Title of Invention	AN IMPROVED PROCESS FOR ANAEROBIC DIGESTION OF AGRO-BASED WASTES WITH SIMULTANEOUS PRODUCTION OF METHANE AND FERTILIZER.
Abstract	An improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer: The invention provides an improved process for anaerobic digestion wherein hydrogen produced during anaerobic digestion is rapidly metabolism by methanogens due to the thermodynamic shift in the reaction towards methane formation. In this work, the H2 metabolism has been regulated by the use of microbes, H2- producers, fungi and the hydrolytic enzymes. It has been possible to shift H2 production process and employ microbes to control methane production.

Title of Invention

AN IMPROVED PROCESS FOR ANAEROBIC DIGESTION OF AGRO-BASED WASTES WITH SIMULTANEOUS PRODUCTION OF METHANE AND FERTILIZER.

Abstract

An improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer: The invention provides an improved process for anaerobic digestion wherein hydrogen produced during anaerobic digestion is rapidly metabolism by methanogens due to the thermodynamic shift in the reaction towards methane formation. In this work, the H2 metabolism has been regulated by the use of microbes, H2- producers, fungi and the hydrolytic enzymes. It has been possible to shift H2 production process and employ microbes to control methane production.

Full Text	The invention relates to an improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer The present process particularly relates to a process for the regulation of anaerobic digestion of waste biological materials. The main utility of the present invention is to provide an environment-friendly method for degradation of waste by controlling anaerobic digestion resulting in t6 biogas - a source of energy and fertiliser simultaneously. Another utility of this process is to relieve stressed anaerobic digesters and revive sick digesters. Another utility of this process is to reduce the release of green house gases into the environment and to mitigate global warming. Anaerobic digestion is gaining importance as a process to treat and recover energy from organic matter rich biological wastes (R.B.Dean, Biocycle, 2: 75-77, 1998). Anaerobic digestion is a multi-step process. It involves the consorted effort and participation of diverse groups of bacteria. In the syntrophic association, the growth and activities of these microbes are very much dependant on the physiological conditions and their abilities to transform different organic compounds. (H.J.Thiele, M.Chartrain and J.G.Zeikus, Applied Environ. Microbiol., 54(1): 10-19, 1988). In addition to organised anaerobic digestion of biological waste materials, such a process operates in landfills, rice cultivation, domestic ruminant rearing, biomass burning, coal mining, and natural gas venting. Currently CH4 concentration in the atmosphere is increasing at a rate of about 0.015 ppmv (0.9%) per year. Global annual release of CH4 from rice paddy has been 12 estimated to be 150 Tg (Tg, giga tons = 1x10 ). The impact of these Green House Gases (GHGs) on environment is evident by the rising temperature, termed as Global Warming. (A.Holzapfel-Pschorn, R.Conrad and W.Seller, Plant Soil, 92: 223-233, 1986; B.S.Rajgopal, N.Beley and L.Daniels, FEMS Microbiol. Ecology, 53: 153-158,1988; K.Sipila, A.Johansson and K.Saviharju, Bioresource Technol., 43:7-12, 1993). Complex organic matter present in the biological wastes is hydrolysed into soluble compounds under partially fermentative conditions. Acidogens and acetogenic bacteria convert these intermediates into volatile fatty acids, hydrogen, carbon dioxide and other simpler compounds. (H.J.Thiele, M.Chartrain and J.G.Zeikus, Applied Environ. Microbiol., 54(1): 10-19, 1988). Methanogens, under strict anaerobic conditions produce methane from acetic acid and from H2 and C02. In nature, sulphate reducers and nitrate reducers compete with methanogens for Ha, which leads to reduced methane production. (M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988; D.M.McCartney and J.A.OIeszkiewicz, Water Environ. Res., 65: 665, 1993). Hydrogen production and fatty acid production are linked to lowering of pH. Under certain acidic conditions, generated as a result of H2 producing bacteria, the growth and metabolism of methanogens is adversely affected, which causes sickening of the digester. (R.I.Mackie and M.P.Bryant, Applied Microbiol. Biotechnology, 43: 346-350, 1995). The reversal of this downward process becomes very difficult. Similarly, the generation and accumulation of certain intermediates like propanoic acid and butyric acid can lower the pH in a reactor and raised H2 levels introduce instability in to the microbial community (F.E.Mosey, Water Pollut. Control, 81: 540, 1982; D.B.Archer, M.G.Hilton, P.Adams and H.Wiecko, Biotechnol. Lett., 8: 197, 1986) due to heavy stress on the anaerobic digestion process. This kind of situation is caused by the chemical composition of the feed material also. Moreover, sulphate reducing bacteria out compete methanogens at elevated levels of propanoic and butyric acid even at low acetate concentrations (F.Omil, P.Lens, P.LHulshoff and G.Lettinga, Process Biochem., 31(7): 699-710, 1996). Inhibition of sulphate reduction declines the quantity of H2S produced. However, it is accompanied by increase in propionic acid ahead of acetic acid. Sulphate reducing bacteria can metabolize propionate directly or in association with proton reducing acetogenic species, by H2 removal (F.W.J.Hoeks, H.J.G. TAN Hoopen, J.A.Roels and J.G.Kuenen, Anaerobic Waste Water Treatment, W.J.VANDEN Brink, Ed. THO Corporate Communications Department, The Hague, pp. 554-555, 1983; Z. Isa, S.Grusenmeyer and W.Verstraete, Appl. Environ. Microbiol., 51: 572, 1986; M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988). Sulphide inhibits propionate utilization (A.Rinzema and G.Lettinga, Environ. Technol. Lett., 9: 83, 1988) and proipionate buildup if not rectified may lead to process failure. A 50% inhibition of methanogenesis from acetate occurs at a HaS concentration of 35.2 mM (1200 mg/L) (Z.lsa, S.Grusenmeyer, W.Verstrate, Appl. Environ. Microbiol., 51: 572, 1986) at a total solid concentration of 100 to 224 mg/L (P.P.Karhadkar, J.M.Audic, C.M.Faup, P.Khanna, Water Res. (G.B.), 21:1061,1987). Hydrogen produced during anaerobic digestion is rapidly metabolised by methanogens due to the thermodynamic shift in the reaction towards methane formation. This H£ transfer reaction is very important in this complex anaerobic digestion process. (S.S.Ozturk, B.O.Palsson and J.H.Thiele, Biotech. Bioeng., 33:745-757, 1989). The process of anaerobic digestion is influenced by a wide array of environmental conditions. The rate of microbial methane production increases with rise in temperature. However, the amount of free ammonia also increases with temperature, the bio-digestive performance could be inhibited or even reduced as a result. (G.FIueh and P. Hubes, Patent No. De 4341713 dt. 08.06.95; O.P.Chawla, in: Advances in Biogas Technology, PID, ICAR, New Delhi, 1986). Higher concentration of available nutrients like copper, 10-250 mg/L; calcium and sodium, 8000 mg/L; magnesium, 3000 mg/L; nickel, 10-1000 mg/L; zinc, 350-1000 mg/L; chromium, 200-2000 mg/L; sulphide (as sulphur), 200 mg/L and cyanide, 2 mg/L usually have inhibitory effect. (I.P.Pankhania and J.P.Robinson, FEMS Microbiol. Ecology, 38: 309-312, 1986; A.Rinzema, J. vanLier and G.Lettinga, Enzyme Microbiol. Technol., 10: 24-32, 1988). The methaneproducing bacteria live best under neutral to slightly alkaline conditions. If