Oil palm empty fruit bunch (OPEFB) was hydrolyzed with dilute sulfuric acid (6% v/v; 8 mL acid per g dry OPEFB) at 120 degrees C for 15-min to release the fermentable sugars. The hydrolysate contained xylose (23.51 g/L), acetic acid (2.44 g/L) and glucose (1.80 g/L) as the major carbon components. This hydrolysate was used as the sole carbon source for photofermentive production of hydrogen using a newly identified photosynthetic bacterium Rhodobacter sphaeroides S10. A Plackett-Burman experimental design was used to examine the influence of the following on hydrogen production: yeast extract concentration, molybdenum concentration, magnesium concentration, EDTA concentration and iron concentration. These factors influenced hydrogen production in the following decreasing order: yeast extract concentration > molybdenum concentration > magnesium concentration > EDTA concentration > iron concentration. Under the conditions used (35 degrees C, 14.6 W/m(2) illumination, initial pH of 7.0), the optimal composition of the culture medium was (per L): mixed carbon in OPEFB hydrolysate 3.87 g, K2HPO4 0.9 g, KH2PO4 0.6 g, CaCl2 center dot 2H(2)O 75 mg, L-glutamic acid 795.6 mg, FeSO4 center dot 7H(2)O 11 mg, Na2MoO2 center dot 2H(2)O 1.45 mg, MgSO4 center dot 7H(2)O 2.46 g, EDTA 0.02 g, yeast extract 0.3 g). With this medium, the lag period of hydrogen production was 7.65 h, the volumetric production rate was 22.4 mL H-2/L medium per hour and the specific hydrogen production rate was 7.0 mL H-2/g (xylose + glucose + acetic acid) per hour during a 90 h batch culture of the bacterium. Under optimal conditions the conversion efficiency of the mixed carbon substrate to hydrogen was nearly 29%. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.