Bio-fertilizers And Its Application In Aquaculture

BIOFERTILIZERS Dr. Subhendu Datta Sr. Scientist CIFE, Kolkata Centre

Background: Why bio-fertilizers?


With the introduction of green revolution technologies the modern agriculture is getting more and more dependent upon the steady supply of synthetic inputs (mainly fertilizers) which are products of fossil fuel (coal+ petroleum).




Excessive dependence of modern agriculture and the supply of these synthetic inputs and the adverse effects being noticed due to their excessive and imbalanced use has compelled the scientific fraternity to look for alternatives.

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(i). Availability and cost of commercial fertilizers


Demand is much higher then the availability.




It is estimated that by 2020, to achieve the targeted production of 321 million tones of food grain, the requirement of nutrient will be 28.8 million tones, while their availability will be only 21.6 million tones being a deficit of about 7.2 million tones.




Increasing costs are getting unaffordable by small and marginal farmers.

(ii) Effect of Chemical fertilizers in soil and environment


Excessive and imbalanced use of chemical fertilizers has adversely affected the soil causing decrease in organic carbon, reduction in microbial flora and fauna of soil, increasing acidity and alkalinity and hardening of soil.




Moreover, excessive use of nitrogenous and phosphatic fertilizers are contaminating water bodies (eutrophication) thus affecting fish fauna and causing health hazards for human beings and animals.




Production of chemical fertilizers adds to the pollution.

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To overcome the deficit in nutrient supply and to overcome the adverse effects of chemical cultivation, it is suggested that efforts should be made to exploit all the available resources of nutrients under the theme of integrated nutrient management.



Under this approach the best available option lies in the complimentary use of Biofertilizers, Biofertilizers, organic manures in suitable combination of chemical fertilizers.

What are bio-fertilizers 

“Biofertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host Plant [Vessey, J.K. (2003). Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255: 571-586].



This definition separates biofertilizer from organic manure.



The latter contains organic compounds which directly, or by their decay, increase soil fertility.

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 Likewise the term biofertilizer should not be used interchangeably with the terms, green manure  Not all plant growth promoting rhizobacteria (PGPR) can be considered biofertilizers.  Bacteria that promote plant growth by control of deleterious organisms are biopesticides, but not biofertilizers.  Similarly bacteria can enhance plant growth by producing phytohormones and are regarded as bioenhancers, not biofertilizer.

 The importance of cyanobacterial biofertilizers was recognized as early as 1939. Since then good deal of literature on these aspects has appeared.  Although biofertilizers are now being used in agriculture fields, it can equivocally be stated that biofertilizers may also be used in fish culture practices.  Field studies shown that about 30 kg N/ha can be saved by the use of cyanobacterial biofertilizers.

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Indian Definition - Biofertilizers are ready to use live formulates of such beneficial microorganisms which on application to seed, root or soil mobilize the availability of nutrients by their biological activity in particular, and help build up the micromicro-flora and in turn the soil health in general. Leguminous oilseed Crops: Soybeans and peanut Leguminous pulses: Arhar, Letils, Mung, Urad, Pea, Cowpea, Gram,

Bio-fertilizer pack

Benefits of biofertilizers Increase crop yield by 2020-30% Replace chemical N & P by 25 %  Stimulate plant growth  Activate soil biologically  Restore natural fertility  Increases soil organic matter and maintains a good soil texture.  Environmentally friendly - Don’t pollute the environment  Cheaper than synthetic fertilizers  Believed to have growth promoting substances.  



