The aim of the study is biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated up-flow packed bed reactor. For this purpose, the effect of hydraulic retention time (H-RT) on the rate (R-H2) and yield (Y-H2) of hydrogen gas formation were investigated. In order to determine the most suitable hydraulic retention time yielding the highest hydrogen formation, the reactor was operated between HRT = 1 h and 8 h. The substrate was the acid hydrolyzed wheat powder (AHWP). Waste wheat was sieved down to 70 mu m size (less than 200 mesh) and acid hydrolyzed at pH = 2 and 90 degrees C in an autoclave for 15 min. The sugar solution obtained from hydrolysis of waste wheat was used as substrate at the constant concentration of 15 g/L after neutralization and nutrient addition for biohydrogen production by dark fermentation. The microbial growth support particle was aquarium biological sponge (ABS). Heat-treated anaerobic sludge was used as inoculum. Total gas volume and hydrogen percentage in total gas, hydrogen gas volume, total sugar and total volatile fatty acid concentrations in the feed and in the effluent of the system were monitored daily throughout the experiments. The highest yield and rate of productions were obtained as Y-H2 = 645.7 mL/g TS and R-H2 = 2.51 L H-2/L d at HRT = 3 h, respectively. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.