The starch-utilizing co-culture Clostridium butyricum and Rhodobacter sphaeroides demonstrated the higher H-2 yield than C. butyricum (4.9 and 1.5 mol/mol hexose, respectively). Two species in co-culture were probably in balance since volatile fatty acids were not accumulated. The co-culture was as efficient in H-2 production from glucose as R. sphaeroides monoculture. A short-term test was suggested to determine the current activity of either species in co-culture. During the early phase, the H-2 production was mediated by both species, or C. butyricum accompanied with H-2 consumption by R. sphaeroides. Glucose addition did not influence the current rates of Clostridium-mediated and Rhoclobacter-mediated H2 production arguing against the limitation/competition for glucose between two species. During the late phase, the H-2 production was completely attributed to R. sphaeroides. The current rates of Clostridium-mediated H-2 production by co-culture were definitely reduced as compared to C. butyricum monoculture due probably to adverse effect of purple bacteria. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.