Study On Ethanol-Type Fermentation Community & Functional Gene Expression Of Ethanoligenens

Biological hydrogen production using microorganisms is a promising method of utilizing low-value waste as raw materials to produce an energy source. In the previous, researchers used many methods to enhance hydrogen production efficiency, such as reactor and operation parameter optimization, hydrogen producing microbial community directional establishment, high efficient hydrogen producer isolation, accelerator application and so on. Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen (H2), and play a critical role in microbial energy metabolism. Hydrogen production can be stimulated by influencing the hydrogenase expression and activity. Based on the functional enzyme gene of hydrogen production, this study analyzed the anaerobic fermentation microbial community structure, investigated the molecular mechanism of hydrogen production in different environmental factors, and made an important practical significance and value for enhancing hydrogen production capability and realizing the ethanol-type fermentative bio-hydrogen production.In this study, 16S rRNA and hydA gene clone libraries were used for comparing the microbial community structure diversity of different methods pretreated sludge samples, and found that butyrate-type fermentation was formed by heat-shock pretreatment and all the functional bacteria belonged to Clostridium, the untreated sludge formed ethanol-type fermentation with Ethanoligenens harbinense as the dominant population. Different amplification capability and efficiency were obtained when amplifying the same fermentation samples using different hydA gene degenerate primer pairs, which were caused by the conserved regions selection, amplification efficiency design and bacteria preference. A further completely view and more exactly information of the genomic diversity of anaerobic bacteria need optimized primers and cooperation of different primer pairs.The hydrogenase gene from E. harbinense was amplified. It consists of an open reading frame of 1743 bp encoding 580 amino acids with the GenBank accession No. DQ177326. It was heterologous expressed in non-hydrogen-producing bacterium strain E. coli BL-21 using constructed vector pET28a-hydA, and build a foundation for genetic engineering strain. Three new characteristic sequence signatures within the H-cluster domain of [FeFe]-H2ases were found and first described.A new method fitting for isolating total RNA from bacteria with plenty of EPS, such as E. harbinense, was founded. The RT-PCR primers for hydA II, AK, ADH I, ADH II and LDH of E. harbinense, and hydA of C. beijerinckii were designed. Seven functional gene standard plasmids were constructed, including hydA I gene of E. harbinense.The effects of Fe²⁺, Mg²⁺ and L-cysteine concentration on cell growth and hydrogen production by strain YUAN-3 was investigated in batch culture experiments, and found that suitable concentrations of them stimulated hydrogen production and cell growth. The optimal culture medium supplemented with 80 mg/L FeSO₄·7H₂O, 600 mg/L MgCl₂·6H2O and 800 mg/L L-cysteine was consistent with previous studies.The relationship between the three environmental factors of Fe²⁺, Mg²⁺ and L-cysteine concentrations and six functional genes expression was built by qRT-PCR technology. The enhancement of hydrogen production by Fe²⁺ and L-cysteine was resulted from their facilitate effect on cell growth and functional gene expression. Mg²⁺ increased cell growth and hydrogen evolution, but negatively facilitated the hydA gene expression. The functional genes expression obtained different affected by the environmental factors. The copy numbers of hydA I and ADH I gene were always in 100 times more than hydA II and ADH II gene, respectively.The effects of different Fe²⁺, Mg²⁺ and L-cysteine concentrations on hydA gene expression, cell growth and hydrogen production by ethanol-type bacterium of E. harbinense YUAN-3 and butyrate-type bacterium of C. beijerinckii RZF-1108 were different. Fe²⁺ in low concentration stimulated the cell growth, hydrogen production and hydA gene expression of the two strains. Both of them have a strong dependence on Mg²⁺, and significantly increased with the Mg²⁺ concentration increasing. But Mg²⁺ negatively facilitated the hydA gene expression. L-cysteine slightly stimulated hydA gene expression, but not hydrogen production in strain RZF-1108. Effects of metal ions and L-cysteine on hydrogen production by the two fermentative strains were complicated. And hydrogen production was highly dependent on the cooperation of cell growth and hydrogenase gene expression.