A two-stage dark-fermentation and electrohydrogenesis process was used to convert the recalcitrant lignocellulosic materials into hydrogen gas at high yields and rates. Fermentation using Clostridium thermocellum produced 1.67 mol H(2)/mol-glucose at a rate of 0.25 L H(2)/L-d with a com stover lignocellulose feed, and 1.64 mol H(2)/mol-glucose and 1.6S L H(2)/L-d with a cellobiose feed. The lignocelluose and cellobiose fermentation effluent consisted primarily of: acetic, lactic, succinic, and formic acids and ethanol. An additional 800 +/- 290 ml H(2)/g-COD was produced from a synthetic effluent with a wastewater inoculum (fermentation effluent inoculum; FEI) by electrohydrogensis using microbial electrolysis cells (MECs). Hydrogen yields were increased to 980 +/- 110 mL H(2)/g-COD with the synthetic effluent by combining in the inoculum samples from multiple microbial fuel cells (MFCs) each pre-acclimated to a single substrate (single substrate inocula; SSI). Hydrogen yields and production rates with SSI and the actual fermentation effluents were 980 +/- 110 mL/g-COD and 1.11 +/- 0.13 L/L-d (synthetic); 900 +/- 140 mL/g-COD and 0.96 +/- 0.16 L/L-d (cellobiose); and 750 +/- 180 mL/g-COD and 1.00 +/- 0.19 L/L-d (lignocellulose). A maximum hydrogen production rate of 1.11 +/- 0.13 L H(2)/L reactor/d was produced with synthetic effluent. Energy efficiencies based on electricity needed for the MEC using SSI were 270 +/- 20% for the synthetic effluent, 230 +/- 50% for lignocellulose effluent and 220 +/- 30% for the cellobiose effluent. COD removals were similar to 90% for the synthetic effluents, and 70-85% based on VFA removal (65% COD removal) with the cellobiose and lignocellulose effluent. The overall hydrogen yield was 9.95 mol-H(2)/mol-glucose for the cellobiose. These results show that pre-acclimation of MFCs to single substrates improves performance with a complex mixture of substrates, and that high hydrogen yields and gas production rates can be achieved using a two-stage fermentation and MEC process. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.