Thermoanaerobacterium-rich sludge acclimated with palm oil mill effluent (POME) in an anaerobic sequencing batch reactor operating at 60 degrees C was used as a seed in batch experiments to investigate the effects of C/N (carbon/nitrogen) ratio, C/P (carbon/phosphate) ratio and iron concentration in POME on fermentative hydrogen production. A central composite design was performed with the aim of optimizing the hydrogen yield together with POME degradation using response surface methodology (RSM). The RSM results indicated that the presence of 257 mg Fe2+/1, a C/N ratio of 74 and a C/P ratio of 559 were optimal for simultaneous hydrogen production and COD (chemical oxygen demand) removal. C/N ratio, C/P ratio and iron concentration all had an individual effect on hydrogen production and COD removal, while iron concentration and C/N ratio had the greatest interactive effect on hydrogen production (P<0.05) while C/N and C/P ratio gave more profound interactive effect on COD removal (P<0.05). The predicted maximum simultaneous hydrogen production and COD removal were 6.51 H-2/l-POME and 58%, respectively. in a confirmation experiment under optimized conditions highly reproducible results were obtained, with a hydrogen production and COD removal efficiency of 6.33 +/- 0.1421 H-2/l-POME and 55 +/- 1.5%, respectively. The total carbohydrate conversion was 92 +/- 2.7%. The hydrogen production rate reached 25.9 mmol H-2/l/day and increased by 60% as compared with the use of raw POME. Thermoanaerobacterium spp. were found to be dominant and present at a higher population density under optimized conditions than in raw POME fermentation. Optimization of the culture cultivation conditions in POME resulted in a simultaneous increase in biohydrogen production and COD reduction. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.