The optimum condition for hydrolysis of oil palm trunk (OPT) by microwave-H2SO4 method and hydrogen production from OPT hydrolysate were explored using response surface methodology (RSM) with central composite design (CCD). The optimum condition for hydrolysis of OPT was 1.56% (w/v) H2SO4 and 7.50 min reaction time at 450 W. At this condition, 8.95 +/- 0.99 g/L glucose/L, 8.29 +/- 0.43 g/L xylose, 4.57 +/- 0.14 g/L arabinose and 2.61 +/- 0.11 g/L acetic were obtained. The individual and interactive effects of the initial pH, initial substrate concentration and NaHCO3 concentration on the hydrogen production potential (P-s) from OPT hydrolysate by hot spring enriched culture were further investigated using RSM with CCD. The initial pH and initial substrate concentration had a statistically individual effect on P-s, while the interactive effect on P-s was found between the initial pH and initial substrate concentration. The optimum condition obtained was an initial pH of 6.03, an initial substrate concentration of 22.07 g/L and a NaHCO3 concentration of 6.71 g/L in which a P-s of 1947 mL H-2/L medium was achieved. Microbial community analysis by PCR-DGGE indicated that Clostridium sp. and Lactobacillus sp. were the predominant species at the optimum condition. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.