BIOFERTILIZERS

Done by C.Deepika,
P8MB102
II M.Sc Applied Microbiology
Introduction

 Commercial preparation of microorganisms added to the soil to enrich the soil

fertility is called biofertilizers.
 They are often known as microbial fertilizers or microbial inoculants.
 A biofertilizer may contain nitrogen fixing microbes or phosphate solubilizing
microbes or spores of VAM fungi.
 It is supplied to the soil either by seed treatment or by spreading it over the
field during cultivation.
 Biofertilizers reduce the use of chemical fertilizers in agriculture and cost of
production.
 the nitrogen biofertilizer mah have nitrogen fixing bacteria or blue-green
algae.
 The nitrogen fixing bacteria include Rhizobium, Azospirillum, Azotobacter,
Azotococcus, etc. blue-green algae such as Anabaena, Aulosira, Nostoc,
Plectonema and Tolypothrix are used as nitrogen biofertilizers.
 Preparation of phosphate solubilizing bacteria such as Bacillus megaterium,
B.subtilis, Xanthomonas and Pseudomonas, are used as phosphate
biofertilizers.
 The spores of VAM fungi like Glomus, Gigaspora, Acaulospora, Sclerocystis and
Endogone are used as VAM biofertilizers.
Benefits of using biofertilizers
Biofertilizers reduce the use of chemical fertilizers in agriculture.

They never cause pollution in air, water and land.

They secrete plant growth hormones to increase the plant growth.

They reduce the attack by soil-borne pathogens.

They improve the quality of soil for more productivity.

They can be mass produced by using renewable wastes.

No special care is required while using biofertilizers.

The farmers themselves can grow biofertilizers and Azolla biofertilizer in their own
lands.
Application methods

There are four types of methods for application of biofertilizers. They are:
 Seed treatment
 Seedling treatment
 Set treatment
 Soil treatment
Seed treatment

 For inoculation of cereals like rice, wheat, sorghum, maize,etc and oilseeds like groundnut,
sunflower, safflower, mustard; pulses like cowpea, green gram, black gram, soybean, etc.,
seed treatment of biofertilizers is recommended. One packet(200g) is sufficient to treat 10-12
kg seed. On this basis, the dose of biofertilizer per acre can be worked out, based on the seed
rate.
Method:
1. Keep the seeds required for sowing one acre in a heap on a clean cemented floor or gunny
bag.
2. Prepare culture suspensions by mixing 1 packet(200g) biofertilizer in approximately 400ml
water (1:2).
3. Sprinkle the culture suspension on the heap of the seeds and mix by hand so that thin coating
is uniformly applied to the seeds.
4. Spread the seeds under shade for sometime for drying and then sow.
5. In place of water, rice glue(kanji) can also be used for better results.
Seedling treatment

 This method is recommended for crops like paddy, tobacco, tomato, chilly, onion,
cabbage, cauliflower,etc.
 Method:
 Prepare the suspension by mixing 1kg of biofertilizer culture in 10-15 litres of
water.
 Get the seedlings required for one acre and make small bundles of seedlings.
 Dip the root portion of these seedlings in this suspension for 15-30 mins and
transplant immediately.
 Generally the ratio of inoculant and water is 1:10(approx.) i.e., 1kg biofertilizer in
10kg of water.
 For vegetables like chilly,tomato, cabbage, cauliflower, 1 packet of biofertilizer is
sufficient for 0.1ha (10000m² land)
Set treatment

 This method is generally recommended for treating the sets of sugarcane, cut
pieces of potato and the base of banana suckers.
 Method:
 Prepare culture suspension by mixing 1kg of biofertilizers in 40-50liters of
water.
 The cut pieces of planting material required for sowing one acre are kept
immersed in the suspension for 30 minutes.
 Bring out the cut pieces and dry them in shade for sometime and then plant it.
 After planting the field is irrigated within 24 hours.
 For set treatment, the ratio of biofertilizers and water is approximately 1:50.
soil treatment

 This method vary crop to crop depending on its duration. Generaly for a short
duration(6 months or less) crop, 10-15 packets of biofertilizer are mixed with
40-60kg of well decomposed cattle manure or with 40-60kg of soil for one
acre of land. The mixture of biofertilizer and cattle manure/soil are sprinkled
with water and then broadcasted into the soil at the time of sowing or at the
time of irrigation for standing crops.
 For long duration crops(perennial crops), 20-30 packets of biofertilizer are
mixed with 80-120kg cattle manure or soil per acre.
Rhizobium

 Rhizobium is a gram negative, aerobic, rod shaped bacterium.