Provide protection against drought and some soil borne diseases

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Types of Biofertilizers Most biofertilizers belong to one of these two categories: (a). Nitrogen fixing (a). Phosphate solubilising. For Nitrogen:  Rhizobium for legume crops  Azotobacter/Azospirillum for non legume crops  Acetobacter for sugarcane only  BGA and Azolla for low land paddy & aquaulture For Phosphorous  Phosphatika for all crops to be applied with Rhizobium, Rhizobium, Azotobacter, Azotobacter, Azospirillum and Acetobacter For enriched compost  Cellulolytic fungal culture  Phosphotika and Azotobacter culture

METHOD OF APPLICATION 

Seed treatment :



Seedling root dip:



Soil treatment:

Suspend 200 gm N biofertilizer and 200 gms Phosphotika in 300300-400 ml of water and mix thoroughly. Mix this paste with 10 kg seeds & dry in shade. Sow immediately. For vegetables 1 kg each of two biofertilisers be mixed in sufficient quantity of water. Dip the roots of seedlings in this suspension for 3030-40 min before transplanting. For paddy make a bed in the field and fill it with water. Mix biofertilisers in water and dip the roots of seedlings for 88-10 hrs. Mix 4 kg each of biofertilisers in 200 kg of compost and leave it overnight. Apply this mixture in the soil at the time of sowing or planting. planting. In plantation crops apply this mixture near root zone and cover with soil.

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Nitrogen fixing biofertilizers  Free-living or symbiotic bacteria and blue-green algae (Cyanobacteria) fix atmospheric gaseous nitrogen as ammonia and release it, increasing the fertility of soil and water.  These include Rhizobium, Azotobacter, Acetobacter, Azospirillum, Blue Green Algae (BGA) and Azolla.  While Rhizobium, A. azollae requires symbiotic association to fix nitrogen, others can fix nitrogen independently.  Anabaena azollae living in leaf cavities of Azolla (aquatic fern) are very efficient nitrogen fixers, and contribute about 500 kg N/ha/year.

Phosphate solubilising biofertilizers 

Phosphate solubilising micro-organisms (PSM) secrete organic acids which enhance the uptake of phosphorus by plants by dissolving rock phosphate and tricalcium phosphates.



PSMs (e.g. Phosphatika) Phosphatika) are particularly valuable as they are not crop specific and can benefit all crops.

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Composting bio-fertilizers o Compositing biofertilizers are used for hastening the process of composting and for enriching its nutrient value. o Composting process is enhanced by enzymes secreted by microorganisms (Cellulolytic fungal culture) to hydrolyse pectins, xylans, hemicellulose, cellulose releasing beneficial micronutrients for the plants.

Nitrogen fixing biofertilizers Rhizobia (Soil bacteria of the genus Rhizobium) produces root nodules in legumes. Legume/Rhizobium Nodules are Red. This is due to the production of Leghaemoglobin which sequesters oxygen. This helps to create a low oxygen environment. The enzyme which fixes nitrogen (Nitrogenase) needs an anaerobic environment.

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Another microsymbiont with nitrogen fixing capacity the principal genus of microbes is Frankia.



Morphology of Frankia is similar to that of actinomycetes and produces nodules in woody non-legumes, like Alnus, Casuarina, Myrica etc. Many of these are "pioneer" species which colonize barren sites.



These nodules fix more atmospheric nitrogen than legume/Rhizobium Nodules..



Root nodules of Alnus

Some of these are alien species like Myrica Myrica,, grows much faster than native competitors, and they alter the soil nitrogen levels levels markedly compared to native stands.

Azolla-Anabaena azollae relationship o

An Azolla plant floating on the surface of the water is roughly triangular or circular in shape and rarely exceeds 3–4cm (except in the species Azolla nilotica).

o

The cyanobacterium Anabaena azollae occurs as filaments located on the stem apexes of Azolla (aquatic fern) and inside the leaf cavities in Symbiotic association, association which are inoculated during their formation with some Anabaena from the apex.

Reference: C. Van Hove and A. Lejeune (2002). Biology And Environment: Proceedings of the Royal Irish Academy, Vol. 102B (1): 23–26.

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The Azolla–A. azollae association can develop on a medium devoid of nitrogen compounds because of the ability of A. azollae to reduce N2 to NH3. Some of the ammonia is supplied to the fern, and the fern supplies the cyanobacterium with photosynthetic assimilates.

