| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 99 === Hydrogen is one of the most developmental clean energy under the oil shortage of today.Some microorganisms can break down cellulose to produce biofuels like hydrogen,ethanol and butanol. Using microorganisms to produce hydrogen is the least regional, lower-cost,and sustainable way among various hydrogen production methods. The genus clostridium are one of the bacteria most often used for studying hydrogen production, they are strict anaerobes, when they carry out fermentation, NADH is produced in the glycolytic pathway and its electrons are transferred to hydrogenase by ferredoxin. In hydrogenase, Fe-S cluster transfers electrons to active site, H-cluster, to form hydrogen with proton. In this hydrogen production pathway, both hydrogenase and ferredoxin are indispensable. Clostridium xylanolyticum Ter3 is an early isolate of our lab, it has good hemicellulolytic ability but moderate hydrogen-producing ability. In this study an attempt was made to improre its hydrogen production, the strategy was to transform the hydrogenase gene and ferredoxin gene of C.pasteurianum F40, which is a very good hydrogen produces isolated by our lab, to C.xylanolyticum Ter3. After both whole genes were obtained from C.pasteurianum F40 chromosome and sequenced, they were cloned into a E.coli / Clostridim shuttle vector pIMP1, the resulting recombinant plasmid pIMP1F40D was used to transform E.coli DH5A. In order to protect pIMP1F40D from degradation by a specific clostridial restriction endonuclease, pIMP1F40D was also introduced into E.coli strains ER2275(pAN1) and JM110 to carry out in vivo methylation. The results of RT-PCR indicated that both hydrogenase gene and ferredoxin gene could be transcribed in E.coli DH5A, but SDS-PAGE did not clearly show the translating products of both genes. When they were grown anaerobically in LB medium containing 10 g/l glucose for 16 hours, these transformants produced 4.7%~108% more hydrogen than these wild type strains.
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