the pH value drops below 6.2, the medium has a toxic effect on the methanogenic bacteria. Similarly, for higher pH values, even a relatively low nitrogen concentration of roughly 1700 mg ammonium-nitrogen (NH4-N) per litre substrate methanogenesis get affected. Various experiments have shown that the metabolic activity of methanogenic bacteria can be optimized at a C/N ratio of approximately 8 to 20. High carbohydrate concentration has an inhibitory effect on methanogenesis (S.Roychaudhury, D.Cox and M.Levandowsky, Int. J. of Hydrogen Energy, 13(7): 407-410, 1983). The presence of heavy metals, antibiotics (Bacitracin, Flavomycin, Lasalocid, Monensin, Spirmycin, etc.) and detergents used in livestock husbandry can have inhibitory effect on the process of biomethanation. (O.P.Chawla, in: Advances in Biogas Technology, PID, ICAR, New Delhi, 1986; R.E.Khabibullin, N.I.Krylova and R.P.Naumova, Bioteckhnologiya, 2: 43-46, 1995). Similarly, high loading rates invariably lead to the accumulation of volatile fatty acids like propanoic acid and butyric acid. This stress results in an imbalance in the metabolisms of different microbes and consequently, the biomethanation process stops operating. (D.T.Hill and J.P. Bolte, Transactions of the ASAE 30(2): 502, 1987; D.T.Hill, S.A.Cobb and J.P. Bolte, Transactions of the ASAE 30(2): 496, 1987; B.K.Ahring and P.Westermann, Appl. Environ. Microbiol., 53(2): 434, 1987). Hydrolytic conversion of organic matter to the available substrate for the anaerobic microflora is a rate limiting step for the organic waste degradation (J.J.Zabranska, M.Bosanka, P.Krempa, P.Jenicek and M.Rohananyos, Meded. Fac. Landobounwet Rijsunsi. Gent., 60(4b): 2217; 1995). In waste containing high lignin content, the lignocellulosic portion present in waste is the major substrate for methane production and it is believed that it's hydrolysis is the limiting step in the overall anaerobic digestion process as well (R.K.Malik and P.Tauro, Indian J. Microbiol., 35(3): 205; 1995). The problem of reduction in methane production or even complete failure of the digester can be solved to a very limited extent by drastic reduction in loading rates and or prolonged retention periods. (C.Ramakrishna and J.D.Desai, World J. Microbiol. Biotech., 13: 329-334, 1997). This method has a major limitation since it demands larger reactor volume for smaller waste material quantities i.e. it is an uneconomical proposition. Dilution of the feed material. (M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988) can result in lowering the concentration of certain toxic materials but will also lead to lowering the availability of food to the microbes. Hence, the digester can be revived but the limitation is that the efficiency of the process goes down dramatically. The potential biodegradable organic matter (methane production) in certain feeds with imbalance in C:N ratio can be improved, by mixing different wastes but the method has its limitation since microbes may not be able to metabolize the feed material in an efficient manner. Similarly, use of micro-nutrients like nickel, iron, cobalt and molybdenum have been shown to stimulate anaerobic fermentation (D.J.Hoban and L. van den Berg, J. Applied Bacteriol., 47: 153-159, 1979; R.E.Speece, G.F.Parkin and D.Gallaghes, Water Resource, 17(6): 677-683, 1983; B.Frostell, Water Sci. Tech., 23: 1179-1188, 1985; M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888,1988;N.R.Raju, S.S.Devi and Krishna Nand, Biotech. Letters, 13: 46- 465, 1991; E.P.S.Hernandez, Bioresource Technol., 47: 189-190, 1994; P.P.Rao and G.Seenayya, World J. Microbiol. Biotech., 10: 211-214, 1994; M.H.Wong and Y.H.Cheung, Bioresource Technol., 54(3): 261-268, 1995) but their affect on sick and stressed digesters has not been shown and the process is limited to a small scale studies only. • The major limitations with all these methods to regulate methane generation is the damage caused to the environment due to accumulation of chemicals in the atmosphere and the reduced economic viability of the process. In the present invention we have over come the major limitations involved in the balanced degradation of biological waste material through anaerobic digestion, which obviates the limitations listed above. The novelty of the present invention is in the use of microbial populations to develop a simple and effective method for the regulation of anaerobic digestion of biological wastes such as agro-based solid or liquid. For the first time biological agents such as fungi and H2 producing microbes have been used for relieving the stressed anaerobic digesters. The main object of the present invention is to provide a process for the regulation of anaerobic digestion of biological waste. Another object of the present invention is to provide an effective process for relieving stressed and reviving sick anaerobic digesters. Another object of the present invention is to biologically reduce the production and release of green house gas (methane) in to atmosphere. Yet another object of the present invention is to control hydrogen production from biological waste materials using micro-organisms and hydrolytic enzymes. Accordingly the present invention provides an improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer which comprises: i) preparing slurry by mixing water with the agro-based waste so as to have 0.1 to 20% solids concentration, ii) subjecting the slurry so obtained to enzymatic hydrolysis at temperature range 25° - 30°C for a period of 1 h to 10 day, with enzymes comprising cellulase, amylase and protease ( 0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in the concentration ranging from 1mg to 200 mg enzyme solids / g feed material, optionally by treating with enzyme producing fungus preferably by A. nigar, to get partially digested slurry, Hi) treating the slurry obtained in step (ii) with hydrogen producing bacteria such as herein described, so as to have 150 mg bacterial protein / L slurry at neutral pH, till hydrogen production ceases, iv) subjecting the said treated slurry in step (iii) to methanogenesis using 5 to 10% (w/w) cattle dung having methanogenic bacteria such as herein described, under anaerobic conditions for at least 6 h, at a temperature ranging 37° - 40°C, v) collecting methane produced by conventional water displacement and solids as fertilizer. In an embodiment of the present invention, biological waste may be preferably agro-bases waste used is either solid or liquid form. In yet another embodiment of the present invention, the selected agro-based waste used contains carbohydrate, protein, cellulose containing wastes. In yet another embodiment of the present invention, water used is pure water, distilled water, potable water, treated effluent. In yet another embodiment of the present invention, enzyme used for hydrolysis may be combination of cellulose, amylase and protease (0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in the concentration ranging from 1 mg to 200 mg enzyme solids / g feed material. In yet another embodiment of the present invention, enzyme used may be obtained from fungus preferably Aspergillus niger, Trichoderma viride. In yet another embodiment of the present invention, enzymatic hydrolysis may be effected at a temperature in the range of 25° to 35°C. In yet another embodiment of the present invention, the hydrogen producing bacteria may be Bacillus lichenifonnis or Bacillus subtilis. n yet another embodiment of the present invention, the days of hydrogen production may range from 4 to 12 days. In yet another