Title of Invention

"A PROCESS FOR THE PRODUCTION OF BLUE GREEN ALGAL BIOFERTILIZER"

Abstract

"A process for the production of blue green algal biofertilizer" involves the production of BGA biofertilizer strains individually in the formulated medium by inoculating about 3-10g wet biomass per multiplication unit with a production of 3-10 days production cycle. The medium formulation is achieved by mixing single super phosphate, mureate of potash and magnesium sulphate at 0.02-0.09 g/L water, once BGA cultures grown to a desired biomass say 15-18 g/unit is mixed with wheat/rice straw as carrier at a final population density of 106 -109 cells per gram carrier containing Aulosira, Tolypothrix, Anabaena and Nostoc strains. The multiplication units are constructed in a single/double tier inside the glass or polyethylene house. The multiplication units are made of cement and mortar. In a two-tier system the lower tier is illuminated by artificial light. The temperature of these units is maintained between 25-45°C during the entire year by providing heat convertors in winters and exhaust fans with suitable cloth filters in summer. The dried inoculum containing all the four strains mixed in 1:1:2:2 or 1:1:1:1 (w/w) proportion and packed in suitable size packets. The shelf life of this inoculum (biofertilizer) is two years if stored in dry and well-ventilated area. The straw based biofertilizer to be applied by broadcasting immediately after transplantation of paddy seedlings in a puddled field. Normally 400-g straw-based BGA biofertilizer is enough for 1 acre.

