Title of Invention

AN IMPROVED PROCESS FOR BIOMETHANATION USING FERMENTATIVE HYDROGEN PRODUCERS.

Abstract

A process for improved biomethanation using fermentative hydrogen producers by preparing slurry of the biological waste with water, mixing the said slurry with fresh cattle dung in the ratio of 9:0.5 to 9:1% by weight,incubating the mixed slurry under fermentative conditions in known manner for 2 to 4 days, homogensing the fermented slurry and optionally removing ligno-cellulosic fibres by sieving, to obtain liquefied slurry, subjecting the liquefied slurry obtained in step to fermentative hydrogen producers by adding novel strains of Bacillus and methanogens by adding to it 5 to 10% (w/w) digested cattle dung in the form of slurry,incubating the mixture obtained in step (v) for 6 h at pH in the range of 6.8 to 7.2, for biomethanation under strict anaerobic conditions in a digester,collecting the biogas produced in gas holders by water displacement.

Full Text

The present invention relates to a process for propation of biogen using fermentative hydrogen producers. The main utility of present invention is to provide an effective method to overcome the problem of process instability during both start-up and operation of biomethanation. The present invention makes use of fermentative hydrogen producers to improve biomethanation process. Anaerobic treatment process is increasingly recognized as the core method of an advanced technology for environmental protection and resource preservation. (L. Seghezzo, G. Zeeman, J.B. van Lier, H.V.M. Hamelers and G. Lettinga, Bioresource Technology, 65: 175; 1998). It becomes imperative to look for renewable energy sources due to increased environmental problems like Global warming and rapidly depleting fossil fuel reserves. (A. Hansel and P. Lindblad, Applied Microbiol. and Biotechnology, 50: 153; 1998). Anaerobic digestion has been applied to derive energy from different wastes (I.J.Callander and J.P.Barford, Process Biochem., 18(8): 24;1996). Anaerobic digestion is a process by which most organic wastes (except lignocellulosic content rich waste) can be biologically converted into energy rich methane gas. This complex process, therefore requires specific environmental conditions and various bacterial populations (N. Sharma and G.Pellizzi, Energy Convers. Mgmt., 32 (5): 447:1991). The National Dairy Development Board (NDDB) Fruit and Vegetable Unit, Mangolpuri, New Delhi, INDIA, alone generates about 8000-10000 tonnes of green pea-shells annually. These waste are till now sent to landfills but in recent years their disposal has become a problem, due to increasing cost of transportation and scarcity of landfill sites for quick disposal. (V.C.Kalia and A.P.Joshi, Bioresource Technology, 53:165;1995; S.K.Madhukar, H.R.Srilatha, K. Srinath, K.Bharthi and K.Nand, Process Biochem., 32(6):509;1997). The major limitations encountered in anaerobic digestion of biological wastes is due to high organic matter over loading into the digester. Under these conditions, accumulation of higher volatile fatty acids results in physiological stress on microbial population and consequent digester failure. (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 & P.Westermann, Appl. Environ. Microbiol., 53(2): 434; 1987). Methane production during anaerobic digestion is preceded by hydrogen production, which serves as substrate for the former. However, thermodynamically methane production is favoured, no net evolution of hydrogen is observed in these syntrophic populations. (W.Verstraete, D. de Beer, M. Pena, G. Lettinga and P.Lens, World J. Microbiol. and Biotechnol., 12: 221; 1996). Hydrogen producers have a very active role to play in this metabolism. H2 has the potential for feed back control of acidogenic reaction. (I.S.Kim, J.C.Young and H.H.Tabak, Water Environment Research, 66 (3) : 119; 1994). It thus becomes another limitation for active methanogenesis leading to stress conditions. Hydrogen is also quenched by other bacteria like nitrate reducers, sulphate reducers, etc. (A.Visser, Y.Gao and G. Lettinga, Bioresource Technology, 44: 113; 1993). Thirty per cent of the total methane production is due to H2 and CO2 combination while the rest is via acetic acid route. Normally, propionate and butyrate accounts for 20% of total methane produced in the digester (P. Fongastitkul, D.S.Mavinic and K.V. Lo, Water Environ. Research, 66 (3): 243; 1994) but under stress conditions there is an accumulation of these acids. Consequently, there is excess