Here is a list of microorganisms specially used for production of biofertilizers:- 1. Rhizobium 2. Azolla 3. Azospirillum 4. Azotobacter 5. Blue-Green Algae 6. Photosynthetic Bacteria 7. Phosphate Solubilizing 8. Mycorrhizae,
Quite a good number of microorganisms both symbiotic and non-symbiotic are being identified and exploited as potential source of biofertilizers. Attempts are being made to large scale production and mass inoculation of these organisms to the soil.
1. Rhizobium:
The role of legumes and part played by species of Rhizobium in enriching the fertility of soil was scientifically demonstrated only in the latter half of the 19th century. With subsequent understanding of legume – Rhizobium interactions Rhizobium as legume inoculant was first developed and marketed in USA during early part of this century.
During subsequent three decades, work on the methodology of mass cultivation of rhizobia, preparation of carrier based inoculants and application of rhizobia to soil or seeds of legume have been carried out in Australia which has been adopted in Asian, Latin American, African countries with modifications to suit their local needs and conditions.
(i) Cultivation and Mass Production of Rhizobium Inoculant:
Rhizobia are maintained on yeast extract mannitol (YEM) agar medium (Yeast extract-1 g; Mannitol 10 g.; K2HPO4 3.5 g, MgSO4. 7 H2O – 0.2 g, NaCl-0.1 g; agar 15 g, D. water -1 litre pH -6.5-7.0) either by subculturing at frequent intervals or by lyophilization to be reconstituted into agar-based cultures whenever necessary. The primary culture is designated as the Mother Culture. For large scale cultivation YEM liquid medium is employed.
The selection of suitable strains of Rhizobium species dependent upon many criteria, a single strain or more than one strains for a particular cultivar or a group of cultivars or crops. The selected strain is grown for 3-4 days on YEM agar slants depending on the fast or slow growing nature of the strain. The culture is tested for purity by well-known tests and transferred to large flasks containing sterile solid or liquid medium for 4-9 days.
This is called ‘starter culture’ which is transferred to a seed tank fermentor and incubated for 4-9 days. By about the same time, a large quantity of liquid broth is formulated in the fermentor, pH adjusted to 6.5 to 7.0 and sterilized. After cooling to 30°C, inoculum from the seed tank fermentor is transferred aseptically to the production fermentor at the rate of 1 percent by volume. The factors influencing the output of cells are aeration, volume, initial inoculum level, bacterial strain, and temperature and incubation time. The main objective is to attain high populations in the minimum time.
In USA large automatic fermentors are used, whereas in Australia a container such as drum of 10 to 100 litre capacity is used. In India, most manufacturers use indigenously made shakers which can hold many conical flasks to produce inoculants on a small scale. The resulting broth from fermentors is checked for purity according to methods outlined by Vincent. The general method of Rhizobial inoculum production is outlined in Fig. 20.1.
(ii) The Product:
The product is free flowing carrier based preparation containing live cells of specific rhizobia. In the USA diluted broth having rhizobial cell population in excess of 109 cells/ml is blended with the peat carrier so as to bring its final moisture level to 35-40 percent on wet basis.
Azolla:
Can also be grown with rice seedlings after transplantation, then the ferns are buried with hands in the soil and the process is repeated as often as necessary so that the Azolla mat may not cause choking of rice plants causing oxygen starvation.
The final product should have atleast 300 million rhizobia per g of peat. In the process of blending, the broth is sprayed to powdered peat and left for curing. The final product is again milled and packed in polythene sheets where the number of rhizobia multiplies. Finely powdered farmyard manure and charcoal powder are good alternatives to peat.
The expiry date of the product is six months in India. Indian Standards Institution (ISI) is entrusted with the task of quality control. The rhizobial inoculum is applied to seeds by adding gum Arabic (40%) or Carboxymethyl cellulose (20%) is added to inoculum slurry before mixing seeds (Fig. 20.2).
(iii) Benefits of Inoculation:
Trials in farmer’s fields in India have proved a great success and serve to depict the importance of legume inoculation in the improvement of grain legume yields (Table 20.3). There have been many advances in the method of inoculating seeds with rhizobia. Adhesive and seed pelleting agents such as gum Arabic, lime etc. have been advocated to improve the survival of bacteria on seed.
2. Azolla:
Azolla is a free floating aquatic fern plant with branched stem, bilobed leaves and true roots which penetrate water. The dorsal, fleshy chlorophyll containing lobe has an algal symbiont, Anabaena azollae within a cavity which fixes nitrogen. It naturally grows in tropical fresh water ponds and rice fields. The value of Azolla as a biofertilizer in rice cultivation was first demonstrated in North Vietnam, where A.pinnate is grown in 400,000 ha. In recent years, it has become a common input in rice cultivation in Thailand, Indonesia, China and Phillippines.