embodiment of the present invention, methanogenesis may be effected at a temperature in the range of 37 to 40°C. In yet another embodiment of the present invention, the digested cattle dung may contain methanogens preferably : Methanobacterium suboxvdans. Methanobacterium formicum. Methanobacterium mobilis, Methanobacterium prooionicum. Methanocococcus mazei. Methanosarcina barkerii. Methanosarcina methanica. Methanobrevibacter. Methanomicrobium. Methanoaenium. spp. In yet another embodiment of the present invention, the agro-based waste originating from plants such as Orvza sativa. Horidium vulaare. Zee mays. Cicer arietinum. Beta vulaaris may be used as feed. Details of the invention: The process of anaerobic digestion is a rnulti-step process. In addition, it also involves the consorted effort and participation of diverse groups of bacteria. In the syntrophic association, the growth and activities of these microbes are very much dependent on the physiological conditions and their abilities to transform different organic compounds. Complex organic matter present in the biological wastes is hydrolysed into soluble compounds under partially fermentative conditions. Acidpgens and acetogenic bacteria convert these intermediates into volatile fatty acids, hydrogen, carbon dioxide and other simpler compounds. Methanogens, strict anaerobic conditions produce methane from acetic acid or from H2 and CO2- In nature, sulphate reducers and nitrate reducers compete with methanogens for H2, which leads to reduced methane production. Under certain acidic conditions, generated as a result of H2 producing bacteria, the growth and metabolism of methanogens is adversely affected, resulting in sickening of the digester. The reversal of this downward process becomes very difficult. Similarly, the generation of certain intermediates like propanoic acid and butyric acid also lead to heavy stress on the anaerobic digestion process. This kind of situation is caused by the chemical composition of the feed material. ,, Hydrogen produced during anaerobic digestion is rapidly metabolised by methanogens due to the thermodynamic shift in the reaction towards methane formation. This H2 transfer reaction is very important in this complex anaerobic digestion process. In this work, the H2 metabolism has been observed to be regulated by the use of microbes, H2-producers, fungi and the hydrolytic enzymes. It has been possible to shift H2 production process and employ microbes to control methane production. The invention has been described herein below with examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner. Example 1: Damaged wheat grains (DWG), 220 g were mixed with 200 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asoeraillus niaer (Twelve days old culture, 1.31 mg protein / ml culture) and 1.0 mL of Trichoderma viride (Twelve days old culture, 1.556 mg protein / mL culture), separately. It was then incubated at 27°C for 1 day. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 275 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (45 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 40°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly, H2 production was monitored for 10 to 11 days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 5 and 57% reduction in H2 production, respectively. However, Trichoderma viride in the presence of bacteria caused 43 to 48% reduction in H2 production compared to 77 to 80% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly. Digested sludge can be used as fertilizer. Table 1: Effect of fungal (One Day) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A niaer TV: ~L virdie Bs: R subtilis Bl: R licheniformis Example 2: Damaged wheat grains (DWG), 185 g were mixed with 180 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asperaillus niaer (Fifteen days old culture, 1.54 mg protein / ml culture) and 4.0 mL of Trichoderma viride (Ten days old culture, 0.60 mg protein / mL culture), separately. It was then incubated at 28°C for 3 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 270 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg protein), separately.. Controls were run without H2 producers and fungal treatment. DWS was incubated at 05°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 9 to 11 days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 58 and 69% reduction in H2 production, respectively. However, Trichoderma viride in the presence of bacteria caused 76 to 92% reduction in H2 production compared to 89 to 90% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly and prolonged exposure to fungi leads to greater inhibition to H2 metabolism. Digested sludge can be used as fertilizer. Table 2: Effect of fungal (Three Days) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A niaer TV: T. virdie Bs: 8. subtilis Bl: B. licheniformis Example 3: Damaged wheat grains (DWG), 200 g were mixed with 250 mL of water. Batches of 20 g feed were inoculated with 5 mL of Asperaillus niaer (Ten days old culture, 2.064 mg protein / ml culture) and 2.5 mL of Trichoderma viride (Fifteen days old culture, 0.97 mg protein / mL culture), separately. It was then incubated at 27°C for 10 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 280 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (40 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 38°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly, H2 production was monitored for 5 to 9 days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 65 and 12% reduction in H2 production, respectively. However, Trichoderma viride in the presence of bacteria caused 37 to 42% reduction in H2 production compared to 67 to 83% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly and prolonged exposure to fungi leads to greater inhibition to H2 metabolism. Digested sludge can be used as fertilizer. Table 3: Effect of fungal (Ten Days) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A. niaer TV: T. virdie Bs: B. subtilis Bl: B. licheniformis Example 4: Damaged wheat grains (DWG), 185 g were mixed with 200 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asoeraillus niaer (Fifteen days old culture, 1.032 mg protein / ml culture) and 4.0 mL of Trichoderma viride (Ten days old culture, 0.60 mg protein / mL culture), separately. It was then incubated at 28°C for 10 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 265 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 35°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 4 to 10 days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 7 and 43% reduction in H2 production, respectively. However, Trichoderma viride in the presence of bacteria caused 43 to 59% reduction in H2 production compared to 69 to 82% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly and prolonged incubation periods also reduce the abilities of the fungi to inhibit H2 metabolism. Digested sludge can be used as fertilizer. Table 4: Effect of fungal (Ten Days) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2+ C02+ H2S TS: Total solids. OS: Organic solids. An: A niaer TV: T, virdie Bs: a subtilis Bl: B. licheniformis Example 5: Damaged wheat grains (DWG), 110 g were mixed with 150 ml of water. Batches of 20 g feed were inoculated with 10 ml of Asperaillus niaer (ten days old culture, 1.32 mg protein / ml culture). It was then incubated at 25°C for 1 h. