Ground wheat solution was used for bio-hydrogen production by dark fermentation using heat-treated anaerobic sludge in a completely mixed fermenter operating in fed-batch mode. The feed wheat powder (WP) solution was fed to the anaerobic fermenter with a constant flow rate of 8.33 mL h(-1) (200 mL d(-1)). Cumulative hydrogen production, starch utilization and hydrogen yields were determined at three different WP loading rates corresponding to the feed WP concentrations of 10, 20 and 30 g L(-1). The residual starch (substrate) concentration in the fermenter decreased with operation time while starch consumption was increasing. The highest cumulative hydrogen production (3600 mL), hydrogen yield (465 mL H(2) g(-1) starch or 3.1 mol H(2) mol(-1) glucose) and hydrogen production rate (864 mL H(2) d(-1)) were obtained after 4 days of fed-batch operation with the 20 g L(-1) feed WP concentration corresponding to a WP loading rate of 4 g WP d(-1). Low feed WP concentrations (10 g L(-1)) resulted in low hydrogen yields and rates due to substrate limitations. High feed WP concentrations (30 g L(-1)) resulted in the formation of volatile fatty acids (VFAs) in high concentrations causing inhibition on the rate and yield of hydrogen production. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.