In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 degrees C and pH 7.5, giving a yield of 0.46-0.53 g reducing sugar/g starch. Five H-2-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H-2 from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H-2 production rate of 116 and 118ml/gVSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H-2 yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H-2/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H-2 production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H-2 production rate increased from 250 to 534ml/g VSS/h, whereas the H-2 yield decreased from 2.03 to 1.50 mol H-2/mol glucose. While operating at 2h HRT, the volumetrie H-2 production rate reached a high level of 1.5 l/h/l. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.