Study On Metabolic Pathway Of Hydrogen Production In Efficient Hydrogen-producing Enterobacteriaceae Strains

Hydrogen is considered as one of the most developing potential alternative energies in the future.Dark fermentation is much favored by scientists because of the hydrogen-producing efficiency,simple equipment requirement and raw materials abundance.With the development of society,the fresh and high-salt organic wastewater caused environmental problems more and more serious,such as industrial wastewater and domestic sewage.The combination of wastewater treatment and hydrogen production by fermentation can not only greatly reduce the cost of wastewater treatment,but also produce clean energy.Thus,the screening and conducting systematic study of efficient bacterial strains was the primary issue for the technology applications of fermentative hydrogen production utilized organic wastewater.In the study,the metabolic pathways on hydrogen production of freshwater and seawater efficient strains were thoroughly researched respectively,for as to provide the theoretical basis for the development and utilization as industrial strains.The efficient hydrogen-producing strain Enterobacter cloacae C13 in freshwater culture was analyzed the differences of metabolic pathways relative to hydrogen production in the process of hydrogen production.The hydrogen production of strain C13 was analyzed by the method of gas chromatography,the result showed the hydrogen yield of 707 ml·L-1,the hydrogen content of 30%-35%and the overall hydrogen production rate of 70.7 ml·h-1·L-1.The proteomics and anaerobic products analysis were performed using LC-MS and HPLC method,respectively.These results indicated that the hydrogen-producing fermentation pathway of E.cloacae was the 2,3-butanediol pathway.NiFe hydrogenase could convert H+ to hydrogen using NAD(P)H.The metabolic flux analysis was performed by GC-MS methods,the results showed that the pentose phosphate pathway(HMP)was not involved in carbon metabolism or had a weaker role.And glycolytic pathway(EMP)was the major carbon metabolic pathway,which hydrolyzed glucose to pyruvate.The efficient hydrogen-producing strain Pantoea agglomerans BH-18 both in freshwater and seawater culture was analyzed the differences in three levels of transcription,translation and metabolites using RT-PCR,proteomics and metabolite products analysis,respectively.The results indicated that the bacterial metabolic rate in freshwater culture was about 1.8 times higher than in seawater culture,which was the major reason of higher hydrogen production in freshwater culture.In freshwater culture,EMP pathway provides ATP and NADPH for hydrogen production and HMP pathway was inhibited due to lack of glucose-6-phosphate dehydrogenase.In seawater culture,EMP and HMP pathways were both the major pathways to provide ATP and NADPH.Comprehensive analysis of LC-MS and RT-PCR results indicated the expression missing of a series of tRNA ligase in high salt condition resulted in the genes translation reduce of HMP and hydrogen-producing pathway,nor affect the genes transcription and translation of EMP pathway.