Microbial community structure of ethanol type fermentation in bio-hydrogen production

Three continuous stirred-tank reactors (CSTRs) were used for H₂ 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 H₂ production rate was the highest for ethanol type fermentation, 0.40 l (g VSS)^-1 day^-1 or 0.45 l H₂ (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. H₂-producing bacteria were affiliated with Ethanoligenens, Acetanaerobacterium, Clostridium, Megasphaera, Citrobacter and Bacteroides. An ethanol-based H₂-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.