Morphology of Azolla stem (longitudinal section). 1. Stem; 2. Stem apex; 3. Apical Anabaena colony without heterocysts; 4. Other bacteria; 5. Leaf primordium; 6. Young leaf; 7. Branched hair; 8. Single hair; 9. Upper leaf lobe; 10. Lower leaf lobe; 11. Leaf cavity (showing a central gaseous region and a peripheral mucilaginous region); 12. Involucre; 13. Indusia; 14. Microsporocarp; 15. Microsporangia; 16. Megasporocarp; 17. Megasporangium; 18. Akinetes of Anabaena; 19. Vegetative cell of Anabaena; 20. Heterocyst.

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Importance of Azolla– Azolla A. azollae symbiosis 

The Azolla–A. azollae symbiosis is has long been used by farmers, mainly in Asia, as feed for their animals and as green manure.



Azolla is one of the most nutritive aquatic plants, owing to its high crude protein and carotenoid contents (anthocyanine ) and generally good aminoacid profile.



It can be incorporated into the feed of fish, pigs, poultry, rabbits and even humans.



A number of laboratory and field studies have shown beyond any doubt the beneficial effect of Azolla as an organic nitrogen fertiliser, mainly in terms of increasing rice grain yield.



The presence of an Azolla mat on the surface of the water body has been shown to significantly reduce weed development, limit evapotranspiration, reduce volatilisation of applied N fertilisers and purify water.



Recent research has focused on the use of Azolla in integrated farming systems, mainly rice– fish–Azolla and pig–poultry–fish–Azolla.

Azotobacter species are free-living (mostly root associated), aerobically nitrogen fixing bacteria. Nostoc, Calothrix, Gloeotrichia, Stigonema, etc are free-living aerobically nitrogen fixing Cyanobacteria. In addition, Vesicular Arbuscular Mycorhizae (VAM fungi) are free-living soil forms that increase nutrient uptake (specially by converting organic phosphorus into inorganic phosphorus), plant growth, nodulation and nitrogen fixation in legumes.

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 In coastal areas of some countries, seaweeds are also used as biofertilizers.  However, all these life forms may be grown artificially and inoculated in seed, root or soil as biofertilizer.  The nitrogen fixers releases nitrogen during their life time and also add other elements after their death and decay, essential for the growth of crops.

Biofertilizers generally used in Aquaculture 1. Azolla 2. Phosphatase bacteria 3. Phosphate solubilizing bacteria 4. Nitrogen fixing bacteria 5. Nitrogen fixing cyanobacteria 6. Enriched compost

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What is Azolla?  Azolla is a dichotomously branched (dichotomy is a mode of branching by repeated bifurcation) free floating aquatic fern which is naturally available mostly on moist soils, ditches marshy ponds and is widely distributed in tropical belt of India.  The shape of Indian species is typically triangular measuring about 1.5 to 3.0 cm in length 1 to 2 cm in breadth.

1. Azolla… Roots emanating from growing branches remained suspended in water. The dorsal lobe which remains exposed to air is having a specific cavity containing its symbiotic partner, a Blue Green Algae (BGA), the Anabaena Azolae. The fern is capable of fixing atmospheric nitrogen in the soil in the form of NH4+ and becomes available as a soluble nitrogen for the wet land rice crop, which is the major cereal for the people of the North East. Owing to the poor economic conditions of the farmers of the North Eastern States, rice crop is mostly grown under natural soil fertility with minimum inputs and amelioratives.

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Azolla…  But for taking a good crop of rice, judicious application of nutrients is necessary.  Besides, this, the farmers of the state of Meghalaya and other north eastern states have apathy in using chemical fertilizers in crop production.  For sustainable crop production, there is a practice to supply some quantity of nutrients through organic manure, viz; FYM and composted plant residues and bio-fertilizers.

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Azolla… In the context of depletion of soil fertility and high prices of chemical fertilizer, it has become imperative to use biofertilizer which is a cheaper and renewable source of low cost plant nutrient and playing a major role in Integrated Plant Nutrient Supply System. Use of Azolla fern as a bio-fertilizer is advocated to minimize the dependency of chemical fertilizer. Azolla supplements nitrogen to rice crop by fixing atmospheric nitrogen in the soil for crop growth, crop production and maintain soil fertility.

