Hydrogen (H-2) production using mixed anaerobic cultures often suffers severe yield reduction due to the syntrophic association between H-2 consumers (methanogens and homoacetogens) and H-2 producers (acidogens). The objective of this study was to uncouple the syntrophic association between H-2 producers and consumers by optimizing conditions for minimum H-2 consumption using a Box-Behnken design approach. The factors investigated in this study include temperature, pH and linoleic acid (LA) concentration. A quadratic response surface model was developed to predict the H-2 consumed by mixed anaerobic cultures and the optimum conditions for minimum H-2 consumption were 38 degrees C, pH 5.5 and 2 g L-1 LA. Methanogenesis was inhibited in cultures fed 2 g L-1 LA and maintained at pH 6.0 and 53 degrees C. In comparison, both methanogenesis and homoacetogenesis were inhibited in cultures fed 1-2 g L-1 LA and maintained at a pH of 4.5 (Fig. 2B and 2E and Table 2 Expt. # 1, 2 and 11). Microbial diversity analysis revealed that LA fed cultures was dominated by spore forming Clostridium sp. in addition to Syntrophus aciditrophus. In comparison, control cultures were dominated by Eubacterium sp., Methanocalculus halotolerans and Methanococcoides alaskense. This study described an approach for regulating H-2 consumption in mixed cultures by optimizing process and environmental factors. Understanding the effects of these individual factors and their interaction is important in the full-scale operation of H-2 production facilities. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.