Production of biohydrogen from dark fermentation is an interesting alternative to producing renewable fuels because of its low cost and various usable substrates. Cellulosic content in plentiful bagasse residue is an economically feasible feedstock for biohydrogen production. A statistical experimental design was applied to identify the optimal condition for biohydrogen production from enzymatically hydrolyzed bagasse with 60-min pre-heated seed sludge. The bagasse substrate was first heated at 100 degrees C for 2 h and was then hydrolyzed with cellulase. Culture of the pretreated bagasse at 55 degrees C provided a higher H(2) production performance than that obtained from cultures at 45 degrees C, 65 degrees C, 35 degrees C and 25 degrees C. on the other hand, the culture at pH 5 resulted in higher H2 production than the cultures at pH 6, pH 4 and pH 7. The optimal culture condition for the hydrogen production rate was around 56.5 degrees C and pH 5.2, which was identified using response surface methodology. Moreover, the pretreatment of bagasse under alkaline conditions gave a thirteen-fold increase in H(2) production yield when compared with that from preheatment under neutral condition. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.