A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H(2) production with glucose as sole carbon source. The H(2) production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3-0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100-150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H(2) yield and specific hydrogen production rate were estimated to be 2.26 mol H(2)/mol glucose and 27.74 mmol H(2)/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.