Economic Value On dry weight basis Azolla contains the following chemical compositions: Nitrogen Phosphorous Potassium Calcium Magnesium Manganese Iron Crude Fat Sugar Starch Chlorophyll Ash

: : : : : : : : : : : :

5.0 % 0.5 % 2.0-4.5 % 0.1-1.0 % 0.65 % 0.16 % 0.26 % 3.0-3.3 % 3.4-3.5 % 6.5 % 0.34-0.55 % 10.0 %

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Classification (Taxonomy)     

Class Order Family Genus Sub Genus

: : : : :

Pteridophyta Salvinales Azollaceae/Salvinaceae Azolla Eu-Azolla

Common Names: Azolla, water velvet, mosquito fern

Different species of Azolla

A. caroliniana

A. mexicana

A. Microphylla

A. Pinnata (SE Asia)

A. japonica

A. filiculoides

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Adaptability Azolla Caroliniana, is identified as a cold tolerant species and survives well even at very low winter temperature of 5ºC during the months of December to February in mid hills of Meghalaya. Azolla pinnata, is a local isolate found widely in the entire North Eastern Region, but does not survive under mid hills of Meghalaya. However, Azolla caroliniana, has shown its adaptability in hills and other similar locations.



Azolla caroliniana, can be preserved in shallow pond having 15 cm of standing water and by providing shade 10-15 cm above the pond water surface through weeds or paddy straw.



For raising Azolla inoculum a pond size of 3 m x 2 m x 1 m is most desirable.



Under such weed or straw mulch cover, the Azolla multiplies rapidly and inoculum will be ready within a period of 20-25 days for further releasing in the main multiplication ponds on the onset of monsoon in the month of April-May.

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How to grow Azolla? 

Azolla can easily be cultivated separately for periodic application in fish ponds.



The system of cultivation involves a network of earthen raceways (20X3.0X0.6m) with water supply and drainage facilities.



In each raceways, 6 kg of Azolla is incorpoprated. 65grams of single super phosphate is added.



Water depth of 10-15 cm to be maintained. Azolla is harvested @25kg/raceway.



It has been estimated that about 1 ton of Azolla can be harvested every week from a water area 650 m2



It has also been reported that application of Azolla @ 20 tons/ha gives 50kg N, 13kg P, 45kg K.

How Azolla fixes atmospheric nitrogen? 

The remarkable feature of Azolla is that its symbiotic relationship with Cyanobacterium (Anabaena azollae) which remained on the dorsal leaf cavity of Azolla.



The fern provides protein substances to Anabaena (BGA).



The BGA then absorbed the atmospheric nitrogen and decomposes it through enzymic activity and converted in to soluble ammonia (NH4+). It can fix 3-7 kg N/ha daily.



It contains 4 % N on a dry-weight basis and is an excellent source of nitrogen fertilizer

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Favourable condition for higher efficacy of Azolla 1. Water 2. pH 3. Salinity 4. Light & Shade 5. Herbicide level 6. Nutrition

Water 



Azolla must grow in water or wet mud to survive. It dies in a few hours if it becomes dry. Water control is critical, especially for for year round production. A water level which allows the roots to touch the soil surface will often cause mineral deficiencies to appear. 10-15 cm fresh current water is necessary in multiplication pond. Temperature: the day/night temperatures ranging between 32ºC and 20ºC have found to be most favorable. The optimum temperature for luxurious growth of Azolla is 25-30ºC.



Wind and wave action can eventually fragment and kill azolla. azolla. Maintaining low water levels and rough plowing can protect azolla from wind. In Africa, hedges, bunds, and mixed culture (with crop crop plants) are used to prevent wind damage. (Von Hove et al., al., 1983).

