Using low value lignocellulosic feedstocks to produce hydrogen (H-2) could be a more economically viable option in comparison to grains derived from agriculture crops. The glucose and xylose composition of lignocellulosic feedstocks is relatively high and can vary from 55-65% to 35-45%, respectively. Mixed anaerobic cultures which are suitable for fermentative H-2 production are capable of utilizing a variety of substrates. However, the H-2 yields from mixed anaerobic cultures are low because of the syntrophic association between H-2 producers and H-2 consumers. In this study, oleic acid (OA) was used to control the growth of H-2 consumers and hence, increase the H-2 yield. The H-2 yield was affected by changing the glucose to xylose ratio. In control cultures, the H-2 yield (1.6 +/- 0.32 mol H-2 mol(-1) glucose) was low because the electron flux was diverted towards the formation of propionate and methane in addition to acetate and butyrate. In presence of 2000 mg L-1 OA, 60-70% of the electron fluxes were diverted towards acetate and butyrate production and this resulted in a maximum H-2 yield of 2.84 +/- 0.24 mol H-2 mol(-1) glucose. This study revealed that the addition of OA to the mixed anaerobic cultures is an effective method for diverting the electron fluxes to H-2 instead of CH4. Based on a principal component analysis 1 (PCA 1), the control cultures were related; however, in case of the OA treated cultures, they were related in terms of PCA 1 and PCA 2. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.