of H2 generation resulting in digester failure. (B.K.Ahring and P.Westermann, Applied Environ. Microbiol., 53 (2): 434; 1987). Till date some conventional process parameters employed for the improving biomethanation of bio-waste materials are briefly described below: For improving the biomethanation process efficiency, recycling of biogas through the digester or stirring of waste matter in digester was done (M.Hunziker and A.Schildknecht, U.S. Patent No. 4,511,370 dt Apr.16,1985, D.O.Hitzman, U.S.Patent No. 4,798,801 Dt Jan.17,1989, M.F.Peters and W.T.Peters, U.S. Patent No. 4,238,337 dt Dec.9,1980, D.W.Grabis, U.S. Patent No. 4,394,136 Dt Jul.19,1983). Another proposal to improve the waste handling and processing is to produce a mixture of hydrogen and methane under 2 stage process (J.Benemann, Nature Biotechnology, 14: 1101, 1996). However, fermentative hydrogen producers have not yet been employed to improve biomethanation. For increasing digestibility of such ligno-cellulosic wastes, various chemical and physical pre-treatments, e.g., acid and alkali treatment, high temperature exposure and selective microbial environment (R.K. Malik and P.Tauro, Indian J. Microbiol., 35 (3) :205; 1995; P.A.Scherer and R.Vollmer,1995. Patent No. DE-; DE 19516378; 26.10.95 Germany), have added only to higher energy inputs and reduced process efficiency. (A.Gupta and D.Madhukar, Biotechnology Prog., 13 :166;1997). However, we have recently shown bio-separation of ligno-cellulosic content from pea-shells by microbial action. The major limitations encountered in the various physical and chemical methods for improving biomethanation process efficiency are in terms of high energy input and environmental degradation. Among the physical methods, recycling of biogas through the digester and stirring of waste material in the digester add to the energy input of the over all treatment. It implies reduced process efficiency. Similarly, use of chemicals cause environmental degradation since the resultant effluent will have certain chemical nickel, iron, cobalt, etc. in excess. These trace elements on accumulation become toxic for other organisms. Ensiling to separate ligno-cellulosic content from wastes is time consuming as it can take up to 6 months to complete. In the present invention we have overcome the major limitations involved in the biomethanation process of bio wastes. The novelty of the present invention is in using fermentative hydrogen producers to improve biomethanation process. In the present invention, no toxic materials are added in the process. In the present invention, the microbes used grow easily under prevailing ambient conditions. The present invention overcomes the problems of process instability during start-up and operation of biomethanation. The main object of present invention is to provide a process for improved biomethanation using fermentative hydrogen producers, which obviates the drawbacks as detailed above. Another object of the present invention is to provide an effective method to overcome the problem of instability during both start-up and operation of biomethanation. Yet another object of present invention is to establish parameters for improved biomethanation by the use of fermentative hydrogen producing bacterial culture. Accordingly the present invention provides A process for preparation of biogas using fermentative hydrogen producers, which comprises. i) preparing slurry of the biological waste such as herein described with water. ii) mixing the said slurry with fresh cattle dung in the ratio of 9:0.5 to 9:1% by weight. iii) incubating the mixed slurry under fermentative conditions in known manner for 2 to 4 days. iv) homogenising the fermented slurry as obtained in step (iii) and optionally removing ligno-cellulosic fibres by sieving, to obtain liquefied slurry, v) subjecting the liquefied slurry obtained in step (iv) to fermentative hydrogen producers by adding novel strains of Bacillus having characteristics such as here in described and methanogens by adding to it 5 to 10% (w/w) digested cattle dung in the form of slurry. vi) incubating the mixture obtained in step (v) for 6 h at pH in the range of 6.8 to 7.2, for biomethanation under strict anaerobic conditions in a digester, vii) collecting the biogas produced in gas holders, by water displacement. In an embodiment o the present invention, the H2 producers used may be Bacillus licheniformis or Bacillus subtilis, In another embodiment of the present invention, the bacterial populations of H2 