16s rRNA gene sequence analysis is applied in the semi-continuous fermentation of Chlorella pyrenoidosa to monitor changes in the microbial community of HPB (hydrogen-producing bacteria). The microbial community of HPB on the 4th day is mainly composed of one genus (Clostridium) including one primary species (Clostridium butyricum). The microbial community of HPB on the 22nd day is mainly composed of three genera (Clostridium, Sporanaerobacter and Eubacterium) including three primary species (Clostridium acetireducens, Clostridium cochlearium and Sporanaerobacter acetigenes). Fermentation results show that production of hydrogen and soluble metabolites by the simple microbial community is unstable with fluctuating pH values at earlier stages (days 1-4). By contrast, the production of hydrogen (51.7-62.0 mL/g-total volatile solids) and soluble metabolites (acetate/butyrate: 0.4-0.5 mol/mol) by complex microbial communities is fairly stable with a steady pH value of approximately 5.9 at later stages of fermentation (days 11-22). (C) 2014 Elsevier Ltd. All rights reserved.