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 275 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (45 mg protein) separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 37°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 8 days. After 8 days when H2 production ceased, 25 ml enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 37°C for methanogenesis. Biogas was collected by water displacement. Biogas produced (2.5 to 3.4 L) was collected and analysed at the interval of 2 to 3 days over a period of 50 to 120 days. Digested sludge was analysed for total solids and organic solids content and used as a fertilizer. Short term fungal (A niaer) treatment of feed material resulted in 26% higher H2 production. However, in the presence of a subtilis there has been a 11% reduction in H2 production. On the other hand, the presence of a licheniformis did not influence the enhancing effect of A niaer. Subsequently, A. niaer treatment lead to 10% reduction in CH4 production i.e., from 2515 to 2275 ml. Addition of a licheniformis or a subtilis to A niaer treatment lead to 20 to 22% reduction in CH4 production. On the other hand, simultaneous addition of a licheniformis and a subtilis to A niaer lead to 75% reduction in CH4 production i.e., from 2515 to 640 ml. It can be seen that maximum reduction in CH4 production can be achieved through a combination of bacteria. Digested sludge can be used as fertilizer. Table 5: Effect of fungal (One hour) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) Example 6: Damaged wheat grains (DWG), 120 g were mixed with 125 mL of water. Batches of 20 g feed were inoculated with 10 ml of Asoeraillus niaer (ten days old culture, 2.68 mg protein / ml culture). It was then incubated at 28°C for 3 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 280 mL DWS was taken in BOD bottles and subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 40°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 8 to 12 days. After 8 to 12 days when H2 production ceased, 30 mL enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 42°C for methanogenesis. Biogas was collected by water displacement. CH4 production was analysed at the interval of 2 to 3 days over a period of 63 to 95 days. Digested slurry was analysed for total solids and organic solids content. Three day fungal (A niaen treatment of feed material resulted in 78% reduction in H2 production. However, the presence of 8. subtilis or B. licheniformis did not significantly influence the effect of A niaer. During methanogenesis of DWS, the presence of R. subtilis or a licheniformis leads up to 30% reduction in CH4 production i.e., from 3740 to 2635 mL. A niaer treatment lead to only 6% reduction in CH4 production. A niqer treatment in addition of a licheniformis or a subtilis lead up to 12% recovery in CH4 production. It can be seen that recovery in CH4 production can be achieved through longer fungal treatment. Digested sludge can be used as fertilizer. Table 6: Effect of fungal (Three days) and bacterial treatment on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S Biogas: CHU + CO2 + H2S TS: Total solids. OS: Organic solids. An: A niaer Bs: R subtilis Bl: a licheniformis. nd: not determined. Example 7: Damaged wheat grains (DWG), 185 g were mixed with 200 mL of water. 60 mL enzyme solution was prepared in phosphate buffer by adding 12 g enzyme solids. Batches of 20 g feed material were incubated with enzyme solution at the final concentrations of 1, 50 and 200 mg enzyme solids/ g feed material. It was then incubated at 37°C for 1 day. Damaged wheat grain slurry (DWS) 275 mL, at 20 g feed material was prepared by mixing with water in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis culture (40 mg protein). Controls were run without H2 producers and enzyme treatment. DWS was incubated at 35°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 6 to 8 days. After 6 to 8 days when H2 production ceased, 20 mL enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 40°C for methanogenesis. Biogas was collected by water displacement. CH4 production was analysed at the interval of 2 to 3 days over a period of 100 days. Enzyme treatment of feed material resulted in 40 to 77% reduction in H2 production. Even in the presence of B. licheniformis. the inhibitory effect of enzyme treatment on H2 production was evident. There was 63 to 70% reduction in H2 production. During methanogenesis of DWS, enzyme treatment at 1mg/ g feed material led to 11% reduction in CH4 production in comparison to control. At higher enzyme concentrations, the effect on CH4 production was quite significant, resulting in 27 to 40% improvement. The presence of 8. licheniformis along with the enzyme treatment led to reversal of inhibitory effect of enzyme (1 mg/ g feed). The recovery in CH4 production was up from 2715 ml to 3030 mL i.e., 11% higher. Thus inhibition in CH4 production caused low enzyme concentrations could be reversed by treatment with 8. licheniformis . Digested sludge can be used as fertilizer. Table 7: Effect of enzyme treatment (One day) on anaerobic digestion of damaged wheat grains. (Table Removed) DAI: Days after incubation. Biogas-H: H2 + C02 + H2S Biogas: CH4 + H2S E1: 1 mg enzyme solids / g feed material. E50: 50 mg enzyme solids / g feed material. E200: 200 mg enzyme solids / g feed material. Bl: B licheniformis. The main advantages of the present invention are: 1. The main advantage is the environment-friendly nature of the method. 2. The process can be employed to relieve stressed anaerobic digesters. 3. The process helps to revive sick digesters. 4. The process can be used to reduce the release of green house gases into the environment. We Claim: 1. An improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer which comprises: i) preparing slurry by mixing water with the agro-based waste so as to have 0.1 to 20% solids concentration, ii) subjecting the slurry so obtained to enzymatic hydrolysis at temperature range 25° - 30°C for a period of 1 h to 10 day, with enzymes comprising cellulase, amylase and protease ( 0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in the concentration ranging from 1mg to 200 mg enzyme solids / g feed material, optionally by treating with enzyme producing fungus preferably by A. nigar, to get partially digested slurry, iii) treating the slurry obtained in step (ii) with hydrogen producing bacteria such as herein described, so as to have 150 mg bacterial protein / L slurry at neutral pH, till hydrogen production ceases, iv) subjecting the said treated slurry in step (iii) to methanogenesis using 5 to 10% (w/w) cattle dung having methanogenic bacteria such as herein described, under anaerobic conditions for at least 6 h, at a temperature ranging 37° - 40°C, v) collecting methane produced by conventional water displacement and solids as fertilizer. 2. An improved process as claimed in claims 1 wherein agro-based waste used is selected from plants preferably O/yza sative, Horidium vulgare. Zee mays, Cicer arietinum, Beta vulgaris . 3. An improved process as claimed in claims 1-2 wherein the hydrogen producing bacterium is Bacillus licheniformis or Bacillus subtilis. 4. A process as claimed in claims 1-3 wherein methanogenesis is effected at a temperature in the range of 37° to 40°C. 5. a process as claimed in claims 1-4 wherein the digested cattle dung contains methanogens such as : Methanobacterium suboxydans, Methanobacterium formicum, methanobacterium mobilis, Methanobacterium propionicum, Methanocococcus mazei, Methanosarcina barkerii, Methanosarcina methanica, Methanobrevibacter, Methanomicrobium, Methanogenium, spp.. 6. An improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer as here in described with reference to examples accompanying this specification.

