Abstract Waste ground wheat was subjected to acid hydrolysis (pH = 3.0) at 90 °C for 15 min using an autoclave. The sugar solution obtained from acid hydrolysis was subjected to dark fermentation for hydrogen gas production after neutralization. In the first set of experiments, initial total sugar concentration was varied between 3.9 and 27.5 g L −1 at constant biomass (cell) concentration of 1.3 g L −1. Biomass concentration was varied between 0.28 g L −1 and 1.38 g L −1 at initial total sugar concentration of 7.2 ± 0.2 g L −1 in the second set of experiments. The highest hydrogen yield (1.46 mol H 2 mol −1 glucose) and the specific formation rate (83.6 ml H 2 g −1 cell h −1) were obtained with 10 g L −1 initial total sugar concentration. Biomass (cell) concentration affected the specific hydrogen production rate yielding the highest rate (1221 ml H 2 g −1 cell h −1) and the yield at the lowest (0.28 g L −1) initial biomass concentration. The most suitable X o / S o ratio, maximizing the yield and specific rate of hydrogen gas formation was X o / S o = 0.037. Dark fermentation of acid hydrolyzed ground wheat was found to be more beneficial as compared to simultaneous bacterial hydrolysis and fermentation.