 It is a soil bacterium present in large numbers in rhizosphere of legume roots.
 Beijerinck first isolated and cultivate a microorganism from the roots of
legumes in 1888 and he named this as Bacillus radicola and latter modified as
Rhizobium.
 Rhizobium invades roots of legumes and forms nodules on the roots.
 Inside the root nodule, the bacteria exist in various pleomorphic forms called
bacterioids.
 The bacterioids fix the atmospheric nitrogen into ammonia.
 They provide the fixed nitrogen for plant’s use and draw nourishments from
the root cells. This type of association is called symbiosis.
 Slow growing Rhizobium is known as “Bradyrhizobium”.
 Rhizobium can fix 50-300kg/ha.
Production:
Phosphate solubilisers

 Though most soils contain appreciable amounts of inorganic P, most of it

being insoluble forms, cannot be utilized by crops unless they are solubilzied.
 Soils also contain organic P that could not be utilized by plants only when it is
mineralized.
 Phosphate solubilizing microorganisms not only able to solubilize insoluble
forms of inorganic P but are also capable to mineralize organic forms of P,
thus improving the availability of native soil P making their P available to
plants.
 PSM can also solubilize P from rock phosphate (RP), slag or bone meal making
their P available to plants.
 They secrete organic acids such as formic acid, acetic acit, lactic acid, succinic acid, propionic
acid and hydroxy acids to solubilize the bound phosphates in the soils.
 The phosphate biofertilizers can save 30-50kg of super phosphate per hectare and increase the
yield upto 200-500kg/ha.
The following are important phosphate biofertilizers:
 Bacillus megaterium
 B. subtilis
 B. striata
 Pseudomonas striata
 Pseudomonas liquifacians
 Aspergillus flavus
 A. awamori
 A. fumigatus
 A. niger,etc
Isolation of phosphate solubilizers:
 Pikovskaias’s medium is a specific medium for the phosphate solubilizing microbes. The
formulated pikovskaia’s medium is treated with 0.5% gum Arabic solution and distributed into
petri dishes.
 One gram of rhizosphere soil is suspended in 10ml of distilled water and the tube is shaken well.
Serial dilutions are made in distilled water and are inoculated into the petri dishes. The petri
dishes are incubated at 28±2°C for two weeks.
 Phosphobacteria grow alone by solubilizing the bound phosphate present in Calcium phosphate
and form clear zones around them. But the other microbes starve for phosphorous and fail to
grow. The microbial colonies are picked up and Pikovskaia’s medium in a flask to form a starter
culture.
Field application:
 The phosphate biofertilizer is diluted with water and then treated with gum Arabic and calcium
carbonate to make slurry.
 Seeds to be sown are soaked in the slurry for some time and dried in shade. By this process, the
inoculant is made to form pellet on the seed. The seeds are then sown in the main field.
Uses:
 Suits for all types of crops.
 In groundnut, B. megaterium together with Rhizobium boosts the yield upto 28%.
 Aspergillus awamori increases the groundnut yield upto 25%.
 By providing 10kg of phosphobacteria per hectare, phosphate fertilizers can be avoided for
sugarcane.
 Co inoculation of B. megaterium and P. striate with Azotobacter increases the yield in rice,
sorghum and cotton.
VAM- Vesicular Arbuscular Mycorrhizal
fungi
 Mycorrhiza is the product of an association between a fungus and plant root. Vesicular-arbuscular
mycorrhiza (VAM) is formed by the symbiotic association between certain phycomycetous fungi and
angiosperm roots.
 The mycelia are aseptate or septate.
  The arbuscules are the most characteristic structures, formed intracellularly and probably having an
absorptive function.
 The vesicles are terminal swellings of hyphae formed inter and intracellularly having a storage function.
 There are six genera of fungi belonging to Endogonaceae which have been shown to form mycorrhizal
associations: Glomus, Gigaspora, Acaulospora, Entrophospora Sclerocystis and Scutellospora.
 These are mainly identified by their characteristic spores and sporocarps which are formed mostly in
the soil surrounding the roots and rarely inside the roots.
 The identification of VAM fungi directly from roots has been difficult.
 One of the striking features of VAM fungi is their very wide host range which includes angiosperm
species belonging to almost all the families. Even the roots of some aquatic plants are colonized by VAM
fungi.
 VA mycorrhizal infection develops from either spores, root fragments, or
hyphal networks in close proximity to host plant roots. Fungal spores, in
general, are spherical reproductive structures which remain dormant during
periods of unfavorable conditions. 
 These fungi are scientifically well known for their ability to uptake and
transport mineral nutrients from the soil directly into host plant roots. 
 Helps to absorb and mobilize primarily phosphorous along with other
important macro and micro elements and water.
 Compatible with other Biofertilizer of different categories (N, P, K and S -
fixers and solubulisers).
 Gives protection to the plants from pathogenic fungi and nematodes causing
damage to the roots.
 Environment friendly and does not affect ecological balance.
Benefits:
 Better and more balanced growth
 Healthier and more dense root system with a largely improved ability to get
nutrients from soil
 Richer and denser flowers and fruit
 Significantly lower need of watering and fertilizing
 Higher resistance to drought
 Decreased stress during transplanting
 Stabilization of surface
 Protection against some soil pathogenes and diseases
Azolla
 Azolla (Azolla sp.) is an aquatic fern consisting of a short, branched, floating
stem, bearing roots which hang down in the water.
 The leaves are alternately arranged, each consisting of a thick aerial dorsal
lobe containing green chlorophyll and a slightly larger thin, colourless, floating
ventral lobe. 
 Under some conditions, an anthocyanin pigment gives the fern a reddish-
brown colour.
 The most remarkable characteristic of azolla is its symbiotic relationship with
the nitrogen-fixing blue-green alga (cyanobacterium) Anabaena azollae.
 The fern provides nutrients and a protective cavity in each leaf
to Anabaena colonies in exchange for fixed atmospheric nitrogen and possibly
other growth-promoting substances 
They have:
 the capacity to fix atmospheric nitrogen,
 a very high productivity in the right environment,
 a high protein content,
 an herbicide effect and the capacity to decrease N-fertilizer volatilization.

