Achievements

B. Liquid formulations of biofertilizers

In recent past, biofertilizers were prepared as carrier based formulations and lignite is the most widely used carrier material. The improper sterilization of carrier material and handling methods during the blending of bacteria with carrier serve as source of contaminations. Because of these, the carrier based inoculant could not hold desired biofertilizer organism for long time, which reduced the shelf-life of the biofertilizers. Further, the quality of the biofertilizer gets deteriorated. Because of these reasons, biofertilizer application is not able to give consistent results in the fields. To avoid these constraints and to increase the quality and shelf-life of bio inoculants, liquid formulation of biofertilizer was developed. Liquid inoculants are available in broth culture in which the cells are suspended in a buffer with suitable cell protectants. The protectants as additive in liquid formulations serve to overcome heat transfer, water retention property, absorption of bacterial toxins and protect enzyme system. Liquid biofertilizer has certain advantages and disadvantages over conventional carrier based biofertilizer as mentioned below:

Advantages

• No contamination
• Easy handling
• Better shelf life (12 – 24 months)
• Better survival on seeds and soil
• Cost saving on carrier material, pulverization, neutralization and sterilization
• Identified by typical fermented smell
• Easy quality control
• Dosages is 10 times less than carrier bio fertilizers
• Greater potentials to fight with native population

Method of application

Seed treatment – 500 ml each Azospirillum and Phosphobacteria per ha seeds; Seedling dip – 500 ml each Azospirillum and Phosphobacteria per ha seedlings; Soil application - 500 ml each Azospirillum and Phosphobacteria per ha seedlings; Tree crops – 100 ml each Azospirillum and Phosphobacteria per tree once in six months. Biofertigation: One ml per lit of water mix in the fertigation tank (Capacity: 60 lit) trice at 30 days interval.

C. Pink-Pigmented Facultative Methylotrophs (PPFM)

Methylobacterium are ubiquitous in nature and have been detected in soil, dust, freshwater, lake sediments, on leaf surfaces and nodules, air, hospital environments, as well as on other solid surfaces. They are aerobic, Gram-negative bacteria and although they are able to grow on a wide range of multi-carbon substrates, they are characterized by the capability to grow on one carbon compounds such as formate, formaldehyde, methylamine and C₂, C₃, and C₄ compounds, including the methanol emitted by the stomata of plants as the sole carbon and energy source. Methylobacterium is a facultative methylotroph, meaning it has the ability to grow by reducing carbon compounds with one or more carbon atoms but no carbon-carbon bonds. Because of their distinctive pink pigmentation, they are also referred as PPFMs (pink-pigmented facultative methylotrophs). These PPFMs are especially abundant on leaves of field-grown crops averaged about10⁶ cfu of PPFMs per leaflet, and typically >80% of the viable bacteria recovered from leaves were PPFMs. Methylobacterium strains have been localized as endosymbionts within plant cells. However, in some plants species, phyllosphere colonization also occurred via seeds. In addition to their ability to colonize the phyllosphere, members of Methylobacterium were also able to colonize the rhizosphere of plant species. Spraying on the aerial parts of plant, bacterial cells were localized in the grooves between the leaf epidermal cells, which may give them protection and access to methanol.

The benefits of application of PPFM to crop plants are:

Fasten seed germination and seedling growth

Accelerate vegetative growth

Increase leaf area index and chlorophyll content

Earliness in flowering, fruit set and maturation

Improves fruit quality, color and seed weight

Yield increase by 10%

Mitigate drought.

Method of application:

Spraying of PPFM at 500 ml/ha with 500 litres of water twice at active growth stage of crops with 15 days interval.

