In this study, the operation of an upflow anaerobic sludge blanket reactor (UASBR) producing hydrogen (H-2) from a steam-exploded switchgrass (SWG) liquor was statistically optimized. The factors consider included pH, hydraulic retention time (HRT) and linoleic acid (LA) concentration. Under optimal operational conditions (pH 5.0, 10 h HRT and 1.75 g L-1 LA), which were close to the predicted conditions using the D-optimality index, the maximum H-2 and methane yield observed were 99.86 +/- 5.6 mL g(-1) TVS and 0.5 +/- 0.1 mL g(-1) TVS, respectively. Under maximum H-2-producing conditions, high levels of acetate plus butyrate were observed with low levels of ethanol and lactate. A principal component analysis revealed that clustering of the samples was based on the operating conditions and fermentation metabolites. The microbial profiles revealed that by lowering the HRT from 16 to 8 h or decreasing the pH from 7.0 to 5.0 in the controls caused a 50% reduction in the relative abundance of the terminal restriction fragments belonging to the methanogenic population (Methanobacteria, Methanomicrobia, Methanococci). With LA treatment, H-2 producers (Ruminococcaceae and Clostridiaceae) were dominant and methanogens were inhibited and/or washed-out from the UASBR. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.