Full Text

The invention relates to an improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer
The present process particularly relates to a process for the regulation of anaerobic digestion of waste biological materials.
The main utility of the present invention is to provide an environment-friendly method for degradation of waste by controlling anaerobic digestion resulting in t6

biogas - a source of energy and fertiliser simultaneously. Another utility of this process is to relieve stressed anaerobic digesters and revive sick digesters. Another utility of this process is to reduce the release of green house gases into the environment and to mitigate global warming.
Anaerobic digestion is gaining importance as a process to treat and recover energy from organic matter rich biological wastes (R.B.Dean, Biocycle, 2: 75-77, 1998). Anaerobic digestion is a multi-step process. It involves the consorted effort and participation of diverse groups of bacteria. In the syntrophic association, the growth and activities of these microbes are very much dependant on the physiological conditions and their abilities to transform different organic compounds. (H.J.Thiele, M.Chartrain and J.G.Zeikus, Applied Environ. Microbiol., 54(1): 10-19, 1988).
In addition to organised anaerobic digestion of biological waste materials, such a process operates in landfills, rice cultivation, domestic ruminant rearing,
biomass burning, coal mining, and natural gas venting. Currently CH4 concentration in the atmosphere is increasing at a rate of about 0.015 ppmv (0.9%) per year. Global annual release of CH4 from rice paddy has been
12
estimated to be 150 Tg (Tg, giga tons = 1x10 ). The impact of these Green
House Gases (GHGs) on environment is evident by the rising temperature, termed as Global Warming. (A.Holzapfel-Pschorn, R.Conrad and W.Seller, Plant Soil, 92: 223-233, 1986; B.S.Rajgopal, N.Beley and L.Daniels, FEMS Microbiol.
Ecology, 53: 153-158,1988; K.Sipila, A.Johansson and K.Saviharju, Bioresource Technol., 43:7-12, 1993).
Complex organic matter present in the biological wastes is hydrolysed into soluble compounds under partially fermentative conditions. Acidogens and acetogenic bacteria convert these intermediates into volatile fatty acids, hydrogen, carbon dioxide and other simpler compounds. (H.J.Thiele, M.Chartrain and J.G.Zeikus, Applied Environ. Microbiol., 54(1): 10-19, 1988). Methanogens,
under strict anaerobic conditions produce methane from acetic acid and from H2 and C02. In nature, sulphate reducers and nitrate reducers compete with
methanogens for Ha, which leads to reduced methane production. (M.G.Hilton
and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988; D.M.McCartney and J.A.OIeszkiewicz, Water Environ. Res., 65: 665, 1993). Hydrogen production and fatty acid production are linked to lowering of pH. Under certain acidic
conditions, generated as a result of H2 producing bacteria, the growth and
metabolism of methanogens is adversely affected, which causes sickening of the digester. (R.I.Mackie and M.P.Bryant, Applied Microbiol. Biotechnology, 43: 346-350, 1995). The reversal of this downward process becomes very difficult. Similarly, the generation and accumulation of certain intermediates like propanoic
acid and butyric acid can lower the pH in a reactor and raised H2 levels introduce
instability in to the microbial community (F.E.Mosey, Water Pollut. Control, 81: 540, 1982; D.B.Archer, M.G.Hilton, P.Adams and H.Wiecko, Biotechnol. Lett., 8: 197, 1986) due to heavy stress on the anaerobic digestion process. This kind of situation is caused by the chemical composition of the feed material also. Moreover, sulphate reducing bacteria out compete methanogens at elevated levels of propanoic and butyric acid even at low acetate concentrations (F.Omil, P.Lens, P.LHulshoff and G.Lettinga, Process Biochem., 31(7): 699-710, 1996).
Inhibition of sulphate reduction declines the quantity of H2S produced. However, it is accompanied by increase in propionic acid ahead of acetic acid. Sulphate
reducing bacteria can metabolize propionate directly or in association with proton reducing acetogenic species, by H2 removal (F.W.J.Hoeks, H.J.G. TAN Hoopen, J.A.Roels and J.G.Kuenen, Anaerobic Waste Water Treatment, W.J.VANDEN Brink, Ed. THO Corporate Communications Department, The Hague, pp. 554-555, 1983; Z. Isa, S.Grusenmeyer and W.Verstraete, Appl. Environ. Microbiol., 51: 572, 1986; M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988). Sulphide inhibits propionate utilization (A.Rinzema and G.Lettinga, Environ. Technol. Lett., 9: 83, 1988) and proipionate buildup if not rectified may lead to process failure. A 50% inhibition of methanogenesis from acetate occurs
at a HaS concentration of 35.2 mM (1200 mg/L) (Z.lsa, S.Grusenmeyer,
W.Verstrate, Appl. Environ. Microbiol., 51: 572, 1986) at a total solid concentration of 100 to 224 mg/L (P.P.Karhadkar, J.M.Audic, C.M.Faup, P.Khanna, Water Res. (G.B.), 21:1061,1987).
Hydrogen produced during anaerobic digestion is rapidly metabolised by methanogens due to the thermodynamic shift in the reaction towards methane
formation. This H£ transfer reaction is very important in this complex anaerobic
digestion process. (S.S.Ozturk, B.O.Palsson and J.H.Thiele, Biotech. Bioeng., 33:745-757, 1989).
The process of anaerobic digestion is influenced by a wide array of environmental conditions. The rate of microbial methane production increases with rise in temperature. However, the amount of free ammonia also increases with temperature, the bio-digestive performance could be inhibited or even reduced as a result. (G.FIueh and P. Hubes, Patent No. De 4341713 dt. 08.06.95; O.P.Chawla, in: Advances in Biogas Technology, PID, ICAR, New Delhi, 1986). Higher concentration of available nutrients like copper, 10-250 mg/L; calcium and sodium, 8000 mg/L; magnesium, 3000 mg/L; nickel, 10-1000 mg/L; zinc, 350-1000 mg/L; chromium, 200-2000 mg/L; sulphide (as sulphur), 200 mg/L and cyanide, 2 mg/L usually have inhibitory effect. (I.P.Pankhania and
J.P.Robinson, FEMS Microbiol. Ecology, 38: 309-312, 1986; A.Rinzema, J. vanLier and G.Lettinga, Enzyme Microbiol. Technol., 10: 24-32, 1988). The methaneproducing bacteria live best under neutral to slightly alkaline conditions. If the pH value drops below 6.2, the medium has a toxic effect on the methanogenic bacteria. Similarly, for higher pH values, even a relatively low nitrogen concentration of roughly 1700 mg ammonium-nitrogen (NH4-N) per litre
substrate methanogenesis get affected. Various experiments have shown that the metabolic activity of methanogenic bacteria can be optimized at a C/N ratio of approximately 8 to 20. High carbohydrate concentration has an inhibitory effect on methanogenesis (S.Roychaudhury, D.Cox and M.Levandowsky, Int. J. of Hydrogen Energy, 13(7): 407-410, 1983). The presence of heavy metals, antibiotics (Bacitracin, Flavomycin, Lasalocid, Monensin, Spirmycin, etc.) and detergents used in livestock husbandry can have inhibitory effect on the process of biomethanation. (O.P.Chawla, in: Advances in Biogas Technology, PID, ICAR, New Delhi, 1986; R.E.Khabibullin, N.I.Krylova and R.P.Naumova, Bioteckhnologiya, 2: 43-46, 1995).
Similarly, high loading rates invariably lead to the accumulation of volatile fatty acids like propanoic acid and butyric acid. This stress results in an imbalance in the metabolisms of different microbes and consequently, the biomethanation process stops operating. (D.T.Hill and J.P. Bolte, Transactions of the ASAE 30(2): 502, 1987; D.T.Hill, S.A.Cobb and J.P. Bolte, Transactions of the ASAE 30(2): 496, 1987; B.K.Ahring and P.Westermann, Appl. Environ. Microbiol., 53(2): 434, 1987).
Hydrolytic conversion of organic matter to the available substrate for the anaerobic microflora is a rate limiting step for the organic waste degradation (J.J.Zabranska, M.Bosanka, P.Krempa, P.Jenicek and M.Rohananyos, Meded. Fac. Landobounwet Rijsunsi. Gent., 60(4b): 2217; 1995). In waste containing high lignin content, the lignocellulosic portion present in waste is the major substrate for methane production and it is believed that it's hydrolysis is the
limiting step in the overall anaerobic digestion process as well (R.K.Malik and P.Tauro, Indian J. Microbiol., 35(3): 205; 1995).
