Abstract
Summary Three continuous stirred‐tank reactors (CSTRs) were used for H 2 production from molasses wastewater at influent pH of 6.0–6.5 (reactor A), 5.5–6.0 (reactor B), or 4.0–4.5 (reactor C). After operation for 28 days, the microbial community formed ethanol type (C), propionate type (A) and ethanol‐butyrate‐mixed type (B) fermentation. The H 2 production rate was the highest for ethanol type fermentation, 0.40 l (g VSS) −1 day −1 or 0.45 l H 2 (g COD removed) −1 . Microbial community dynamics and diversity were analysed using double‐gradient denaturing gradient gel electrophoresis (DG‐DGGE). Denaturing gradient gel electrophoresis profiles indicated that the community structures changed quickly in the first 14 days. Phylogenetic analysis indicated that the dominant bacterial groups were low G+C Gram‐positive bacteria, Bacteroides , γ‐Proteobacteria and Actinobacteria ; α‐Proteobacteria , β‐Proteobacteria , δ‐Proteobacteria and Spirochaetes were also presented as minor groups in the three reactors. H 2 ‐producing bacteria were affiliated with Ethanoligenens , Acetanaerobacterium , Clostridium , Megasphaera , Citrobacter and Bacteroides. An ethanol‐based H 2 ‐producing bacterium, Ethanoligenens harbinense CGMCC1152, was isolated from reactor C and visualized using fluorescence in situ hybridization (FISH) to be 19% of the eubacteria in reactor C. In addition, isoenzyme activity staining for alcohol dehydrogenase (ADH) supported that the majority of ethanol‐producing bacteria were affiliated with Ethanoligenens in the microbial community.