This study investigated pathways for anaerobic hydrogen production from biomass at low pH values, also known as dark fermentation. A 200 1 bench scale anaerobic baffled reactor with four internal compartments was used for hydrogen production from wheat starch. The liquid fermentation products and hydrodynamic characteristics were analyzed using high performance liquid chromatography, tracer studies, and gas analysis. A mean residence time of 29 h and a feed strength of 4 g(coD)/l resulted in a total gas production of 230 l/d containing 42% hydrogen and 11% methane. The gas collected from the different compartments highly differed in composition showing a partial phase separation, with maximum H-2 concentrations of up to 60% observed in the first compartment. 49% and 44% of the total H-2 produced were derived during the formation of acetic and butyric acid respectively. Just 8% of the H-2 was produced during propionic acid synthesis. Concentrations up to 1 g/l lactic acid built by the bifidum pathway was also observed. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.