| As a clean, efficient and renewable energy carrier, hydrogen draws more and more attention with the growing shortage of fossil fuels and gradual increase in environmental requirement. It has strong prospects in the future to produce hydrogen by fermentation using biomass, while it is very popular to modify the hydrogen producing bacteria in the research field of bio-hydrogen. The gene of a hydrogen-promoting protein (HPP) was cloned and over-expressed in E. cloacae CICC10017for the first time in this study, and the hydrogenase activity of the recombinant strain is increased by97.4%of the wild strain. After the microwave-assisted acid pretreatment of water hyacinth, the hydrogen production was increased by111.1%using the domesticated mixed bacteria and detoxification with active carbon. Semi-continuous cogeneration of hydrogen and methane from water hyacinth was carried out in a500L pilot-scale bioreactor and gave an energy conversion efficiency of51.3%.In this paper, efficient hydrogen producing bacteria E. cloacae CICC10017, and E. aerogenes ATCC13408are used for gene modification, and their hydrogenase activity, hydrogen producing capacity and metabolic pathways after the over-expression of foreign genes were studied. After the over-expression of the HPP gene in E. cloacae CICC10017, the hydrogenase activity of the recombinant strain is534.78±18.51ml/(g-DW·h), increased by97.4%of the wild strain, and the hydrogen yield of the recombinant strain is2.54mol-H2/mol-glucose, which is97.9%higher than the wild strain. Compared to the wild strain, the recombinant strain produces more acetate and butyrate during hydrogen fermentation, but less ethanol, which is corresponding to the metabolism of NADH in the cell. After the over-expression of the large and small subunit genes of hydrogenase hyc in E. aerogenes ATCC13408, the hydrogenase activities of the recombinant strains are increased by124%and67%, respectively, and the hydrogen yields of the recombinant strains are increased from1.16mol-H2/mol-glucose to2.16mol-H2/mol-glucose and1.97mol-H2/mol-glucose, respectively. After the over-expression of the hydrogenase gene hoxUYH of Synechocystis sp. PCC6803in E. aerogenes ATCC13408, the hydrogenase activity is increased by153.8%and the hydrogen yield is increased from1.27 mol-H2/mol-glucose to2.39mol-H2/mol-glucose. Based on these results, experiments of hydrogen production from pretreated water hyacinth were carried out using gene modified bacteria, among which E. cloacae/HPP has the strongest capacity to produce hydrogen from water hyacinth, with the yield of74.9ml-H2/g-TVS.Cogeneration of hydrogen and methane from three kinds of complex organic compounds including cellulose, proteins and fats are investigated in this paper. In order to promote the hydrogen yield from biomass, the hydrogenogens were acclimated with biomass for many times to enrich the strains, which are strong hydrogen producers. Comparison of hydrogenogenic16S rRNA gene sequences before and after domestication with xylose shows that by enrichment with xylose, strains which decompose xylose well become dominant in the sludge, and are expected to give higher hydrogen production rate and hydrogen yield. The hydrogen yield was increased by45%when domesticatied strains were used. From xylose, the maximum hydrogen yield is190.6ml H2/g-xylose with a methane yield of216.5ml CH4/g-xylose, and the energy conversion efficiency of the cogeneration process is67.48%. Lactoprotein in DMP cogenerates hydrogen yield of136.5ml/g-TVS and methane yield of157.8ml/g-TVS by two-phase fermentation, and the energy conversion efficiency is41.9%.After the microwave-assisted acid pretreatment, the hydrolysis rate of water hyacinth can reach91.36%using cellulase, which siginificantly enhanced the hydrogen yield from water hyacinth by62.8%. After that, when domesticated bacteria were used, the hydrogen yield was increased by95.5%. When active carbon was further used for detoxification, the hydrogen yield was increased by111.1%. The maximum hydrogen yield from water hyacinth is134.9ml-H2/g-TVS, and the methane yield from the residual of hydrogen fermentation is107.7ml-CH4/g-TVS. Semi-continuous cogeneration of hydrogen and methane from water hyacinth was carried out using two pilot-scale bioreactors with the volume of50L and500L, respectively. The continuous and stable production of hydrogen and methane are340.9±36.6ml-H2/(L·d) and184.3±10.4ml-CH4/(L-d). The energy conversion efficiency of the pilot-scale cogeneration process is51.3%. |