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pH Soil pH: Azolla grows well in slightly acidic soil having pH 5.2 to 5.8. Water pH: Since azolla lives in water the following refers to the pH of the water only. only. Azolla can survive within a pH range of 3.5 to 10, but optimum growth is in the range of 5 to 7.  The relative growth rate is influenced by a direct relationship between light intensity and pH with the highest growth rates achieved at high pH (9(9-10) and high light intensity and low pH (5(5-6) and low light.  Nitrogen fixation is optimal at pH 6 and 200C.  Deficiency problems can be caused in neutral to alkaline water because because ferric ions precipitate.  There can also be competition between ferrous and manganous ions in water with a neutral pH and reduction in absorption of both iron and managanese with high calcium concentrations.  At pH 4, ferric ions are so readily available that a high concentration concentration of calcium is required to balance the increased absorption of iorn, iorn, otherwise azolla suffers from iron toxicity. (Lumpkin and Plucknett, Plucknett, 1980). 

Salinity Tolerance 

The growth rate of azolla gradually declines as salinity increases.



At about 1.3% salt (33% of sea water) the growth of azolla stops and higher concentrations will kill it.



In rice fields where salt concentration reaches 148014801872 mg/l during the dry season azolla wilts.



Salinity is a factor which should be looked wherever azolla is being considered (Lumpkin and Plucknett, Plucknett, 1980).

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Shade Tolerance & Herbicide Sensitivity Shade Tolerance:





Azolla prefers to grow well under partial shade. As dual cropping Azolla gets partial shade from rice plant and therefore as dual cropping with rice is most successful. Relative growth and nitrogenase activity is at a maximum at 50% of full sunlight although the difference between growth at 50% and 100% sunlight is not that great. Heavy shading is known to decrease azolla growth to almost zero (Lumpkin and Plucknett, Plucknett, 1980).

Herbicide Sensitivity: 

Most rice herbicides kill or inhibit azolla growth. Differences in sensitivity are specific to the different azolla species (Moody and Janiya, Janiya, 1992).

Nutrition Being an N fixing fern Azolla does not require nitrogenous fertilizer for its growth. Phosphorus is the most common limiting factor in the growth of azolla. Fronds placed in P deficient solution decrease or stop growth, become red, and develop curled roots. The minimum P requirement is not known but it thrives on as little as 1.1 mg P/liter. Problems due to iron deficiency or toxicity are fairly frequent. Azolla fronds turn yellow when iron is lacking. Rapid growth is achieved with 1 ppm iron. 

Yield: Azolla produces around 300 tons of green biomaas/ha/year under normal sub tropical climate which is comparable to 800 kg of N (1800 kg of urea).

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Contribution of Azolla Basal application on green Azolla manure @ 10-12 t/ha increases soil nitrogen by 50-60 kg/ha and reduces 30-35 kg of nitrogenous fertilizer requirement of rice crop.  Release of green Azolla twice as dual cropping in rice crop @ 500 kg/ha enriches soil nitrogen by 50 kg/ha and reduces N requirement by 20-30 kg/ha. Use of Azolla increases rice yield by 20 to 30 %. Rice varieties like DR-92, RCPL-1-87-8, Mendri, H-2850 and Manipuri produced more than 30 q/ha rice when grown with Azolla as dual cropping under natural soil fertility.

Under low land condition a thick Azolla mat does not allow the weeds to grow in rice filed thus, Azolla suppresses the weed growth and creates congenial condition for rice production. Azolla reduces evaporation from water surface and increases water use efficiency in rice. Dry Azolla flakes can be used as poultry feed and green Azolla is also a good feed for fishes. Azolla is used in carp ponds at @ 40 ton/ha/yr proving the full complement of nutrients.

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Ridged-field rice-azolla-fish model 

This design was originally developed for swampy areas with the objectives of improving soil properties and increasing rice yield. Later it was, stepwise, integrated with azolla and fish.



Rice is planted on the ridge, azolla as a feed for fish as well as a biofertilizer, and green manure and fish are stocked in the trenches (Pl. see the diagram in the next slide).



In tropical Asia azolla is traditionally cultivated as a green manure for rice in two ways.



One way is to set aside 5-10% of the crop area for year-round production. The cultivated azolla is later added to crop fields as compost.



In the second way, azolla is cultivated in the rice fields and incorporated before and/or after the rice crop and between crops. Ideally azolla is grown several times before rice transplanting.

