Biologically produced hydrogen using biomass and mixed bacterial cultures is one approach to generate renewable H-2. Response surface methodology (RSM) was used to study the effect of initial pH (3.88-8.12) and initial substrate concentration (0.86-29.14 g/L) on both hydrogen molar yield (HMY) and volmuetric H-2 production rate (VHPR). Lactose, cheese whey powder (CWP) and glucose were used as substrates and heat-treated anaerobic granular sludge as inoculum. For lactose, 3.6 rnol H-2/mol lactose and 5.6 mmol H-2/L/h were found at pH 7.5 and 5 g lactose/L. CWP yielded 3.1 mol H-2/mol lactose at pH 6 and IS g CWP/L while 8.1 rnmol H-2/L/h were attained at pH 7.5 and 25 g CWP/L. Glucose yielded 1.46 mol H-2/mol substrate (pH 7.5, 5 gglucose/L), with a VHPR of 8.9 mmol H-2/L/h, atpH 8.12 and 15 g glucose/L. Acetic and butyric acids were the main organic metabolites detected. HMY and VHPR obtained in this study were found at initial pH above the reported optimum pH value for hydrogen production. These findings could be of significance when alkaline pretreatments are performed on organic feedstock by eliminating the need to lower the pH to acidic levels before fermentation start-up. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.