Mass Cultivation:
The Chinese grow small nurseries where Azolla initially grown for four weeks. At the time of rice cultivation, Azolla is seeded in the flooded rice fields at the rate of 7.5 t/ha. After 5-10 days, the water in the field is drained and the Azolla is ploughed. This process of flooding the field, draining water and ploughing is repeated before transplanting the rice seedlings.
3. Azospirillum:
Azospirillum lipoferum (earlier known by Spirillum lipoferum) as a nitrogen fixer was known since 1963. It was Deobereiner and his associates in Brazil who in 1975 highlighted and attributed the nitrogen fixation potential of some tropical sorghum, wheat and rye to the activity of A. lipoferum in their roots.
Subsequently, the bacterium has been isolated from many tropical countries, found near roots and aerial parts of plants. Dobereiner coined the name ‘Associate symbiosis’ to denote the occurrence of nitrogen fixing Azospirillum near the roots in plants. Three more species viz., A.amazonense, A.barsilense and A.serapedica are added to this genus.
Azospirillum is a gram negative and contains poly-B-hydroxy butarate granules. It shows polymorphism and spirillar movements. For fixation of molecular nitrogen, it needs microaerophilic conditions. The ability of the organism to fix nitrogen has been demonstrated by the acetylene reduction test and uptake of 15N2, gas. This organism is also known to produce growth substances such as IAA, kinetins and gibberellins.
(i) Mass cultivation of Azospirillum and Inoculant Preparation:
For large-scale cultivation of Azospirillum, bottles or flasks with ammonium chloride containing liquid media are being used. The composition of the medium is as follows (g/l).
A solution-
(a) K2HPO4 – 6.0, KH2PO4 – 4.0, Dist. Water – 500 ml.
(b) MgSO4 – 0.2, NaCl – 0.1, CaCl2 – 0.02, NH4Cl – 1.0, Malic acid – 5.0,
NaOH – 3.0, Yeast extract – 0.05, Na2Mo O4 – 0.0002, MnSO4 – 0.001,
H3BO3 – 0.0014, Cu(NO3)2 – 0.0004, ZnSO4 – 0.0021, FeCl3 – 0.002,
Dist. Water – 500 ml, Bromothymol blue – 2 ml
The phosphate buffer portion of the medium was made in half of the total volume require part- (a) and (b) were sterilized separately, mixed while hot and poured into plates. These containers are incubated at 35°C on a rotary shaker and cells are harvested after three days. The broth is incorporated into a carrier material consisting of powdered, sterilized farmyard manure and soil in the ratio of 1:1 then packed in polythene packets. This carrier is the most suitable among different, tested carriers.
(ii) Benefits of Inoculation:
Several field experiments have been carried out in India by inoculating seeds of different crop plants with carrier-based culture of strains of Azospirillum brasilense. The response of sorghum and pearl millet to A.brasilense inoculant under several agroclimatic conditions of India is presented in Table 20.5. Such responses to Azospirillum inoculant have also been confirmed in Israel.
4. Azotobacter:
It is a heterotrophic free-living nitrogen fixing bacterium belonging to the family Azotobacteriaceae. Several species of Azotobacter are recognized such as A.chroococcum, A.agilis, A.uinelandii and A.beijerinckii. Among different species of Azotobacter, A.chroococcum and A.Vinelandii are the most intensively investigated species that have been commercially exploited.
Bacterial preparations containing Azotobacter cells under the name ‘Azotobacterin’ are being produced, used in USSR, East European Countries such as Czechoslovakia, Rumania, Poland, GDR, Bulgaria and Hungary where bacterization of seeds with Azotobacterin has proved beneficial in increasing crop yield.
(i) Mass Cultivation of Azotobacter and the Preparation of Inoculants:
In India large scale cultivation of Azotobacter chroococcum has been carried out either by in large flasks on rotary shakers or in batch fermentors. The medium used for Azotobacter chroococcum is as follows (g/l) Sucrose – 20, K2HPO4 – 01, MgSO4.7H2O – 0.5, NaCl – 0.5, FeSO4 – 0.1, CaCO3 – 2.
At the end of the desired incubation period, the broth is poured into a powdered carrier and mixed uniformly to bring the level of moisture in the carrier to 40 percent. After curing for 2-5 days, the carrier based inoculant is packed in polythene bags for transport. Seed inoculation is done in the same way as have done for Rhizobium.
