Bio-hydrogen production by combined dark and light fermentation of ground wheat starch was investigated using fed-batch operation. Serum bottles containing heat-treated anaerobic sludge and a mixture of Rhodobacter sp. was fed with a medium containing 20 g dm(-3) wheat powder (WP) at a constant flow rate. The system was operated at different initial dark/light biomass ratios (D/L). The optimum D/L ratio was 1/2 yielding the highest cumulative hydrogen (1548 cm(3)), yield (65.2 cm(3) g (1) starch), and specific hydrogen production rate (5.18 cm(3) g (1) h (1)). Light fermentation alone yielded higher hydrogen production than dark fermentation due to fermentation of volatile fatty acids (VFAs) to H-2 and CO2. The lowest hydrogen formation was obtained with D/L ratio of 1/1 due to accumulation of VFAs in the medium. (C) 2010 Elsevier Ltd. All rights reserved.