The problem of reduction in methane production or even complete failure of the digester can be solved to a very limited extent by drastic reduction in loading rates and or prolonged retention periods. (C.Ramakrishna and J.D.Desai, World J. Microbiol. Biotech., 13: 329-334, 1997). This method has a major limitation since it demands larger reactor volume for smaller waste material quantities i.e. it is an uneconomical proposition. Dilution of the feed material. (M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888, 1988) can result in lowering the concentration of certain toxic materials but will also lead to lowering the availability of food to the microbes. Hence, the digester can be revived but the limitation is that the efficiency of the process goes down dramatically. The potential biodegradable organic matter (methane production) in certain feeds with imbalance in C:N ratio can be improved, by mixing different wastes but the method has its limitation since microbes may not be able to metabolize the feed material in an efficient manner. Similarly, use of micro-nutrients like nickel, iron, cobalt and molybdenum have been shown to stimulate anaerobic fermentation (D.J.Hoban and L. van den Berg, J. Applied Bacteriol., 47: 153-159, 1979; R.E.Speece, G.F.Parkin and D.Gallaghes, Water Resource, 17(6): 677-683, 1983; B.Frostell, Water Sci. Tech., 23: 1179-1188, 1985; M.G.Hilton and D.B.Archer, Biotechnol. and Bioengn., 31: 885-888,1988;N.R.Raju, S.S.Devi and Krishna Nand, Biotech. Letters, 13: 46- 465, 1991; E.P.S.Hernandez, Bioresource Technol., 47: 189-190, 1994; P.P.Rao and G.Seenayya, World J. Microbiol. Biotech., 10: 211-214, 1994; M.H.Wong and Y.H.Cheung, Bioresource Technol., 54(3): 261-268, 1995) but their affect on sick and stressed digesters has not been shown and the process is limited to a small scale studies only.
•
The major limitations with all these methods to regulate methane generation is the damage caused to the environment due to accumulation of chemicals in the atmosphere and the reduced economic viability of the process.
In the present invention we have over come the major limitations involved in the balanced degradation of biological waste material through anaerobic digestion, which obviates the limitations listed above.
The novelty of the present invention is in the use of microbial populations to develop a simple and effective method for the regulation of anaerobic digestion of biological wastes such as agro-based solid or liquid.
For the first time biological agents such as fungi and H2 producing microbes have been used for relieving the stressed anaerobic digesters.
The main object of the present invention is to provide a process for the regulation of anaerobic digestion of biological waste.
Another object of the present invention is to provide an effective process for relieving stressed and reviving sick anaerobic digesters.
Another object of the present invention is to biologically reduce the production and release of green house gas (methane) in to atmosphere.
Yet another object of the present invention is to control hydrogen production from biological waste materials using micro-organisms and hydrolytic enzymes.
Accordingly the present invention provides an improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer which comprises: i) preparing slurry by mixing water with the agro-based waste so as to have 0.1 to 20%
solids concentration, ii) subjecting the slurry so obtained to enzymatic hydrolysis at temperature range 25° -
30°C for a period of 1 h to 10 day, with enzymes comprising cellulase, amylase and
protease ( 0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in
the concentration ranging from 1mg to 200 mg enzyme solids / g feed material,
optionally by treating with enzyme producing fungus preferably by A. nigar, to get
partially digested slurry, Hi) treating the slurry obtained in step (ii) with hydrogen producing bacteria such as
herein described, so as to have 150 mg bacterial protein / L slurry at neutral pH, till
hydrogen production ceases, iv) subjecting the said treated slurry in step (iii) to methanogenesis using 5 to 10%
(w/w) cattle dung having methanogenic bacteria such as herein described, under
anaerobic conditions for at least 6 h, at a temperature ranging 37° - 40°C, v) collecting methane produced by conventional water displacement and solids as
fertilizer.
In an embodiment of the present invention, biological waste may be preferably agro-bases waste used is either solid or liquid form.
In yet another embodiment of the present invention, the selected agro-based waste used contains carbohydrate, protein, cellulose containing wastes.
In yet another embodiment of the present invention, water used is pure water, distilled water, potable water, treated effluent.
In yet another embodiment of the present invention, enzyme used for hydrolysis may be combination of cellulose, amylase and protease (0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in the concentration ranging from 1 mg to 200 mg enzyme solids / g feed material.
In yet another embodiment of the present invention, enzyme used may be obtained from fungus preferably Aspergillus niger, Trichoderma viride.
In yet another embodiment of the present invention, enzymatic hydrolysis may be effected at a temperature in the range of 25° to 35°C.
In yet another embodiment of the present invention, the hydrogen producing bacteria may be Bacillus lichenifonnis or Bacillus subtilis.
n yet another embodiment of the present invention, the days of hydrogen production may range from 4 to 12 days.
In yet another embodiment of the present invention, methanogenesis may be effected at a temperature in the range of 37 to 40°C.
In yet another embodiment of the present invention, the digested cattle dung may contain methanogens preferably : Methanobacterium suboxvdans. Methanobacterium formicum. Methanobacterium mobilis, Methanobacterium prooionicum. Methanocococcus mazei. Methanosarcina barkerii. Methanosarcina methanica. Methanobrevibacter. Methanomicrobium. Methanoaenium. spp.
In yet another embodiment of the present invention, the agro-based waste originating from plants such as Orvza sativa. Horidium vulaare. Zee mays. Cicer arietinum. Beta vulaaris may be used as feed.
Details of the invention:
The process of anaerobic digestion is a rnulti-step process. In addition, it also involves the consorted effort and participation of diverse groups of bacteria. In the syntrophic association, the growth and activities of these microbes are very much dependent on the physiological conditions and their abilities to transform different organic compounds.
Complex organic matter present in the biological wastes is hydrolysed into soluble compounds under partially fermentative conditions. Acidpgens and acetogenic bacteria convert these intermediates into volatile fatty acids, hydrogen, carbon dioxide and other simpler compounds. Methanogens, strict
anaerobic conditions produce methane from acetic acid or from H2 and CO2- In nature, sulphate reducers and nitrate reducers compete with methanogens for H2, which leads to reduced methane production. Under certain acidic conditions,
generated as a result of H2 producing bacteria, the growth and metabolism of methanogens is adversely affected, resulting in sickening of the digester. The
reversal of this downward process becomes very difficult. Similarly, the generation of certain intermediates like propanoic acid and butyric acid also lead to heavy stress on the anaerobic digestion process. This kind of situation is caused by the chemical composition of the feed material.
,, Hydrogen produced during anaerobic digestion is rapidly metabolised by methanogens due to the thermodynamic shift in the reaction towards methane
formation. This H2 transfer reaction is very important in this complex anaerobic
digestion process. In this work, the H2 metabolism has been observed to be regulated
by the use of microbes, H2-producers, fungi and the hydrolytic enzymes. It has been possible to shift H2 production process and employ microbes to control methane production.
The invention has been described herein below with examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner.
Example 1:
Damaged wheat grains (DWG), 220 g were mixed with 200 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asoeraillus niaer (Twelve days old culture, 1.31 mg protein / ml culture) and 1.0 mL of Trichoderma viride (Twelve days old culture, 1.556 mg protein / mL culture), separately. It was then incubated at 27°C for 1 day. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 275 mL of this DWS was taken in BOD bottles. DWS was
subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (45 mg
protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 40°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were
estimated and recorded regularly, H2 production was monitored for 10 to 11
days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 5 and
57% reduction in H2 production, respectively. However, Trichoderma viride in
the presence of bacteria caused 43 to 48% reduction in H2 production compared to 77 to 80% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly. Digested sludge can be used as fertilizer.
Table 1: Effect of fungal (One Day) and bacterial treatment on anaerobic digestion of damaged wheat grains.

