Batch dark fermentation experiments were performed to investigate the effects of biomass and substrate concentration on bio-hydrogen production from acid hydrolyzed ground wheat at 55 degrees C. In the first set of experiments, the substrate concentration was constant at 20 g total sugar L(-1) and biomass concentration was varied between 0.52 and 2.58 g L(-1). Total sugar concentration was varied between 4.2 and 23.7 g L(-1) in the second set of experiments with a 1.5 g L(-1) constant biomass concentration. The highest cumulative hydrogen formation (582 mL, 30 degrees C, 1 atm), formation rate (5.43 mL h(-1)) and final total volatile fatty acid (TVFA) concentration (6.54 g L(-1)) were obtained with 1.32 g L(-1) biomass concentration. In variable substrate concentration experiments, the highest cumulative hydrogen (365 mL) and TVFA concentration (4.8 g L(-1)) were obtained with 19.25 g L(-1) initial total sugar concentration while hydrogen gas formation rate (12.95 mL h(-1)) and the yield (200 mL H(2) g(-1) total sugar) were the highest with 4.2 g L(-1) total sugar concentration. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.