A novel co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense with pretreated sugarcane bagasse (SCB) under mild alkali conditions for bio-hydrogen production was established, exhibiting a cost-effective and synergetic advantage in bio-hydrogen production over monoculture of C. thermocellum or T. aotearoense with untreated SCB. The optimized pretreatment conditions were established to be 3% NaOH, and a liquid to solid ratio of 25:1 at 80 degrees C for 3 h. A final hydrogen production of 50.05 +/- 1.51 mmol/L was achieved with 40 g/L pretreated SCB at 55 degrees C. The established co-culture system provides a novel consolidated bio-processing strategy for bioconversion of SCB to bio-hydrogen. (C) 2013 Elsevier Ltd. All rights reserved.