In this study, hydrogen (H-2) production from the fermentation of steam exploded corn stalk (CS) liquor was statistically optimized using a fractional factorial design approach. The factors under consideration included temperature, pH and hydraulic retention time (HRT). Under optimal conditions at 53 degrees C, a pH at 4.5 and a 9.5 h HRT, the observed maximum H-2 yield of 98 +/- 2 mL g(-1) TVS together with negligible CH4 were similar to the model predicted responses. A flux analysis revealed negligible homoacetogenic activity in cultures at 53 degrees C and low pH (<= 5.5). Both homoacetogenic (R17) and methanogenic (R28 and R29) fluxes accounted for more than 68-90% of H-2 consumption in cultures at low temperatures. The low H-2 yields observed in cultures maintained at 21 degrees C and 37 degrees C was associated with high lactate and solvent levels. High H-2 yields in cultures at 53 degrees C were associated with a higher abundance of Clostrdium sp. and CH4 production at low temperatures was due to the presence of hydrogenotrophic methanogens (Methanothermobacter marburgensis and Methanobrevibacter ruminatum) and aceticlastic methanogens (Methanosaeta sp. and Methanosarcina sp.). The results obtained from this study (within the factor ranges investigated) indicated that steam exploded CS liquor could be a potential substrate for H-2 production using mixed microbial cultures. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.