producers and methanogens may be used together or separately for degradation of biological waste In yet another embodiment of the present invention, biological waste used may be pea-shells or damaged grains. In still another embodiment of the present invention, the concentration of total solids in the slurry may range form 1 to 5%. In yet another embodiment of the present invention, the anaerobic incubation may be effected fro a period ranging from 40 to 50 days. In yet another embodiment of present invention, the incubation in step (vi) may be effected at a temperature the range of 35 to 42°C. In another embodiment of the present invention the biodegradation of biological waste may be carried out with or without fibrous matter. In yet another embodiment of present invention, the cattle during slurry, may contain acidogenic bacteria such as: Bacillus cereus. Bacillus knelfelkampi. Bacillus megaterium. Bacteroides succinogenes Clostridium camofoetidum. Clostridiumflavifaciens. In yet another embodiment of present invention, the digested cattle dung may contain methanogens such as: Methanobacterium suboxvdans. Methanobacterium formicum, Methanobacterium mobilis. Methanobacterium propionicum. Methanocococcus mazei. Methanosarcina barkerii, Methanosarcina methanica. Details of the invention: Source of the fermentative hydrogen producing bacteria, Bacillus licheniformis strain JK1 were isolated from actively fermenting apple pomace and Bacillus subtilis strain VC2 were isolated from damaged wheat grains, in our laboratory. Both the strains were identified by Microcheck, Inc. Microbial Analysis Laboratory, P.O. Box 456. Northfield, Vermont 05663, U.S.A. Bacillus licheniformis strain JK1 grows well on nutrient agar medium and also on medium specific for Desulfovibrio species at pH 7.0 at 37-40°C. It turns red on Desulfovibrio specific medium. It can also grow at 55°C and in medium containing 7% sodium chloride. Strain JK1 colonies on agar plate have irregular margins, rugose surface, thick density, and pink coloured. Strain JK1 cells are Gram positive, small rods and endospore forming. It can grow and produce acids from glucose, mannitol, xylose, arabinose, cellobiose, fructose and ribose. It can utilize citrate and is able to hydrolyse starch, casein and not urea. It shows catalase and oxidase activities. Strain JK1 evolves 1.4 mole biogas-H / mole glucose consumed and 0.68 mole H2 / mole glucose consumed. Bacillus subtilis strain VC2 grows well on nutrient agar medium and also on medium specific for Desulfovibrio species at pH 7.0 at 37-40°C. It can also grow at 55°C and in medium containing 5% sodium chloride.. Strain VC2 colonies on agar plate are circular, medium sized, translucent and cream coloured. Strain VC2 cells are Gram positive, motile rods and mostly single. It grows also on McCarty agar but cannot utilize citrate and is unable to hydrolyse starch, casein and urea. It shows catalase and oxidase activities. Strain VC2 evolves1.8 mole biogas-H / mole glucose consumed and 0.98 mole H2 / mole glucose consumed. The isolates have ability to produce hydrogen from biological wastes as well. The strains were maintained on nutrient agar plates. These strains have been deposited at CBT, a constitutive laboratory of CSIR (India) and have been given accession numbers CBT-JK1 and CBT-VC2. The biological waste material was allowed to undergo fermentation in step (iii) at pH 7 for 2 to 3 days at 37 to 40°C. A small amount of the actively fermenting slurry was streaked on nutrient agar medium and incubated for 24 h at 37 to 40°C. In the present invention, anaerobic digestion of pea-shells was done in a two stage process. The various plant tissues contain complex organic molecules. The complete degradation of the organic matter is a multi-step process. A syntrophic bacterial consortia is required for effective biodegradation. Hydrolysis of feed into simple monomeric compounds and acidogenesis i.e. production of volatile fatty acids is done in microaerophillic conditions, where fermentative bacteria digested the soft tissues of the pea-shells. The acidified material under anaerobic conditions are acted upon by hydrogen producers. A wide range of food (feed) to microbe ratio are tested by making slurries at different organic solid concentrations. pH adjustment to neutral level is done regularly after each hydrogen evolution cycle of 24 h. Hydrogen producers can convert a portion of the available organic matter in the waste material, the rest of the unutilised organic matter