Full Text

This process for the production of blue green algal biofertilizer involves production of blue green algae strains in glasshouse/polyethylene house in multiplication ponds of RCC containing formulated medium by adding the single super phosphate, mureate of potash and magnesium sulphate in a concentration range of 0.02-0.09 g/L preferably 0.02-0.08 g/L with a final pH of neutral or near neutral in an open area; each strain say Anabaena variabilis or Nostoc muscorum or Aulosira fertilissima or Tolypothrix tenuis with a definite size of inoculum say 1 to 10 or preferably 3 to 5 gram wet biomass/pond inoculated and allowed to grow till finally 12 -30 gram/unit biomass is produced which is mixed in 400-1000 gram riceAvheat straw per multiplication pond to provide 10,000 to 1000,000,000 cells/gram of rice/wheat straw as carrier and sun dried, mixed in definite proportion w/w in a ratio of 1:1:2:2 or 1:1:1:1 to provide 1,000,000 -1,000,000,000 cells/gram carrier or 1,000,000 cells/gram carrier and packed in suitable size of packets with a shelf life of 2 years. 3. TECHNOLOGY PARAMETERS: A process for the production of blue green algal (BGA) biofertilizer. Field of invention: This invention relates to the selection or standardization of multiplication unit - glasshouse/polyhouse, formulation of medium for multiplication of BGA, pH of the formulated medium, quantity of inoculum, duration of mass multiplication of BGA in a multiplication pond, quantity of carrier to be used for the final product, final population of BGA per gram of carrier, and finally developed a technology package for the blue-green algal (BGA) biofertilizers. Biofertilizers have attained an important place in the modem agriculture. BGA biofertilizers are specific to rice and sugarcane crops and depending upon the soil type, availability of water provides 25-30 kg of biologically fixed nitrogen per hectare per cropping season. BGA biofertilizers also increases the availability of phosphorus, provides growth-promoting substances (vitamins and indole acetic acid) and improves soil structure by contributing organic carbon thus improves soil health and fertility. BGA biofertilizers also reclaim degraded wastelands like saline/alkali soils. The results of experimental trials and farmers fields are as follows: i. That in areas where chemical nitrogen use is either nil or in very small quantities, particularly in some of the districts of Assam, West Bengal, Bihar and eastern U.P, application of BGA biofertilizer without any chemical nitrogen fertilizer, a net increase of 15-20% in the crop yield is obtained. ii. In areas where chemical nitrogen fertilizer is used in small quantities (25-75 kg N/ha), a fertilizer dose to the extent of 25 kg N/ha can be reduced without any decrease in crop yield rather an additional 10-15% increase in crop is obtained. iii.Even at higher doses of fertilizer nitrogen, increased rice yield (15-20%) is obtained. BACKGROUND OF INVENTION: Blue green algae (BGA) are photosynthetic organisms. Some of the BGA are capable of fixing atmospheric gaseous nitrogen in to ammonia with the help of enzyme nitrogenase and deriving energy from sunlight. In an area of one hectare it can provide about 25-30 kg of biologically fixed nitrogen in a period of 90 days. During its growth it also releases organic carbon, some growth promoting substances and thus if BGA biofertilizer is applied to rice crop it supplements 54-64 kg urea per hectare per season and an 10-15% increased crop yield. However, no suitable protocol or technology was available to produce it in bulk round the year. This was basically due to non-availability of cheaper growth media and suitable carrier. Similarly physiological growth parameters like pH of growth media, aeration, temperature, inoculum load and duration of growth were not standardized Hence the BGA biofertilizer was not available to the farming community. With the large number of limitations in the production of BGA biofertilizer with the soil-based method, a better protocol or technology was required For want of suitable protocols, the use of BGA biofertilizer was not popular among farming community. The present invention relates to the formulation of growth media, selection of suitable carrier and all the growth parameters pertaining to BGA biofertilizer production. The production should be feasible round the year hence can be produced industrially. PRIOR ART: Prior to straw carrier based blue green algae (BGA) biofertilizer production technology a soil-based rural production methodology was available for which there were no takers. The main drawbacks were that production was done in troughs of GI sheets or directly in the fields either by placing polyethylene sheets or without sheets after digging the pits 6' x 3' x 9". The pits were to be made in an open area so as to receive sunlight for the entire day. To each pit 10 kg soil and 250 g single super phosphate was added and water was filled up to a height of 3-4". Starter culture (mixture) of Anabaena, Nostoc, Aulosira and Tolypothrix was to be inoculated per troughs or multiplication unit. To prevent insect breeding during multiplication carbofuran (3% granules, 20g/tray or unit) or Malathion 5-10ml of 50% was recommended. In hot summer months in about 3-4 weeks a thick mat formed. It was allowed to dry and the dried material along with soil was used as inoculum. The recommended rate of application was 10 kg per hectare. The production was discontinued during rainy and winter seasons. This method (Prior Art) suffered due to: i. Production period was limited to summer months only, ii. Soil in water was medium of growth which varied from region to region hence quality of product was not uniform, iii. Wide fluctuations in population of BGA as exposed to external environment, iv. High levels of contamination due to open production, v. Slow production rate (3-4 weeks per harvest), vi. Multiplication of 1 or maximum 2 strains at a time in mixed culture, vii. Low population density, viii. Bulky nature of final product hence quantity required is high (10 kg/hectare), and ix. Increased cost of transportation. OBJECTIVES OF THE INVENTION: A process for the production of blue green algal (BGA) biofertili/er was to be developed with the following objectives: i. To formulate a growth medium with known constituents and cheaper than the earlier known