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

Abstract

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 a highly 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 (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 is 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.

Optimization of hydrogen production in Enterobacter aerogenes by Complex Ⅰ peripheral fragments destruction and maeA overexpression

“…During dark fermentation for hydrogen production, E. aerogenes produces hydrogen through the formate pathway and the NADH pathway [7,10]. In the formate pathway, pyruvate is converted by pyruvate formate lyase (p ) to produce acetyl coenzyme A and formic acid.…”

Application of Clostridium thiosulphatireducens, Enterobacter aerogenes and their co-culture inoculum for Biohydrogen Production

“…Hydrogen (H 2 ) is the cleanest energy carrier with intrinsic high combustion calori c value of 143 MJ•Kg − 1 that can effectively be produced by dark fermentation using variety of wastes including wastewater, for example palm oil mill e uent (POME) [12], cotton stalk hydrolysate [13], cellulosic biomass [14], food waste [15,16,17,18,12]. In recent years, great efforts have been made in metabolic engineering of hydrogen-producing bacteria to improve their hydrogen production potential.…”

“…Hydrogen-producing bacteria mainly Enterobacter, Clostridia and Bacillus etc, have intrinsic sugar utilization and hydrogen producing properties. The xylose metabolic pathway of Enterobacter cloacae does not even require modi cation for lignocellulose-based hydrogen production [21,13].…”

“…In recent years, great efforts have been made in metabolic engineering of hydrogen-producing bacteria to improve their hydrogen production potential. However, most of the reported engineered hydrogen-producing bacteria can only utilize single carbon sources, like glucose, for hydrogen production [19,20,13]. Hydrogen-producing bacteria mainly Enterobacter, Clostridia and Bacillus etc, have intrinsic sugar utilization and hydrogen producing properties.…”

Genome mining discovery of hydrogen production pathway of Klebsiella sp. WL1316 fermenting cotton stalk hydrolysate

“…However, fermentative hydrogen production by single wild strains is relatively low. Therefore, measurements, such as metabolic pathway analysis and engineering modi cation, are needed for strains with clear genome information (Tran et Zhang et al 2020) in order to reconstruct the hydrogen production metabolic pathways and increase the yield of hydrogen produced by hydrogenproducing strains.…”