E. Zinc solubilizing bacteria

Zinc (Zn) deficiency in major food crops has been considered as an important factor affecting the crop production and subsequently the human health. Rice is sensitive to Zn deficiency and thereby causes malnutrition to most of the rice-eating Asian populations. More than 50% of the Indian soils (50 Mha) are Zn-deficient and rice comes under “high sensitive” crop for Zn nutrition. Zn fertilization corrects Zn-deficiency, but often fails fortification. Sustainable approach, integrating microbial resources and soil biochemical processes with physiological response of rice is essential for Zn-fertilization as well as Zn-fortification in rice. At TNAU, four elite Zn-solubilizing bacteria viz., Achromobacter xylosoxidans (ZSB6), Enterobacter cloacae (ZSB12 and ZSB14) and Pseudomonas chlororaphis (ZSB15) were developed as inoculant for rice. These ZSB inoculants with insoluble amendment (ZnO or Zn₃(PO₄)₂) can increase the available Zn in wetland soil, red laterite and calcareous soil in a range of 5-10 mg/kg of soil. When these bacteria were applied to rice along with zinc phosphate ensures “Zn sufficient” condition throughout the crop period. It enhances the Zn uptake of rice (33 mg/kg of biomass) and also improved the grain-Zn content (25 mg/kg of seeds) along with 10-15% yield increase. ZSB inoculation also reduced the Zn/phytate ratio in grains and thereby improved the Zn fortification of rice.

Method of application:

Seed treatment: 20 mL/kg of seed

Seedling dip: 20 mL/lit of water (Roots should be soaked for 30 min before transplanting)

Soil application: Mix 120 mL/6 kg of vermicompost and broadcast at 1kg/plot (Total of 6 plots) a day after transplanting.

G. Azolla Biofertilizer technology

Azolla is a floating water fern, commonly present in lakes and tanks. They harbor the nitrogen fixing cyanobacteria, Anabaena azollae as micro-symbiont in the leaf cavities. Azolla is widely used as a potential biofertilizer for increasing the grain yield of rice. A layer of Azolla mat covering 1 ha of rice field will produce about 10 tonnes of green matter which containing 30 kg of nitrogen. Since Azolla can able to fix about 40 to 50 kg of nitrogen per ha per season, through its partner cyanobacteria, it can be applied as biofertilizer or green manure to rice fields. Azolla should be multiplied as nursery separately and applied to rice crop. For Azolla multiplication, puddled soil, water, phosphorus, low temperature (25 to 35ºC); shady sunlight are essential. Application of Azolla to rice fields contributes about 50 kg of nitrogen per ha; increases the organic matter content of soil; increases the soil fertility and physico-chemical properties; reduces the weed problems and increases the grain yield of about 25 per cent.

Method of application:

Since Azolla can grow at temperature range of 25 to 35ºC, Samba season (rabi) is ideal for cultivation and application to rice field. Azolla can be applied as green manure or dual crop with rice.

Green manure: Azolla should be grown separately as nursery and can be applied as green manure. For this, 4 tonnes of fresh Azolla biomass is required for one acre of rice field. The Azolla biomass should be broadcasted in the main field during last ploughing and should be incorporated as green manure. This method needs huge amount of fresh Azolla biomass, whereas the dual cropping requires relatively less quantity of Azolla.

Dual cropping: Azolla can be grown along with rice for 45 days as dual crop and can be incorporated in soil. A quantity of 80 kg of fresh Azolla biomass should be broadcasted in the rice main field at 7 days after transplanting. This will form as Azolla mat throughout the rice field within 40 – 45 days. After 45 days, Azolla biomass can be incorporated manually into soil, which will decompose at very faster rate and release the nitrogen and other nutrients to rice crop.

H. Azolla as animal feed

Azolla is distributed widely throughout warm-temperate and tropical regions. Its most remarkable feature is its symbiotic relationship with the nitrogen fixing blue-green alga, Anabaena azollae, within leaf cavities. The host plant Azolla provides the carbon source sucrose to the symbiont while, the symbiont fixes nitrogen and transfer to Azolla which in turn multiplies very rapidly. The growth rate of Azolla is rapid that it doubles its weight in a mere 2-3 days. Due to its higher relative growth rate, the biomass production is heavy in a given time and can be utilized as a feed for livestock. Azolla is rich in protein, essential amino acids, vitamins, growth promoter intermediaries and minerals like calcium, phosphorus, potassium, magnesium etc. Azolla on a dry weight basis consists 25-30% protein and carotenoids including the antioxidant, β carotene. The dry matter of Azolla is 9.2%.Crude fibre content in Azolla ranges from 15-15.8%. Thus Azolla is most promising from the point of view of ease of cultivation, productivity and nutritive value making it an economic and efficient feed substitute for livestock. Moreover, Azolla can be easily digested by livestock owing to its high fibre and low lignin content.

