Ground wheat powder solution (10 g L(-1)) was subjected to combined dark and light fermentations for bio-hydrogen production by fed-batch operation. A mixture of heat treated anaerobic sludge (AN) and Rhodobacter sphaeroides-NRRL (RS-NRRL) were used as the mixed culture of dark and light fermentation bacteria with an initial dark/light biomass ratio of 1/2. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. The highest cumulative hydrogen formation (CHF = 3460 ml), hydrogen yield (201 ml H(2) g(-1) starch) and formation rate (18.1 ml h(-1)) were obtained with a starch loading rate of 80.4 mg S h(-1). Complete starch hydrolysis and glucose fermentation were achieved within 96 h of fed-batch operation producing volatile fatty acids (VFA) and H(2). Fermentation of VFAs by photo-fermentation for bio-hydrogen production was most effective at starch loading rate of 80.4 mg S h(-1). Hydrogen formation by combined fermentation took place by a fast dark fermentation followed by a rather slow light fermentation after a lag period. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.