Biohydrogen production by dark fermentation in a series of batch tests under different environmental control conditions was evaluated to determine the optimal initial cultivation pH and temperature for a continuous-flow kinetic test to validate the kinetic model system. The waste activated sludge (WAS) from fructose-processing manufacturing was used as the model substrate for biohydrogen production. The batch experiments for biohydrogen production were conducted in a 61 bioreactor. Fifteen batch kinetic tests were investigated when pH was controlled at 6, 7, 8 and 9 as well as the temperature was controlled at 37 degrees C, 45 degrees C and 55 degrees C, respectively. The experimental results indicated that the optimal operational condition for hydrogen production occurred while pH was 7 and temperature was 55 degrees C with the highest hydrogen production of 7.8 mmol. The optimal recovery time for hydrogen was 25 h in the batch experiments. Furthermore, the kinetic test of biohydrogen production was performed by anaerobic mixed microbial culture in the continuous-flow experiment when pH and temperature was maintained at 7 and 55 degrees C. Approximately 60% and 7% of substrate solution was converted into acetate and hydrogen, respectively, at the steady state. Roughly only 0.77% and 2.7% of substrate solution was converted into propionate and butyrate, respectively, at a steady-state condition. The experimental and modeling approaches presented in this study could be employed for the design of pilot-scale and full-scale anaerobic biohydrogen fermentors using food-processing waste activated sludge (WAS) as a substrate solution. (C) 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.