growth media to avoid soil as a source of nutrient and carrier. ii. The medium should support faster growth rate than the known growth media, iii. To select a carrier which is lightweight, cheap and better carrying capacity, iv. To give inoculum of known and constant quantity of cells/gm carrier. v. To give product of known quality (all the culture should be present in a packet). vi. To provide inoculum free of contamination. vii. That the technology should be operational round the year so that the labour is fully utilized with industrial implications. viii. To standardize the growth parameters (light, pH, temperature, and growth rate), drying and shelf life of the culture produced by formulated growth media. Overall, it was intended to develop commercially viable technology. THE INVENTION: The most significant aspect of this invention is the formulation of growth medium (food for growth of BGA), selection of carrier, selection of production site and production units, inoculum load, time of harvesting, drying and mixing of different culture before packing. The detail of entire protocol has been worked out 1. The Technology: A process for the production of blue green algal (BGA) biofertilizer. 1.1. Formulation of growth medium: Nonnally the growth media available in literature are designed for specific research work. Such media although support the growth of specific BGA but not suitable for mass cultivation of BGA biofertilizer. These growth media are commercially nonviable, as they are most expensive and not necessarily support fast growth rate for all the BGA biofertilizer strains. Therefore a new growth media was formulated for the cultivation of BGA biofertilizer strains with the objectives mat it is cheaper and higher growth rate per unit time of all BGA biofertilizer strains. The major absolute requirements for the growth of BGA are nitrogen, phosphorus, potassium, sulphur, and magnesium. Elements like iron, manganese and trace metals like cobalt, zinc, molybdenum and copper also play significant role in growth of BGA. To provide major nutrients like phosphorus and potassium, commercially available fertilizers (single super phosphate and mureate of potash) were taken in a definite quantity. Since the biofertilizer strains are capable of fixing atmospheric nitrogen, the formulated medium was devoid of combined/preformed nitrogen source. Most of the trace elements are present as impurities in phosphatic and potassic fertilizers hence the salts for calcium, sulphur, copper, zinc, manganese, cobalt, molybdenum, citric acid and EDTA were not included in formulated medium. Magnesium the essential component of chlorophyll was supplied as magnesium sulphate. Finally growth media was formulated with Mureate of potash, super phosphate and magnesium sulphate. The required quantity per litre of growth media varies between 0.03-0.075 gram. The growth kinetics in the final formulated medium was found to be significantly superior to the earlier known media-BGn (BM). The growth rate of AuIosira sp., Tolypothrix sp., Nostoc sp. and Anabaena sp. in formulated medium (FM) was 0.025, 0.023, 0.018 and 0.026 per hour against 0.012, 0.02, 0.013 and 0.015 per hour respectively in the BG-11 growth media. The cost of 1000 litre Formulated Medium was Rs. 12.60 against Rs. 48.15 for the BG-11 medium; hence, the formulated medium (FM) was much cheaper than the routinely used growth media for BGA biofertilizer strains. The constituent of FM and their quantity is one of the most important aspects and needs to be patented as the part of process/technology. 1.2. Inoculum carrier: To supply BGA biofertilizer strains, soil was being used traditionally. Soil as a carrier is heavy and has poor carrying capacity and varies from area to area thus amendments is needed. A large variety of carriers like riverbed sand, rice straw, wheat straw, sawdust, thermocol and polyurethane foam were investigated From a variety of carrier wheat straw was found to be best. This has been used for the first time as carrier of BGA biofertilizer. The carrying capacity of wheat straw was found to be more than 1,000,000 cells per gram carrier. Hence the use of wheat straw as carrier for BGA biofertilizer needs to be patented as apart of technology. 1.3. Starter culture: The initial inoculum load is very important parameter to determine the duration of multiplication, final biomass buildup as well as time of harvesting to obtain desired population density in the final product. During the development of process for BGA biofertilizer strains production initial inoculum load was standardized. Accordingly each strain of BGA grown in pure cultures in laboratory and multiplied independently. Initially 500 millilitre 10 days old culture (about 3 gram) is inoculated per pond initially for 2 days. Subsequently, dry inoculum from the material produced there itself can be used. Hence, the quantity of wet biomass per multiplication unit of specific size is important to provide final a final yield of 1,000,000 cells per gram carrier and is important aspect needs patenting. 1.4. Strategies to reduce the contamination in final inoculum: To reduce the external aerial contamination in the final product, the multiplication ponds to be constructed in a polyethylene house/ glasshouse. The dimensions of poly- or glasshouse vary depending on the investment proposed. The smallest size of glasshouse can be of 12.6m x 5.6m x 8m. The multiplication ponds are of two types (a) polyethylene lined shallow pits, or (b) made of RCC. Aeration is achieved by providing windows on one-side and exhaust fans on the opposite wall. The air is sucked through muslin cloth filters. The use of such filters in glasshouse for BGA biofertilizer production is for the first time and should be patented as the part of the process/technology. 1.5. Strains used for BGA biofertilizer: Anabaena variabilis, Nostoc muscorum, Aulosira fertilissima and Tolypothrix tenuis. 1.6. Multiplication site: The multiplication unit should be constructed in open away from shade of trees, so as to receive maximum sunlight during the day. 1.7. Physical growth parameters: 1.7.1. pH: By growing the four BGA biofertilizer strains in formulated medium at different pH levels it was observed that the best growth rate (by factor of 2) was achieved in medium with near neutral to mild saline pH. This particular pH is employed in the current technology. 