Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup(-) mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH4+ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H-2/mol sucrose) and maximum volumetric productivity (7.1 mmol H-2/L-c.h) were obtained in the absence of NH4+. The effluent of the dark fermentation containing no NH4+ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H-2/L-c.h) were attained using the hup(-) mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H-2/mol sucrose in dark fermentation to 13.7 mol H-2/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H-2/mole of sucrose) by sequential dark and photofermentation. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.