Immobilized cell bioreactor was operated in batch mode for biohydrogen generation by dark fermentation from acid hydrolyzed waste wheat powder. It was aimed to optimize the fermentation conditions with the purpose of obtaining the highest hydrogen yield (Y-H2) and production rate (HPR) by applying Box Wilson statistical experimental design method. Particle number (PN = 120-240; Xi), initial total sugar concentration (T-S0 = 10-30 g/l; X-2) and fermentation temperature (T = 35-55 degrees C; X-3) were selected as independent variables. Polyester fibers with particle diameter "Dp" = 0.5 cm were used as support material to immobilize microorganisms with heat-pretreated sludge. Quadratic equations for production yield and rate were developed by using experimental results. The maximum Y-H2 (3.21 mol H-2/mol glucose) and HPR (73.3 ml H-2/h) were predicted at the optimum conditions of PN = 240, T-S0 = 10 g/l and T = 44.9 degrees C. Also, analysis of variance, as well as sum of ranking difference test results demonstrated that fitting models were statistically significant. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.