The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 degrees C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 degrees C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 degrees C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H-2/L/d and 0.40 mmol H-2/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively. Crown Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.