The Regulation Mechanism Of Gene Expression In Ethanoligenens Harbinense And Directional Control Of Electron Flow To Enhance Its Hydrogen Production

Ethanol-type fermentation is one of the three main fermentation types in acidogenesis of the anaerobic biological treatment.Ethanol-type fermentative hydrogen-producing bacteria(HPB)obsesses the advantages of high hydrogen-producing efficiency,acidophilia,autoaggregation,co-production of ethanol-H₂ and direct utilization of fermentation products by methanogens.However,the molecular regulation mechanism of ethanol-type HPB and the interaction mechanisms with other microorganisms are still not clear,and recovering energy from wastewater or wastes by ethanol-type fermentation remains a major challenge.In this study,a multi-omics approach was employed to provide comprehensively genetic and evolutionary information of the representative ethanol-type fermentative H₂producer-Ethanoligenens harbinense,and to construct the ethanol-H₂ cooperative metabolic regulation network of E.harbinense.The complete genome sequences and DNA methylome of E.harbinense strains were obtained using the Pac Bio single-molecule real-time sequencing platform.Comparative genomic analysis demonstrated a low level of genetic similarity between E.harbinense and other well-known hydrogen-producing bacteria in phylogenesis.The genome size of E.harbinense strains was about2.97?3.10 Mb.Approximately 3,020?3,153 genes were annotated,and most of which were methylated at specific sites or motifs.The metabolic mechanism of H₂-ethanol coproduction fermentation catalyzed by pyruvate ferredoxin oxidoreductase was proposed.Then the metabolic mechanism of H₂-ethanol coproduction fermentation of E.harbinense at different growth stages was further clarified according to RNA-seq and i TRAQ^TManalysis.The integrated data revealed that all major pathways of E.harbinense were under multi-level transcriptional and translational regulation.In particular,posttranslational modification was crucial to the growth and metabolic regulation of E.harbinense.The key enzymes involved in H₂-ethanol co-metabolism(such as[Fe Fe]-hydrogenases,ferredoxins,alcohol dehydrogenases,and the acetate kinase)changed their expression at the transcription level,the translation level and acetylation/phosphorylation level.Moreover,protein-protein interactions existed among these enzymes which cooperatively regulated the H₂-ethanol co-metabolism of E.harbinense.The pH has a substantial influence on the formation of different types of acidogenesis and significantly affects the stability and efficiency of the hydrogen production system.The effects of different initial pH on ethanol-H₂ co-metabolism of E.harbinense and the response of E.harbinense to the low pH condition were investigated at RNA level.Total of 1753 differentially expressed genes(DEGs)were identified among different initial pH conditions and functionally categorized by using RNA-seq.E.harbinense adapted to low pH by regulating the gene expression networks of cell growth-,basic metabolism-,chemotaxis-and resistance-related genes.Low initial pH(pH 4,pH 5)down-regulated the expression of cell growth-and acidogenesis-related genes but did not affect the expression of H₂ evolution-related hydrogenase and ferredoxin genes.High pH(pH 8)down-regulated the expression of H₂ evolution-and acidogenesis-related genes.The ethanol-H₂ co-metabolism by E.harbinense not only regulated by the expression of hydrogenases,ferredoxins,alcohol dehydrogenases and other key metabolic enzymes,but also depended on the availability of electrons transferred to[Fe Fe]-hydrogenase and the distribution of electron flow.The coupling system of ethanol-type fermentation and MECs by co-cultures of E.harbinense and electroactive bacteria,and the continuous flow hydrogen-producing MBBR by co-cultures of E.harbinense and facultative aerobes were constructed in this study.An effective strategy to enhance hydrogen production of ethanol fermentation was proposed by means of directional control of electron flow through co-culture.The interspecies syntrophic interactions between the fermentative hydrogen-producing E.harbinense and the electroactive G.sulfurreducens were confirmed in co-cultured systems.The co-cultures formed active biofilms with the intercellular pili-like nanowire structures,which might mediate and facilitate interspecies electron transfer.The maximum hydrogen production rate of co-cultured MEC was about 76 m L/L-medium·h,and the hydrogen yield was 2.01 mol-H₂/mol-glucose which was about 1.3 times as much as that of the non-electrochemical co-culture system and 2 times as much as that of the single-cultured system of E.harbinense.The proportion of ethanol in defined co-culture greatly increased compared to mono-cultures,suggesting that the concentrations of metabolic products of E.harbinense could be directly regulated by the interaction with G.sulfurreducens.In addition,the co-culture of E.harbinense and facultative aerobic Pseudomonas aeruginosa shortened the start-up period of continuous-flow hydrogen-producing MBBR.P.aeruginosa consumes oxygen and can creat an appropriate anaerobic environment for ethanol-type fermentation,which ensure the biomass and hydrogen-producing activity of E.harbinense in the continuous flow MBBR.The cumulative hydrogen production of MBBR reactor inoculated with P.aeruginosa reached71.87%of that in MBBR reactor treated with filter-sterilized N₂ gas.The results suggested that the inoculation of facultative aerobes could be a recommendable oxygen removal approach for the fast start-up of anaerobic reactor to establish strict anaerobic condition and enhance hydrogen production of ethanol-type fermentation.