A strategic method utilizing the co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum has been developed to improve hydrogen production via the thermophilic fermentation of cornstalk waste. The hydrogen yield in the co-culture fermentation process reached 68.2 mL/g-cornstalk which was 94.1% higher than that in the mono-culture. The hydrogen fermentation process was successfully scaled-up from 125 mL anaerobic bottles to an 8 L continuous stirred tank reactor, and the hydrogen production from cornstalk waste was significantly improved in the bioreactor system due to efficient mixing and mass transfer. The hydrogen yield in the bioreactor reached 74.9 mL/g-cornstalk which was 9.8% higher than that in the 125 mL anaerobic bottle. The present work indicates that the direct microbial conversion of lignocellulosic waste by co-culturing C. thermocellum and C. thermosaccharolyticum is a promising avenue for enhancing hydrogen production. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.