(Table Removed)
DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A niaer TV: ~L virdie Bs: R subtilis Bl: R licheniformis
Example 2:
Damaged wheat grains (DWG), 185 g were mixed with 180 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asperaillus niaer (Fifteen days old culture, 1.54 mg protein / ml culture) and 4.0 mL of Trichoderma viride (Ten days old culture, 0.60 mg protein / mL culture), separately. It was then incubated at 28°C for 3 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 270 mL of this DWS was taken in BOD bottles. DWS was
subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg
protein), separately.. Controls were run without H2 producers and fungal treatment. DWS was incubated at 05°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were
estimated and recorded regularly. H2 production was monitored for 9 to 11 days.
Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 58 and
69% reduction in H2 production, respectively. However, Trichoderma viride in
the presence of bacteria caused 76 to 92% reduction in H2 production compared to 89 to 90% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary
significantly and prolonged exposure to fungi leads to greater inhibition to H2 metabolism. Digested sludge can be used as fertilizer.
Table 2: Effect of fungal (Three Days) and bacterial treatment on anaerobic digestion of damaged wheat grains.

(Table Removed)
DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A niaer TV: T. virdie Bs: 8. subtilis Bl: B. licheniformis
Example 3:
Damaged wheat grains (DWG), 200 g were mixed with 250 mL of water. Batches of 20 g feed were inoculated with 5 mL of Asperaillus niaer (Ten days old culture, 2.064 mg protein / ml culture) and 2.5 mL of Trichoderma viride (Fifteen days old culture, 0.97 mg protein / mL culture), separately. It was then incubated at 27°C for 10 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 280 mL of this DWS was taken in BOD bottles.
DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis
cultures (40 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 38°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were
estimated and recorded regularly, H2 production was monitored for 5 to 9 days.
Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 65 and
12% reduction in H2 production, respectively. However, Trichoderma viride in
the presence of bacteria caused 37 to 42% reduction in H2 production compared to 67 to 83% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary
significantly and prolonged exposure to fungi leads to greater inhibition to H2 metabolism. Digested sludge can be used as fertilizer.
Table 3: Effect of fungal (Ten Days) and bacterial treatment on anaerobic digestion of damaged wheat grains.

(Table Removed)
DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S TS: Total solids. OS: Organic solids. An: A. niaer TV: T. virdie Bs: B. subtilis Bl: B. licheniformis
Example 4:
Damaged wheat grains (DWG), 185 g were mixed with 200 mL of water. Batches of 20 g feed were inoculated with 10 mL of Asoeraillus niaer (Fifteen days old culture, 1.032 mg protein / ml culture) and 4.0 mL of Trichoderma viride (Ten days old culture, 0.60 mg protein / mL culture), separately. It was then incubated at 28°C for 10 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 265 mL of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 35°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 4 to 10 days. Digested slurry was analysed for total solids and organic solids content. Trichoderma viride and A. niaer treatment of feed material resulted in 7 and
43% reduction in H2 production, respectively. However, Trichoderma viride in the presence of bacteria caused 43 to 59% reduction in H2 production compared to 69 to 82% reduction caused by A niaer under similar conditions. It can be seen that the abilities of fungal species to influence H2 production vary significantly and prolonged incubation periods also reduce the abilities of the fungi to inhibit H2 metabolism. Digested sludge can be used as fertilizer.
Table 4: Effect of fungal (Ten Days) and bacterial treatment on anaerobic digestion of damaged wheat grains.

(Table Removed)
DAI: Days after incubation. Biogas-H: H2+ C02+ H2S TS: Total solids. OS: Organic solids. An: A niaer TV: T, virdie Bs: a subtilis Bl: B. licheniformis
Example 5:
Damaged wheat grains (DWG), 110 g were mixed with 150 ml of water. Batches of 20 g feed were inoculated with 10 ml of Asperaillus niaer (ten days old culture, 1.32 mg protein / ml culture). It was then incubated at 25°C for 1 h. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 275 mL
of this DWS was taken in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (45 mg protein) separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at
37°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2
production was monitored for 8 days. After 8 days when H2 production ceased,
25 ml enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 37°C for methanogenesis. Biogas was collected by water displacement. Biogas produced (2.5 to 3.4 L) was collected and analysed at the interval of 2 to 3 days over a period of 50 to 120 days. Digested sludge was analysed for total solids and organic solids content and used as a fertilizer. Short term fungal (A niaer) treatment of feed material
resulted in 26% higher H2 production. However, in the presence of a subtilis
there has been a 11% reduction in H2 production. On the other hand, the presence of a licheniformis did not influence the enhancing effect of A niaer. Subsequently, A. niaer treatment lead to 10% reduction in CH4 production i.e., from 2515 to 2275 ml. Addition of a licheniformis or a subtilis to A niaer treatment lead to 20 to 22% reduction in CH4 production. On the other hand, simultaneous addition of a licheniformis and a subtilis to A niaer lead to 75% reduction in CH4 production i.e., from 2515 to 640 ml. It can be seen that
maximum reduction in CH4 production can be achieved through a combination of bacteria. Digested sludge can be used as fertilizer.
Table 5: Effect of fungal (One hour) and bacterial treatment on anaerobic digestion of damaged wheat grains.
(Table Removed)

Example 6:
Damaged wheat grains (DWG), 120 g were mixed with 125 mL of water. Batches of 20 g feed were inoculated with 10 ml of Asoeraillus niaer (ten days old culture, 2.68 mg protein / ml culture). It was then incubated at 28°C for 3 days. Damaged wheat grain slurry (DWS) was prepared by mixing with water. 280 mL DWS was taken in BOD bottles and subjected to H2 producers Bacillus licheniformis and B. subtilis cultures (50 mg protein), separately. Controls were run without H2 producers and fungal treatment. DWS was incubated at 40°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 8 to 12 days. After 8 to 12 days when H2 production ceased, 30 mL enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 42°C for methanogenesis. Biogas was collected by water displacement. CH4 production was analysed at the interval of 2 to 3 days over a period of 63 to 95 days. Digested slurry was analysed for total solids and organic solids content. Three day fungal (A niaen treatment of feed material resulted in 78% reduction in H2 production. However, the presence of 8. subtilis or B. licheniformis did not significantly influence the effect of A niaer.
During methanogenesis of DWS, the presence of R. subtilis or a licheniformis leads up to 30% reduction in CH4 production i.e., from 3740 to 2635 mL. A niaer treatment lead to only 6% reduction in CH4 production. A niqer treatment in addition of a licheniformis or a subtilis lead up to 12% recovery in CH4 production. It can be seen that recovery in CH4 production can be achieved through longer fungal treatment. Digested sludge can be used as fertilizer.
Table 6: Effect of fungal (Three days) and bacterial treatment on anaerobic digestion of damaged wheat grains.