1.7.2. Temperature: The temperature range for growth of all the BGA strains varies from 25- 45°C. However, the optimum temperature for growth is from 32-37°C and for nitrogen fixation from 29-35°C. To maintain the temperature particularly in whiter months in northern India heat convectors (2) are provided whereas in summer temperature is controlled through exhaust fans. The use of either heat convectors or exhaust fans for BGA biofertilizer is for the first time hence as a part of technology should be patented. 1.7.3. Light: BGA biofertilizer strains can grow at a very broad light range i.e. from 1000 lux to 45,000 lux. For indoor cultivation, light intensity of 1800 - 3500 lux would support growth of BGA biofertilizer strains and this is achieved by providing artificial light source. In a multi-tier system one white light and one yellow light illuminate only lower tiers by 100-watt bulb. Providing the artificial light for lower tier has been demonstrated for the first time hi BGA biofertilizer production process hence needs patenting. 1.8. Harvesting: Once BGA strains produce enough biomass in formulated medium i.e. about 5 times in a period of 3-5 days the cultures are harvested. Harvesting is done by mixing the BGA biomass in a known quantity of carrier and removed immediately from the multiplication pits. 1.9. Drying: Natural sunlight is employed for drying the product. Drying should be done mat the final moisture content is low (15%) and does not support the bacterial growth in product. Keeping moisture percentage greater than 15 will lead to the deterioration of product whereas less than 15% moisture will reduce the survival and shelf life of product. 1.10. Packing and Storage of BGA biofertilizer: The individual BGA strains are mixed before packing hi suitable fresh bag. All the four strains are in equal proportion and sealed. 1.11. Population density: The straw based BGA biofertilizer was found to contain more than 1,000,000 propagules or cells per gram of carrier. BGA biofertilizer with lesser population density will not give desired benefits. 1.12 Shelf life: The shelf life of this BGA biofertilizer is 2 years when stored in dry ventilated room. The application: Since the BGA biofertilizer produced by this technology is of assured quality i.e. containing all the four BGA strains and of known population size 1,000,000 cells per gram carrier only 1 kg BGA biofertilizer per hectare (400 gram per acre) is enough to provide the desired benefits. It should be applied by broadcasting immediately after rice seedling transplantation. 4.I CLAIM 1. A process for the production of blue green algal (BGA) biofertilizer, in a novel formulated medium containing known constituents, mureate of potash, super phosphate and magnesium sulphate, each at 0.02-0.08 gram per litre of water, in neutral pH range 6.5-7.3, by inoculating a fixed biomass of 2-3 gram of single blue green algal strain ARM 310 Anabaena variabilis or ARM 221 Nostoc muscorum or ARM 68 Aulosira fertilissima or ARM 76 Tolypothrix tenuis in a multiplication unit/pond each in different pond at one point of time and allowing the blue green algal strains to grow in 10 hours light: 14 hours dark cycle for a specific period of 3-5 days; mixing definite quantity 14- 15 gram of the primary mass of blue green algae is mixed with wheat or rice straw as carrier of known quantity 400-500 gram to provide a particular quantity of 1,000,000 blue green algal cells per gram of the said carrier, drawing individual blue green algal cultures in carrier in a specific manner in sunlight and mixed in a definite proportion so that final populations of all the four blue green algal strains is 1,000,000 cells per gram of carrier. 2. A process as claimed in Claim 1 wherein the said multiplication unit is in two tiers inside a glass house and of single tier in polyhouse, both glasshouse or polyhouse located away from shade of trees, wherein it is filled with water of pH of growth medium 6.9-7.1 or 7.0. 3. A process as claimed in Claim 1 wherein each multiplication pond containing novel formulated medium is inoculated with only one strain of blue green algae ARM 310 Anabaena variabilis or ARM 221 Nostoc muscorum or ARM 68 Aulosira fertilissima or ARM 76 Tolypothrix tenuis per pond with a definite size of inoculum 1 to 10 or preferably 3 to 5 gram fresh biomass of approximately 10 days old culture and allowed to grow in the said novel formulated medium. 4. A process for the production of blue green algal (BGA) biofertilizer as claimed in Claim 3, wherein each blue green algae is allowed to grow in novel formulated medium using sunlight for the upper tier and artificial light for lower tier in glasshouse, provided by fixing 1M long white fluorescent tube light and two bulbs of 100 watts, in a dark/light cycle of 10:14 hours each, wherein in polyhouse using sunlight alone, for a period of 3- 10 days preferably 3-7 days or 5 day till 12 -30 or 15-18 gram per pond/unit biomass is produced; 5. A process for the production of blue green algal (BGA) biofertilizer as claimed in Claim 4, wherein blue green algae grown up to 12 -30 or 15-18 gram per unit biomass is taken out and mixed in 400-1000 gram wheat or rice straw per multiplication pond to provide 10,000 to 1000,000,000 or preferably 1,000,000 cells per gram of wheat or rice straw as carrier; 6. A process for the production of blue green algal (BGA) biofertilizer as claimed in Claim 5, wherein all the four blue green algal cultures mixed with carrier is dried separately preferably under the partial shade up to a final moisture content of 10 to 15 percent; 7. A process for the production of blue green algal (BGA) biofertilizer as claimed in Claim 2-6, wherein the air dried, carrier based, strains of all the four blue green algae mixed in definite proportion weight/weight in a ratio of 1:1:2:2 or 1:1:1:1 to provide 1,000,000 - 1,000,000,000 cells gram carrier or 1,000,000 cells per gram carrier and process as claimed in Claim 1, wherein such produced blue green algal (BGA) biofertilizer has a shelf life of 2 years.

Documents:

444-del-2002-abstract.pdf

444-del-2002-claims.pdf

444-del-2002-correspondence-others.pdf

444-del-2002-correspondence-po.pdf

444-del-2002-description (complete).pdf

444-del-2002-form-1.pdf

444-del-2002-form-19.pdf

444-del-2002-form-2.pdf

444-del-2002-form-3.pdf