However, in transplanted crops such as rice, cauliflower, cabbage etc., the roots of seedlings are dipped for 10 to 30 minutes in the watery slurry of the inoculant before transplanting. For other crops, manufacturer recommends multiple inoculations during the early stage of plant growth by pouring the slurry of the inoculant near the root zone. Mixing the cultures in farmyard manure and broadcasting near the root zone is also recommended.
(ii) Benefits of Inoculation:
Recent work on the response of field crops such as maize and cotton to inoculation with new strains of A.chroococcum has shown the feasibility of using Azotobacter inoculants to minimize the use of nitrogen fertilizer. Besides the ability to fix atmospheric nitrogen, Azotobacter is also known to synthesize biologically active substances such as B-vitamins, indole acetic acids and gibberellins in pure cultures.
The organism possesses fungistatic properties even on certain pathogenic ones such as Alternaria and Fusarium. These attributes of Azotobacter explain the observed beneficial effects of the bacteria in improving seed germination, plant growth, plant stands and vegetative growth (Table 20.6).
5. Blue-Green Algae:
Blue-green algae constitute an important group of microorganisms capable of fixing atmospheric nitrogen. They comprise unicellular, colonial and filamentous forms. Most of the nitrogen fixing BGA belongs to the orders Nostocales and stigonematalses under the genera Anabaena, Anabaenopsis, Aulosira, Chlorogloea, Cylinderospermum, Nostoc, Calothrix, Scytonema, Tolypothrix and Fischerella.
In general, nitrogen fixation is associated with forms possessing heterocysts, although there are reports of N2 fixation by unicellular and filamentous non-heterocystous strains. The number of heterocysts could be taken as a rough parameter to indicate the nitrogen fixing capacity of BGA.
The importance of BGA (Cynobacteria) as biofertilizers was recognized as early in 1939, when De reported the restoration of soil fertility in rice fields by cyanobacteria. Thenceforth agronomic potential of cyanobacteria has been further proved from the studies at various laboratories. Restoration of soil fertility in tropical paddy fields has been attributed mainly to nitrogen fixing, heterocystous forms.
(i) Mass Cultivation of BGA:
In India, a simple farm oriented open air method for bulk production of BGA from a starter culture consisting of a soil based mixture of Aulosira, Tolypothrix, Nostoc, Anabaena and Plectonema has been developed. Shallow trays of galvanized iron sheet (6′ x 3′ x 9′) or brick mortar or its pits lined with polythene sheets are constructed into which 10 kg soil plus 200 g superphosphate, pH of mixture is adjusted to 7.0 by the addition of lime.
When the soil settles down, saw dust is sprinkled along with the starter culture of algae. On hot sunny days, the algal growth in the open air tanks is quick and within a week a thick scum of algae is formed. The water is allowed to drain and the dried algal flakes from the surface are scraped and stored in bags. The production of dried algae on farmer’s fields can be a continuous process.
Dried algae are broadcasted at the rate of 10 kg/h over the standing water in rice fields, one week after the transplantation of rice seedlings. The cost of algal material has been calculated at Rs. 30 for 10 kg sufficient for one hectare of land. Mass production of carrier based immobilized cyanobacterial inoculants are depicted in Fig. 20.3.
(ii) Benefits of Inoculation:
Field trials conducted in different parts of India have shown significant increase in grain yields of rice due to the inoculation of rice fields with BGA.
The mean increase in yield of grain due to algal inoculation works out to be 300 kg for Rs. 30 investment. It is now well known that any form of nitrogen is detrimental to the process of nitrogen fixation by microorganism. The increased yields of rice due to algal inoculation, even under heavy doses of nitrogenous fertilizers could be attributed to the combined effect of biologically fixed nitrogen and the growth substances secreted by BGA (Table 20.7).
6. Photosynthetic Bacteria:
Though some photosynthetic bacteria like Rhodopseudomonas are known to fix the atmospheric nitrogen in a photoautotrophic manner, these are not yet commercially exploited as biofertilizers.
7. Phosphate Solubilizing or Phosphate Augmenting Organisms:
The deficiency of phosphorus may occur in crop plants growing in soils containing adequate phosphates. This may be partly due to the fact that plants are able to absorb phosphorus either by plant roots or by soil microorganism through secretion of organic acids. Therefore, phosphate dissolving soil microorganisms play some part in correcting phosphorus deficiency of crop plants.
They may also release soluble inorganic phosphate (H2PO4) into soil through decomposition of phosphate rich organic compounds. In this regard phosphate solubilizers such as fungi and bacteria and mycorrhizae have been investigated as promising sources of biofertilizers.
(i) Phosphate Solubilizers:
Many fungi (Aspergillus, Penicillium) and bacteria (Bacillus megatherium, Pseudomonas striata) are potential solubilizers of bound phosphates as revealed by experiments in pure culture. Though fungi seem to be better agents in the dissolution of phosphates, bacteria have been used in the commercial preparation of phosphate dissolving cultures to improve the growth of plants.
