter culture for
100 litre of media is transfered to
sterilizedized above said media in a
bioreactor
2. Azotobactor attainded the concentration of
108 cells/ml the inoculant (broth) is
harvested to make career based inoculant.
3. A suitable carrier is ground into fine powder
then the inoculant is allowed for curing for a
week
4. Then packed in polythene bags and
dispatched