is quite suitable as food for methanogens. Although pH is maintained near neutral by the microbial populations however, it may be adjusted to around 7 invariably only once in the beginning of this incubation period. The partially digested feed is then subjected to biomethanation under anaerobic conditions. At very high organic matter load bacterial population become stressed and their metabolism shifts to production and accumulation of higher fatty acids. There is also an increase in partial pressure of hydrogen gas inside the system. It lead to digester failure. Fermentative hydrogen producers can be employed to relieve the acetogens and methanogens of impeding hydrogen stress by selectively producing hydrogen. With reduced partial pressure of hydrogen in the system, there is improvement in methanogenic bacterial activity and consequently in the overall process as well. The invention has been described here in 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: Pea-shell slurry (PSS), 1.0 L, at 1, 3 and 5% Total Solids (TS) concentration was prepared with water. PSS was inoculated with acidogens in 9:0.5 ratio (v/v, 5% fresh cattle dung slurry, CDS) and incubated under fermentative conditions for 4 days at 40°C. After 4 days of acidogenesis, 245 ml of acidogenic PSS was inoculated with 5 ml of H2-producers Bacillus licheniformis strain JK1 and was incubated for 3 - 4 days to observe H2 production. 25 ml of enriched methanogens in 9:1 ratio (v/v, 10% cattle dung slurry, CDS) were added and incubated under fermentative conditions for 45 days at 42°C, at pH 6.8. These experiments on slurries with fibrous materials showed that biomethanation is now effective also at 5% TS level. In 5% TS slurries with fibres, the methane yield being 174 to 218 L / kg TS fed. In controls, methane yield was 125 to 150 I/kg TS fed at 1% TS slurries and highly stressed biomethanation was observed (9 to 96 I CH4/ kg TS fed) at higher %TS slurries. The detailed results are as given below in the Table. Anaerobic digestion of pea-shells slurry (with fibres) at different loading rates: With Bacillus licheniformis strain JK1. LR Observed volume (ml) % Yield (L/kg TS fed) % reduction %TS Biogas Methane CH4 Biogas CH4 TS OS (Table Removed) EXAMPLE 2: Pea-shells slurry (PSS), 1.0 L, at 1, 3 and 5% Total Solids (TS) concentration was prepared with water. PSS was inoculated with acidogens in 9:0.5 ratio (v/v, 5% fresh cattle dung slurry, CDS) and incubated under fermentative conditions for 2 days at 35°C. After 2 days of acidogenesis, 245 ml of acidogenic PSS were inoculated with 5 ml of H2-producers Bacillus subtilis strain VC2 and incubated for 4 days to observe H2 production. At the end of this phase of hydrogen production (25 ml of enriched methanogens in 9:1 ratio (v/v, 10% cattle dung slurry, CDS) were added and incubated under fermentative conditions for 50 days at 35°C, at pH 6.8 to 7.0. These experiments on slurries with fibrous materials showed effective biomethanation even at 5% TS level. In 5% TS slurries with fibres, the methane yield being 174 to 185 L / kg TS fed. In controls, methane yield was 125 to 150 I/kg TS fed at 1% TS slurries and highly stressed biomethanation was observed (9 to 96 I CH4/ kg TS fed) at higher %TS slurries. The detailed results are as given below in the Table. Anaerobic digestion of pea-shells slurry (with fibres) at different loading rates: With Bacillus subtilis strain VC2. LR Observed volume (ml) % Yield (L/kg TS fed) % reduction %TS Biogas Methane CH4 Biogas CH4 TS OS (Table Removed) EXAMPLE 3: Pea-shell slurry (PSS), 1.0 L, at 1, 3 and 5% Total Solids (TS) concentration was prepared with water. PSS was inoculated with acidogens in 9:1 ratio (v/v, 10% fresh cattle dung slurry, CDS) and incubated under fermentative conditions for 4 days at 42°C. After 4 days of acidogenesis, PSS was crushed in a hand operated meat mincer and the sheath like structure (Ligno-cellulosic fibres) were removed from the pea-shell slurry. 