Rice ridge and fish ditch farming system in China

Source Li Kangmin (1992). Rice-fish farming in China: past, present and future, p. 17-26. In C.R. de la Cruz, C. Lightfoot, B.A. Costa-Pierce, V.R. Carangal and M.P. Bimbao (eds.) Rice fish research and development in Asia. ICLARM Conf. Proc. 24, 457 p.

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2. Phosphatase activity with reference to bacteria and phosphorus in tropical freshwater aquaculture pond systems  The bio-geochemical cycle of phosphorus is significantly influenced by microbes in the aquatic environment.  Phosphorus compounds are decomposed and mineralized by enzymatic complexes such as phosphatases produced by microbes.  Enzymatic catalysis results in the production of orthophosphate, which can be used readily by primary producers.  Even the smallest concentration of phosphate in water has an influence over the production process in aquaculture systems.

Phosphatase activity… Extracellular alkaline phosphatase activity was observed in water and sediment media of aquaculture ponds with different management practices. Heterotrophic bacterial populations as well as phosphatase-producing bacterial populations were higher in sediments compared with water.

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Phosphatase activity…
In the freshwater fish ponds, Bacillus spp. were the dominant forms of bacteria producing phosphatase.
The alkaline phosphatase activity of sediment was always higher than that of water.
The partitioning of extracellular alkaline phosphatase in pond water by a 0.22-µm membrane filter revealed that a proportion was often free rather than cell associated and might have originated as free enzymes released by enriched sediments or by fish or microbes.

Phosphatase activity…
In the case of water, although the dissolved alkaline phosphatase activity was lower than the total alkaline phosphatase activity, the former was nevertheless unimportant, as it constitute about 20% of the 'total' activity.
Free alkaline phosphatase activity shared a negative correlation with the orthophosphate concentration of water, whereas gross alkaline phosphatase activity was positively correlated with the total phosphorus and bacterial population of water.

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Seventeen rhizobacteria isolated from different ecological regions, i.e. Brazil, Indonesia, Mongolia and Pakistan were studied to develop inoculants for wheat, maize and rice. Almost all the bacterial isolates were Gram ‘-’ve, fastgrowing motile rods and utilized a wide range of carbon sources. These isolates produced indole-3-acetic acid at concentrations ranging from 0.8-42.1 µg/mL, irrespective of the region. Isolate 8N-4 from Mongolia produced the highest amount of indole-3-acetic acid (42.1 µg/mL), produced siderophores (0.3 mg/mL) and was the only isolate that solubilize phosphate (188.7 µg P/mL).

3. Nitrogen fixing cyanobacteria

o Nostoc, Calothrix, Gloeotrichia, Stigonema, etc are free-living aerobically nitrogen fixing Cyanobacteria. o In addition, Vesicular Arbuscular Mycorhizae (VAM fungi) are free-living soil forms that increase nutrient uptake (specially by converting organic phosphorus into inorganic phosphorus), plant growth, nodulation and nitrogen fixation in legumes. o Rhizobium producing root nodules in legumes and Anabaena azollae living in leaf cavities of Azolla (aquatic fern) are very efficient nitrogen fixers, and contribute about 500 kg N/ha/year.

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Nitrogen fixing cyanobacteria… Technology for production of BGA biofertiliser with simple nutrient medium in polyhouse has been standardized. A thick mat was found to develop within 5 days. A suitable carrier material has been developed where survival percentage of BGA strains was 85% even after two and half years of storage. The carrier material, Montmorillonite Clay has been found to be very promising for BGA biofertilizers where it is essential to store the inoculums for a longer period. Technology for large scale production of BGA inoculum biofertilizers in Flexi bioreactors was developed at Madurai Kamraj University, Tamilnadu.

4. Nitrogen fixing bacteria
Azotobacter species are free-living (mostly root associated), aerobically nitrogen fixing bacteria.
Rhizobium producing root nodules in legumes and thereby fix nitrogen.

5. Enriched compost 

They are used in cellulolytic fungal culture -Phosphotika and Azotobacter culture.



In fish culture they not yet reported.

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