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The effect of different pretreatment methods on anaerobic H2 production from a sucrose-rich synthetic wastewater was investigated in this work. The substrate utilization, formation of aqueous products, H2 production and microbial diversity in anaerobic sludge all markedly depended on these pretreatment methods. The highest H2 production according to the values for the maximum H2 production rate of 27.57 ml h-1, specific H2 production rate of 2.962 ml g-VSS h-1, and H2 yield of 1.617 mol H2 mol glucose-1 was observed in the H2 production by the sludge pretreated with butyrate (pH = 2). Correspondingly, the main microbial communities were Clostridium sp. HPB-16, Clostridium sp. HPB-46, Clostridium sp. HPB-2, Clostridium sp. HPB-4, Oxalobacteraceae bacterium QD1, uncultured bacterium clone HPR93, uncultured Olsenella sp. clone J27, and uncultured bacterium clone SR_FBR_E5. This result demonstrates that acid pretreatment such as butyrate could be also as an effective method to enrich H2-producing bacteria from anaerobic sludge for large-scale biological H2 production. [ABSTRACT FROM AUTHOR]