245 ml of PSS without fibres were inoculated with 5 ml of H2-producers Bacillus licheniformis strain JK1 and incubated for 4 days to observe H2 production. At the end of this phase of hydrogen production, 25 ml of enriched methanogens in 9:1 ratio (v/v, 10% cattle dung slurry, CDS) were added and incubated under fermentative conditions for 45 days at 40°C, at pH 7.0 to 7.2. These experiments on slurries without fibrous materials showed effective biomethanation even at 5% TS level. In 5% TS slurries without fibres, the methane yield being 224 to 262 L / kg TS fed. In controls, maximum methane yield 300 I/kg TS fed at 3% TS slurries and highly stressed biomethanation was observed (24 to 30 I CH4/ kg TS fed) at higher %TS slurries. The detailed results are as given below in the Table. Anaerobic digestion of pea-shells slurry (without fibres) at different loading rates: With Bacillus licheniformis strain JK1. LR Observed volume (ml) % Yield (L/kg TS fed) % reduction %TS Biogas Methane CH4 Biogas CH4 TS OS (Table Removed) EXAMPLE 4: Pea-shell slurry (PSS), 1.0 L, at 1, 3 and 5% Total Solids (TS) concentration was prepared with water. PSS was inoculated with acidogens in 9:1 ratio (v/v, 10% fresh cattle dung slurry, CDS) and incubated under fermentative conditions for 4 days at 40°C. After 4 days of acidogenesis, PSS was crushed in a hand operated meat mincer and the sheath like structure (Ligno-cellulosic fibres) were removed from the pea-shell slurry. 245 ml of PSS without fibres were inoculated with 5 ml of H2-producers Bacillus subtilis strain VC2 and incubated for 4 days to observe H2 production. At the end of this phase of hydrogen production, 25 ml of enriched methanogens in 9:1 ratio (v/v, 10% cattle dung slurry, CDS) were added and incubated under fermentative conditions for 40 days at 37°C, at pH 7.0 to 7.2. These experiments on slurries without fibrous materials showed effective biomethanation even at 5% TS level. In 5% TS slurries without fibres, the methane yield being 251 to 275 L / kg TS fed. In controls, maximum methane yield 300 I/kg TS fed at 3% TS slurries and highly stressed biomethanation was observed (24 to 30 I CH4/ kg TS fed) at higher %TS slurries. The detailed results are as given below in the Table. Anaerobic digestion of pea-shells slurry (without fibres) at different loading rates: With Bacillus subtilis strain VC2. LR Observed volume (ml) % Yield (L/kg TS fed) % reduction %TS Biogas Methane CH4 Biogas CH4 TS OS (Table Removed) Similarly, other biological wastes such damaged grains can also be used for biomethanation. The main advantages of the present invention are 1. It provides a simple and effective biological process for improving biomethanation of biological wastes. 2. The process improves biomethanation of high total solid biological waste slurry by use of fermentative hydrogen producers. 3. It also provides an effective method to overcome the problem of reactor instability during both start-up and operation of biomethanation. 4. It also provides an effective method for disposal of waste with high ligno- cellulosic content. 5. In this process 60 to 80% reduction in organic solids content is achieved. 6. In this process the biogas production is improved by 3.5 to 5.0 folds, with the help of fermentative bacterial culture. We Claim: 1. An process for preparation of biogos using fermentative hydrogen producers, which comprises. i) preparing slurry of the biological waste such as herein described with water. ii) mixing the said slurry with fresh cattle dung in the ratio of 9:0.5 to 9:1% by weight. iii) incubating the mixed slurry under fermentative conditions in known manner for 2 to 4 days, iv) homogensing the fermented slurry as obtained in step (iii) and optionally removing ligno-cellulosic fibres by sieving, to obtain liquefied slurry, v) subjecting the liquefied slurry obtained in step (iv) to fermentative hydrogen producers by adding novel strains of Bacillus having characteristics such as here in described and methanogens by adding to it 5 to 10% (w/w) digested cattle dung in the form of slurry, vi) incubating the mixture obtained in step (v) for 6 h at pH in the range of 6.8 to 7.2,for biomethanation under strict anaerobic conditions in a digester, vii) collecting the biogas produced in gas holders, by water displacement. 2. A process as claimed in claims 1, where in the H2 producers used are Bacillus Lichenlformis or Bacillus subtilis. 3. A process as claimed in claims 1-2, wherein in the biological waste used is pea-shells or damaged grains. 4. A process as claimed in claims 1-3, wherein in the anaerobic incubation is effected for a period ranging from 40 to 50 days. 5. A process as claimed in claims 1-4, where in the anaerobic incubation is effected at a temperature in the range of 35 to 42°C. 6. A process for preparation of biogas using fermentative hydrogen producers substantially herein described with reference to examples 1 to 4.

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272-del-2000-abstract.pdf

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