Utilization of waste materials is one of the most economical approaches to biohydrogen production. Continuous generation of biohydrogen in a bioreactor makes the process more economical with respect to the conventional physical and chemical method. The two main parameters that affect the biohydrogen production in a continuously operated bioreactor are hydraulic retention time (HRT) and influent substrate concentrations. The effect of influent substrate concentration on biohydrogen generation in an up-flow packed bed reactor (UPBR) at HRT = 3 h was investigated in this study. The substrate was waste wheat which was acid hydrolyzed in H2SO4 by adjusting the pH value to pH = 2, under high temperature as T = 90 degrees C in an autoclave to obtain fermentable sugar solution. A natural and porous support particle namely, aquarium biological sponge (ABS) was the microbial immobilization surface in the reactor. Total and hydrogen gas volumes, hydrogen percentage, influent and effluent substrate concentrations, VFA concentrations were monitored. The influent substrate concentration (TSO) was varied between TSO = 10 g/L and TSO = 35 g/L. The process performance was evaluated as biohydrogen volume, percentages, rate and yield under varying operating conditions. The production volume (4275 ml/day) and the rate (3.05 L H-2/L day) were maximum at influent sugar concentration of TSO = 25 g/L, but the yield reached to its maximum value as Y = 1.22 mol H-2/mol glucose at TSO = 19 g/L. Substrate limitation and inhibitions were observed at influent concentrations of TSO = 10 g/L and TSO = 35 g/L, respectively. The results indicated that ABS could be suggested as a microbial support particle for hydrogen generation in immobilized systems. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.