The optimization of culture condition for enhanced hydrogen production by Thermoonaerobacterium thermosaccharolyticum W16 was conducted using statistical experimental design and analysis. Plackett-Burman design was first used to screen the most important variables influencing hydrogen production. and subsequently central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum hydrogen yield. Experimental results showed that xylose, phosphate buffer, and yeast extract had significant influence on hydrogen production and the maximum hydrogen yield of 2.39 mol/mol xylose was predicted when the concentrations of xylose, phosphate buffer, and yeast extract were 12.24 g/L 0.170 M, and 4.11 g/L, respectively. The results were further verified by repeated experiments under optimal conditions. The excellent correlation between predicted and measured values further confirmed the validity and practicability of this statistical optimum strategy. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.