Hydrolyzed sugars from lignocellulosic biomass are primarily glucose and xylose. Efficient dark fermentation of isolated strains of both glucose and xylose to generate hydrogen is of considerable practical and academic importance. This study utilized a newly isolated, moderately thermophilic bacterium, W16, to produce hydrogen from xylose, glucose, and mixed xylose and glucose. The strain W16 was identified and designated as Thermoanaerobacterium thermosaccharolyticum W16. Physiology of strain W16 strain with numerous carbon sources and at pH of 4-7.5 and temperature of 30-70 degrees C was demonstrated. The maximum cumulative H(2) yield and H(2) production rate obtained using the W16 strain in pure glucose and pure xylose tests were 2.42 mol H(2) mol(-1) glucose and 12.9 mmol H(2)L(-1)h(-1), and 2.19 mol H(2) mol(-1) xylose and 10.7 mmol H(2) L(-1)h(-1), respectively. The strain W16 also uptakes mixed xylose and glucose at comparable rates of individual pure sugar tests to produce biohydrogen via an acetate-butyrate-type fermentation. The isolated strain W16 was noted to effectively degrade reducing sugars in hydrolysate of corn stover collected in field. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.