Genome Mining Discovery of Hydrogen Production Pathway of Klebsiella sp. WL1316 Fermenting Cotton Stalk Hydrolysate

Genome sequencing was used to identify key genes for the generation of hydrogen gas through cotton stalk hydrolysate fermentation by Klebsiella sp. WL1316. Genome annotation indicated that the genome size was 5.2 Mb with GC content 57.6%. Xylose was metabolised in the pentose phosphate pathway via the conversion of xylose to xylulose in Klebsiella sp. WL1316. This strain contained diverse formate-hydrogen lyases and hydrogenases with gene numbers higher than closely related species. A metabolic network involving glucose, xylose utilisation, and fermentative hydrogen production was reconstructed. Metabolic analysis of key node metabolites showed that glucose and xylose metabolism influenced biomass synthesis and biohydrogen production. Formic acid accumulated during fermentation at 24–48 h but decreased sharply after 48 h, illustrating the splitting of formic acid to hydrogen gas during early-to-mid fermentation. The Kreb’s cycle was the main competitive metabolic branch of biohydrogen synthesis at 24 h of fermentation. Lactic and acetic acid fermentation and late ethanol accumulation competed the carbon skeleton of biohydrogen synthesis after 72 h of fermentation, indicating that these competitive pathways are regulated in middle-to-late fermentation (48–96 h). This study is the first to elucidate the metabolic mechanisms of mixed sugar utilisation and biohydrogen synthesis based on genomic information.

Enhanced biohydrogen production from cotton stalk hydrolysate of Enterobacter cloacae WL1318 by overexpression of the formate hydrogen lyase activator gene

Background: Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness. Overexpression of the formate hydrogen lyase activator (fhlA) gene is a promising tactic for enhancement of hydrogen production in facultative anaerobic Enterobacter. As a species of Enterobacter, Enterobacter cloacae was reported as high efficient hydrogen-producing bacterium. However, little work has been reported in terms of cloning and expressing the fhlA gene in E. cloacae for lignocellulose-based hydrogen production.Results: In this study, the formate hydrogen lyase activator (fhlA) gene was cloned and overexpressed in Enterobacter cloacae WL1318. We found that the recombinant strain significantly enhanced cumulative hydrogen production by 188% following fermentation of cotton stalk hydrolysate for 24 h, and maintained improved production above 30% throughout the fermentation process compared to the wild strain. Accordingly, overexpression of the fhlA gene resulted in an enhanced hydrogen production potential (P) and maximum hydrogen production rate (Rm), as well as a shortened lag phase time (λ) for the recombinant strain. Additionally, the recombinant strain also displayed improved glucose (12%) and xylose (3.4%) consumption and hydrogen yield Y(H2/S) (37.0%) compared to the wild strain. Moreover, the metabolites and specific enzyme profiles demonstrated that reduced flux in the competitive branch, including succinic, acetic, and lactic acids, and ethanol generation, coupled with increased flux in the pyruvate node and formate splitting branch, benefited hydrogen synthesis. Conclusions: The results conclusively prove that overexpression of fhlA gene in E. cloacae WL1318 can effectively enhance the hydrogen production from cotton stalk hydrolysate, and reduce the metabolic flux in the competitive branch. It’s the first attempt to engineer the fhlA gene in the hydrogen producing bacterium E. cloacae. This work provides a highly efficient engineered bacterium for biohydrogen production from fermentation of lignocellulosic hydrolysate in the future.

Enhanced biohydrogen production from cotton stalk hydrolysate of Enterobacter cloacae WL1318 by overexpression of the formate hydrogen lyase activator gene

Background Biohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness. Overexpression of the formate hydrogen lyase activator ( fhlA ) gene is a promising tactic for enhancement of hydrogen production in facultative anaerobic Enterobacter . However, little work has been reported in terms of the fhlA gene cloning and expression in Enterobacter cloacae as well as the engineered strain for lignocellulose-based hydrogen production.Results The formate hydrogen lyase activator ( fhlA ) gene was cloned and overexpressed in Enterobacter cloacae WL1318, and the cumulative hydrogen production and dynamics, glucose and xylose consumption, cell growth, and soluble metabolites were analyzed in the wild and recombinant strains. The results showed that the recombinant strain significantly enhanced cumulative hydrogen production by 188% following fermentation of cotton stalk hydrolysate for 24 h, and maintained improved production above 30% throughout the fermentation process compared to the wild strain. Accordingly, overexpression of the fhlA gene resulted in an enhanced hydrogen production potential ( P ) and maximum hydrogen production rate ( R m ), as well as a shortened lag phase time ( λ ) for the recombinant strain. Additionally, the recombinant strain also displayed improved glucose (12%) and xylose (3.4%) consumption and hydrogen yield Y(H 2 /S) (37.0%) compared to the wild strain. Moreover, the metabolites and specific enzyme profiles demonstrated that reduced flux in the competitive branch, including succinic, acetic, and lactic acids, and ethanol generation, coupled with increased flux in the pyruvate node and formate splitting branch, benefited hydrogen synthesis.Conclusions The results conclusively prove that overexpression of fhlA gene in E. cloacae WL1318 can effectively enhance the hydrogen production from cotton stalk hydrolysate, and reduce the metabolic flux in the competitive branch. It’s the first attempt to engineer the fhlA gene in the hydrogen producing bacterium E. cloacae . This work provides a highly efficient engineered bacterium for biohydrogen production from fermentation of lignocellulosic hydrolysate in the future.