The individual and interactive effects of pH, temperature and substrate concentration on the biohydrogen production from sucrose by mixed anaerobic cultures were investigated in this study. A central composite design and response surface methodology (RSM) were employed in planning the experiments, in order to determine the optimum conditions for biohydrogen production. Experimental results show that pH, temperature and substrate concentration all had a significant influence on specific hydrogen production potential (P-s) and the maximum hydrogen production rate (R-max) individually. Temperature and sucrose concentration, pH and temperature were interdependent or there was a significant interaction on P-s and R-max. Substrate concentration and pH were slightly interdependent, or their interactive effect on P-s and R-max was not significant. A maximum P-s of 252 mL H-2/g sucrose was estimated under the optimum conditions of pH 5.5, temperature 34.8 degrees C and sucrose concentration of 24.8 g/L, while a maximum R-max of 1511 mL H-2/h was calculated under the optimum conditions of pH 5.5, temperature 35.5 degrees C and sucrose concentration of 25.4 g/L. The experiment results show that the RSM with the central composite design was useful for optimizing the biohydrogen-producing process. (c) 2005 Elsevier Inc. All rights reserved.