Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H(2) fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H(2) from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 degrees C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H(2) production with the starch hydrolysate, the highest H(2) production rate and yield was 0.52 L/h/L and 13.2 mmol H(2)/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH(4)HCO(3)) concentration was 2.62 g/L, attaining a good H(2) production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H2 fermentation while using dark fermentation effluents as the substrate. (c) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.