(ii) Mass Cultivation of Phosphate Solubilizers and the Preparation of Inoculants:
In India large scale cultivation of Bacillus megaterium or pseudomonas striata is being carried out either by growing cultures in large flasks on rotary shakers or in batch fermentors. The carriers that are used for Rhizobium and Azotobacter are also suitable for phosphate – solubilizing bacteria. The medium used for large scale production is as follows, (g/l). Glucose 10, Ca3(PO4)2 5, (NH4)2 SO4 0.5, KCl 0.2, MgSO4 . 7H2O 0.1, MnSO4 trace, yeast extract 0.5. Preparation and application of inoculum are essentially similar to Azotobacter inoculant.
(iii) Benefits of Inoculation:
It has been observed that vegetables respond better than cereal crop to the application of phosphate dissolving microorganism. A commercial preparation under the name ‘Phosphobacterin’ containing bacterial cells of Bacillus magatherium was widely used in USSR. Field trials conducted at IARI, New Delhi with wheat, bersem, maize, arhar and rice have shown significant increase in the yield in 10 out of 37 experiments (Table 20.8).
8. Mycorrhizae:
The symbiotic association between fungi and root systems of higher plants come under the general name, mycorrhiza which literally means ‘fungus roots’. These fungus roots were first discovered by Frank (1855) in pine, but subsequent work has pointed out that such a symbiotic association with fungi exists under natural conditions in root systems of many other economically important crops and plays a very important role in plant nutrition.
The correlation between mycorrhizal formation and mineral deficiency in the soil led to the conclusion that mycorrhizae must help in the absorption of nutrient of plants from the soil. The increase in nutrient absorption has been attributed to several reasons.
There are two kinds of mycorrhizae the ectomycorrhizae and endomycorrhizae. The ectomycorrhizae is not as common as the endomycorrhiza even though the former has been studied more thoroughly. In the ectomycorrhizae, the fungus completely encloses each feeder rottlets in a sheath or mantle of hyphae. The hyphae penetrate only between the cells of the root cortex (Harting net). Ectomycorrhizal associations are common in most forest trees primarily in the families Pinaceae, Betulaceae and Fagaceae.
In endomycorrhizae, the fungus lives within the cells of the root (intracellular) and establishes direct connections between the cells of the roots and the surrounding soil. Endomycorrhiza produced by nonseptate fungi is more commonly known as vesicular arbuscular mycorrhiza (VAM) or arbucular fungi (AM). Throughout the world, its widespread occurrence is found in almost all family’s plants.
The beneficial effects of VAM mycorrhizae on plant growth are numerous. It is now well established that mycorrhizae can improve the P nutrition of host particularly in low fertility due to exploration of the soil by the external hyphae beyond the root hairs and phosphorus depletion zone. Mycorrhizal fungi also stimulate plant, uptake of zinc, copper, sulphur, potassium and calcium.
They play an important role in water economy of plants and bestow the drought tolerance to the plants. VAM infections alleviate heavy metal toxicity, increase the tolerance of the crops to high acidity and temperature. It also decreases the growth depression and root rots caused by fungal pathogens. Mycorrhizal inoculations stimulate rooting and growth, thereby transplant survival of cuttings and seedlings, which is essential for the successful reclamation and forestation programmes.
Due to these beneficial attributes the mycorrhizae are widely used as biofertilizers for improving the growth of agricultural and horticultural plants. They have been exploited to save the costly phosphatic fertilizers. They are also employed in wasteland reclamation and afforestation. However, the most important constraint in their wide application is the commercialization of their inoculum because of their inability to grow and develop in the absence of their host.
At present, VAM fungi are cultured in bulk only on the roots of the living plants grown either in soil (pot culture) or in nutrient solutions. These systems are crude and expensive, require a lot of money and space. Nevertheless, attempts are being made to refine these systems. An outline for VAM inoculum production is given in Fig. 20.4 produced at different centre including Terri, New Delhi and Forest Research Institute, Dehradun. Inocula of these can be procured as needed and used in crop production, horticulture and forestry.
Finally it is concluded that bioinoculants have a tremendous potential in India as a cheap source of plant nutrients provided the quality of product is assured. Another important thing is to discover and use as many bioinoculants as possible rather to evaluate the subject by quibbling or sophiststy. The subject is so challenging that one should approach it hopefully and with optimism, not with skeptism and doubts. As Samuel Johns observed long ago. ‘Nothing will ever be attempted if all possible objections must first be overcome’. Each successful application, no matter how limited increases the level of familiarity.