(Table Removed)
DAI: Days after incubation. Biogas-H: H2 + CO2 + H2S Biogas: CHU + CO2 +
H2S TS: Total solids. OS: Organic solids. An: A niaer Bs: R subtilis Bl: a licheniformis. nd: not determined.
Example 7:
Damaged wheat grains (DWG), 185 g were mixed with 200 mL of water. 60 mL enzyme solution was prepared in phosphate buffer by adding 12 g enzyme solids. Batches of 20 g feed material were incubated with enzyme solution at the final concentrations of 1, 50 and 200 mg enzyme solids/ g feed material. It was then incubated at 37°C for 1 day. Damaged wheat grain slurry (DWS) 275 mL, at 20 g feed material was prepared by mixing with water in BOD bottles. DWS was subjected to H2 producers Bacillus licheniformis culture (40 mg protein). Controls were run without H2 producers and enzyme treatment. DWS was incubated at 35°C. Biogas-H was collected by water displacement. H2 was analysed by gas chromatography and volumes were estimated and recorded regularly. H2 production was monitored for 6 to 8 days. After 6 to 8 days when H2 production ceased, 20 mL enriched methanogenic slurry was mixed with 250 ml of the partially digested slurry. It was then incubated at 40°C for methanogenesis. Biogas was collected by water displacement. CH4 production was analysed at the interval of 2 to 3 days over a period of 100 days. Enzyme treatment of feed material resulted in 40 to 77% reduction in H2 production. Even in the presence
of B. licheniformis. the inhibitory effect of enzyme treatment on H2 production was evident. There was 63 to 70% reduction in H2 production.
During methanogenesis of DWS, enzyme treatment at 1mg/ g feed material led to 11% reduction in CH4 production in comparison to control. At higher enzyme concentrations, the effect on CH4 production was quite significant, resulting in 27 to 40% improvement. The presence of 8. licheniformis along with the enzyme treatment led to reversal of inhibitory effect of enzyme (1 mg/ g feed). The recovery in CH4 production was up from 2715 ml to 3030 mL i.e., 11% higher. Thus inhibition in CH4 production caused low enzyme concentrations could be reversed by treatment with 8. licheniformis . Digested sludge can be used as fertilizer.
Table 7: Effect of enzyme treatment (One day) on anaerobic digestion of damaged wheat grains.
(Table Removed)
DAI: Days after incubation. Biogas-H: H2 + C02 + H2S Biogas: CH4 +
H2S E1: 1 mg enzyme solids / g feed material. E50: 50 mg enzyme solids / g feed material. E200: 200 mg enzyme solids / g feed material. Bl: B licheniformis.
The main advantages of the present invention are:
1. The main advantage is the environment-friendly nature of the method.
2. The process can be employed to relieve stressed anaerobic digesters.

3. The process helps to revive sick digesters.
4. The process can be used to reduce the release of green house gases into the
environment.

We Claim:
1. An improved process for anaerobic digestion of agro-based wastes with simultaneous production of methane and fertilizer which comprises:
i) preparing slurry by mixing water with the agro-based waste so as to have 0.1 to 20% solids concentration,
ii) subjecting the slurry so obtained to enzymatic hydrolysis at temperature range 25° - 30°C for a period of 1 h to 10 day, with enzymes comprising cellulase, amylase and protease ( 0.98 U/mg solids, 0.8 U/mg solids and 0.013 U/mg solids, respectively) in the concentration ranging from 1mg to 200 mg enzyme solids / g feed material, optionally by treating with enzyme producing fungus preferably by A. nigar, to get partially digested slurry,
iii) treating the slurry obtained in step (ii) with hydrogen producing bacteria such as herein described, so as to have 150 mg bacterial protein / L slurry at neutral pH, till hydrogen production ceases,
iv) subjecting the said treated slurry in step (iii) to methanogenesis using 5 to 10% (w/w) cattle dung having methanogenic bacteria such as herein described, under anaerobic conditions for at least 6 h, at a temperature ranging 37° - 40°C,
v) collecting methane produced by conventional water displacement and solids as fertilizer.
2. An improved process as claimed in claims 1 wherein agro-based waste
used is selected from plants preferably O/yza sative, Horidium vulgare.
Zee mays, Cicer arietinum, Beta vulgaris .
3. An improved process as claimed in claims 1-2 wherein the hydrogen
producing bacterium is Bacillus licheniformis or Bacillus subtilis.
4. A process as claimed in claims 1-3 wherein methanogenesis is effected
at a temperature in the range of 37° to 40°C.
5. a process as claimed in claims 1-4 wherein the digested cattle dung
contains methanogens such as : Methanobacterium suboxydans,
Methanobacterium formicum, methanobacterium mobilis,
Methanobacterium propionicum, Methanocococcus mazei,
Methanosarcina barkerii, Methanosarcina methanica, Methanobrevibacter,
Methanomicrobium, Methanogenium, spp..
6. An improved process for anaerobic digestion of agro-based wastes with
simultaneous production of methane and fertilizer as here in described
with reference to examples accompanying this specification.

Documents:

1035-del-2000-abstract.pdf

1035-del-2000-claims.pdf

1035-del-2000-correspondence-others.pdf

1035-del-2000-correspondence-po.pdf

1035-del-2000-description (complete).pdf

1035-del-2000-form-1.pdf

1035-del-2000-form-19.pdf

1035-del-2000-form-2.pdf

1035-del-2000-form-3.pdf

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Patent Number

232357

Indian Patent Application Number

1035/DEL/2000

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

16-Mar-2009

Date of Filing

17-Nov-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	VIPIN CHANDRA KALIA	CENTER FOR BIOCHEMICAL TECHNOLOGY, MALL ROAD, UNIVERSITY CAMPUS, DELHI-110007, INDIA.
2	VIKAS SONAKYA	CENTER FOR BIOCHEMICAL TECHNOLOGY, MALL ROAD, UNIVERSITY CAMPUS, DELHI-110007, INDIA.
3	NEENA RAIZADA	CENTER FOR BIOCHEMICAL TECHNOLOGY, MALL ROAD, UNIVERSITY CAMPUS, DELHI-110007, INDIA.

PCT International Classification Number

C02F 003/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA