Screening Of Hydrogen-Producing Microorganisms From Termite Gut And Heterologous Expression And Characterization Of Hydrogenase

Research in the field of renewable green energy can effectively solve the two major world problems of environmental pollution and energy shortage.Hydrogen energy,as a kind of high-efficiency clean energy,has been regarded as the most promising energy source in recent years,and it has aroused extensive attention worldwide.Biological hydrogen production technology mainly refers to the microbial decomposition of organic matter in the process of growth and metabolism to generate hydrogen.Compared with the traditional physical and chemical hydrogen production technology,the biological hydrogen production technology has a series of outstanding advantages such as green energy saving and recyclability.The advantages of microorganism fermentation hydrogen production technology have gradually become a hot field in the research of biological hydrogen production due to the advantages of simple operation,a wide source of raw materials and low energy consumption.The microorganism fermentation hydrogen production technology can be studied from the aspects of the screening of highly efficient hydrogen-producing microorganisms and the construction of new hydrogen-producing metabolic pathways.The ability of termites to efficiently degrade and transform lignocellulose and the special intestinal hydrogen-producing microbial community have attract researchers'attention.This study was explored through three aspects as follows:isolation and identification of hydrogen-producing bacteria from termite intestines;parameter optimization of hydrogen production,and the characterization of key enzymes in hydrogen-producing pathway.The main research contents and results of this article are as follows:?1?Based on the isolation and screening of the termite intestinal microenvironment and identification of the hydrogen-producing strain Clostridium bifermentans G3,the characterization of hydrogen production by fermentation were studied.On the basis of the morphological characteristics as well as 16S rDNA sequence analysis,the bacterium was identified as a strain of Clostridium bifermentans,and the similarity with Clostridium bifermentans strain W11109B1-1 reached 99.78%.The optimal temperature for hydrogen production by strain G3 is 30?,the optimal pH is 6.9,and the hydrogen production reached1.3 mol H₂/mol glucose.?2?Different hydrogen-producing microorganisms were selected as the research object,and the heterologous expression of hydrogenases in Escherichia coli was analyzed using recombinant expression technology.The hydrogenase from C.bifermentans was a single subunit[FeFe]hydrogenase.The single subunit[FeFe]hydrogenase gene was amplified by PCR using genomic DNA as template and the gene was cloned into the expression vector pBlu10-Htk.At the same time,the[FeFe]hydrogenase from the thermophilic bacterium Thermoaga neapolitana DSM 4359,composed of three subunits,was heterologous expressed in E. coli.The expression level of hydrogenase from C.bifermentans G3 was low.While the[FeFe]hydrogenase from T.neapolitana DSM 4359 was mainly found in inclusion bodies,and only a small proportion was soluble when expressed in E. coli.?3?The key enzymes in the hydrogen production pathway of T.neapolitana DSM 4359:pyruvate ferredoxin oxidoreductase?PFOR?and ferredoxin?Fd?were expressed and purified,and the properties of the PFOR was analyzed.The optimal reaction temperature and pH is 90?and 11,the catalytic activity of PFOR was sensitive to oxygen.In summary,the screening and identification of hydrogen-producing strains and the analysis of the characterization of key enzymes in the hydrogen-producing pathway are helpful to genetic modification of the strains and achieve the purpose of increasing hydrogen production.