Development of bio-production process from biogas

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(1) Breeding of high ethanol producing Moorella sp. HUC22-1:To improve the ethanol productivity of Moorella sp. HUC22-1, development of its genetic transformation system was tried. As the results, an uracil auxotroph that lacks dehydrogenase dehydrogenam gene(pyrD) and orotate phosphonlosyltransferaro gene(pyreE) was obtained by the transformation system using homologous recombination and electroporation. Now, we are carrying on a development of genetic transformation system by using the uracil auxotroph, pyrD and pyrE as a host and selection markers.(2) Analysis of metabolic characterization of Moarella sp. HUC22-1 in various substrates:Moorella sp. HUC22-1 ferments glyoxylate to acetate roughly according to 2 glyoxylate→acetate 2 CO_2. Based on the data obtained in a batch culture, the ATP yield per substrate dissimilated was calculated to be 1.11 mol/mol, which was higher than that obtained from only Wood-Ljungdahl pathway. Crude extracts of glyoxylate-grown cells catalyzed the ADP- … More and NADP-dependent condensation of glyoxylate and acetyl-CoA to pyruvate and CO_2 as well as the conversion of pyruvate to acetyl-CoA and CO_2. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, L-malate, an intermediate in the malyl-CoA pathway, was consumed and acetate was produced in this bacterium. These findings suggest that Moorella sp. HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway. But, ethanol was not produced in all substrates tested including glyoxylate, glycolate, oxalate and malate.(3) Ethanol production in gaseous substrate in batch culture:In this year, we used CO as substrate instead of H_2/CO_2. Although the cell could not grow up to 20% CO in the first batch culture, it can grow 100% CO after repeating the acclimatization in the CO gas. At the 100% CO gas substrate, cell growth could increase 8 times and the product could be sifted to ethanol production by increasing the acetate/ethanol ratio.(4) Conversion of acetate to ethanol in resting cell system:Conversion of acetate to ethanol was carried out in the resting cell system. As the results, ethanol production was increased to 147 g/kg dry cell under mechanical shaking in the presence of methyl-viologen at pH7. Less