Methods of feeding livestock with Azolla

Feeding livestock with Azolla can be done as fresh biomass, dried form, pellets and as ensiled Azolla.

i. Fresh Azolla: Azolla can be fed directly to poultry, swine, ducks, rabbits, goats. With excessive moisture content, it would be very difficult to transport the feed from one place to another. Reduction in moisture content can be achieved by spreading on a floor for few hours. Trials on dairy animals showed an overall increase of milk yield by 15-20% when 1.5 – 2 kg of fresh Azolla was combined with regular feed.

ii. Dried Azolla: Sun dried and powdered Azolla is gaining importance due to its storability and mixibility with concentrate feed. When water is available in plenty, Azolla can be multiplied and can be dried and stored. After complete drying, the dried Azolla is mixed with livestock feed in required proportion and can be used.

iii. Azolla pellets: Azolla can be pelletized after sufficient drying. The dried pellets can be handled in much easier way than the fresh bulky biomass.

iv. Ensiled Azolla: During rainy season, the excess biomass obtained may be ensiled and could be used as feed. This process is very popular with Chinese farmers. The moisture content of Azolla is brought down to 60%. They are filled into the silos made of cement or in plastic bags to 3 cm. Above this layer, salt (5g) and maize flour (50g) are sprinkled for every kg of Azolla and the piling process in repeated. The silo is then covered and made air tight. The fermentation process takes less than a month and its product can be kept for 2 years.

J. High level secretion and method of production of laccase (LccH) by a novel basidiomycetes fungus MSF2 and uses there of

A high level laccase secreting novel basidiomycetes fungus MSF2 isolated from decaying wood samples. Extracellular laccase production by MSF2 was greatly stimulated by the addition of a low cost inducer to simple glucose based medium. Optimization of assay conditions for buffer, pH and temperature resulted in a maximum LccH activity of approximately 1362 U.ml^-1 using citrate phosphate buffer at pH 3.4 and a temperature of 40 °C. Under these optimized conditions, GY medium resulted in enhanced activity (1944.44 U.ml^-1) in presence of an inducer, and this suggests that the fungus can be used for industrial applications. In addition, considerable LccH can also be produced by MSF2 using natural biomass substrates under either SSF or SmF. The LccH thus produced can delignify lignocellulosic substrates, to a level of 16.5 and 28.6 per cent, respectively.

K. Enzolv: A novel process for delignification and simultaneous high value chemical generation from lignocellulosic biomass using laccase

A novel process was developed for delignification of woody (Melia dubia) biomass encompassing steam pretreatment, followed by laccase treatment in the presence of solvent and termed as Enzolv. Pronounced effect on lignin removal by formation of pores upto 7µm and separation of vascular bundles in combination with cellulose enrichment was obvious while monitoring enzolv treated biomass under SEM. Under FT-IR, the intensity of O-H spectra was higher for enzolv treated biomass than all the treatments suggesting significant lignin removal as well as higher intensity reduction in the lignin regions such as 1465, 1425 and 1323 cm^-1 was perceived. As a consequence, intensity of cellulose at 1029 C-O cm^-1 stretching was remarkably higher in the Enzolv treated process. The crystalinity was increased and reached a maximum of 78.39% in Enzolv process which corresponds to the cellulose content 52.12% while observing under the XRD. In addition, the process also generated potential high value chemicals such as benzaldehyde, vanillin and isovanillin which are of industrial interest.