In the present study, the growth and hydrogen production of Rhodobacter sphaeroides O.U. 001, was investigated in media containing five different volatile fatty acids (VIA) individually (malate, acetate, propionate, butyrate and lactate) and in media containing mixtures of these acids that reflect the composition of dark fermentation effluents. The highest hydrogen production rate was obtained in malate (24 ml(hydrogen)/I(reactor)h) and the highest biomass concentration was obtained in acetate containing media (1.65 g/l). The substrate conversion efficiencies for different volatile fatty acids were found to vary between 14 and 50%. The malate and butyrate consumption rates were first order with consumption rate constants of 0.026 h(-1) and 0.015 h(-1), respectively. In the case of substrate mixtures, it was observed that the bacteria consumed acetate first, followed by propionate and then butyrate. It was also found that the consumption rate of the main substrate significantly increased when the minor substrates were depleted. (c) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.