Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H(2)) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett-Burman design identified three factors that significantly influenced H(2) yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H(2) production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H(2)/mol sucrose (5.08 mol H(2)/mol hexose), which was near the expected value of 10.70 mol H(2)/mol sucrose (5.35 mol H(2)/mol hexose). (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.