The effect of individual and combined mixed culture on dark fermentative hydrogen production performance was investigated. Mixed cultures from cow dung (C1), sewage sludge (C2), and pig slurry (C3) were enriched under strict anaerobic conditions at 37 degrees C with glucose as the sole carbon source. Biochemical hydrogen production test in peptone-yeast-glucose (PYG) and basal medium was performed for individual mixed cultures (C1, C2 and C3) and their combinations (C1-C2, C2-C3, C1-C3 and C1-C2-C3) at a glucose concentration of 10 g/L, 37 degrees C and initial pH 7. Maximum hydrogen yields (HY) of 2.0 and 1.86 mol(H2)/mol(glucose) by C2, and 1.98 and 1.95 mol(H2)/mol(glucose) by C2 C3 were obtained in PYG and basal medium, respectively. Butyrate and acetate were the major soluble metabolites produced by all the cultures, and the ratio of butyrate to acetate was similar to 2 fold higher in basal medium than PYG medium, indicating strong influence of media formulation on glucose catabolism. The major hydrogen-producing bacterial strains, observed in all mixed cultures, belonged to Clostridium butyricum, C. saccharobutylicum, C tertium and C. perfringens. The hydrogen production performance of the combined mixed culture (C2-C3) was further evaluated on beverage wastewater (10 g/L) at pH 7 and 37 degrees C. The results showed an HY of 1.92 mol(H2)/mol(glucose-equivalent). Experimental evidence suggests that hydrogen fermentation by mixed culture combination could be a novel strategy to improve the HY from industrial wastewater. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.