 Water is the fundamental requirement for the growth and multiplication of Azolla as the plant is
extremely sensitive to lack of water.
 Although Azolla can grow on wet mud surfaces or wet pit litters, it prefers a free-floating state.
 Azolla can survive within a pH range of 3.5 to 10, but optimum growth is observed in the range
of 4.5 to 7.
 Optimum temperature for growth and nitrogen fixation depend on the species. It is usually in
the 20-30°C range, though Azolla mexicana is more tolerant of temperatures over 30°C.
 Outside this range, growth decreases until the plant begins to die at temperatures below 5°C
and above 45°C. Azolla filiculoides can withstand temperatures as low as -5°C without apparent
harm. 
 Azolla can be fed to livestock either in a fresh or dried form. It can be given
directly or mixed with concentrates to cattle, poultry, sheep, goats, pigs and
rabbits.
 Azolla is usually dried in the shade and stored dry, for example in a plastic
bin, for later use.
 Production of azolla for green manure is done according to 3 systems. It can
be grown as a monocrop and then incorporated as foundation manure before
the rice is transplanted, or transported to another site for use on upland
crops. Monocrop azolla has been used in China and Vietnam during winter and
spring to produce nitrogen for the spring rice crop. 
 Azolla can also be grown as an intercrop, and used as a top dressing manure
after the rice is transplanted. This is done in places where there is no time
available in the cropping system for growing azolla as a monocrop. 
 It can also be grown both as a monocrop and an intercrop. This technique is
designed to grow azolla before planting the rice crop, allowing production of
added nitrogen for the crop through cultivation of intercropped azolla (
benefits

 Its high content in proteins, essential amino acids, vitamins (vitamin A,

vitamin B12, Beta Carotene), growth promoter intermediaries and minerals.
 Its ability to proliferate without inorganic nitrogen fertilization.
 Its high rate of growth in water without the need to displace existing crops or
natural ecological systems.