Dark fermentation of microcrystalline cellulose to produce biohydrogen using monoculture or co-culture of isolated strains was studied. A strain (X-9) with high hydrogen yield from microcrystalline cellulose was isolated and identified to be closely affiliated with Clostridium acetobutylicum, ATCC 824. At 37 degrees C and pH 5.0, the mono-culture of X-9 yields hydrogen with a 5-h time lag and end liquid products primarily of acetate and butyrate. The co-culture of X-9 with another strain, Ethanoigenens harbinense B-49, which can produce hydrogen efficiently from monosaccharides but directly from microcrystalline cellulose, produced more efficiently the bichydrogen via ethanol-type fermentation metabolism compared with mono-culture X-9 test. Bioaugmentation with X-9 + B-49 improved cellulose hydrolysis and subsequent hydrogen production rates as compared with that of monoculture bioaugmentation with X-9. (C) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.