Dark fermentation of carboxymethyl cellulose to produce biohydrogen using mono-culture or co-culture of isolated strains was studied. Three isolates were noted to effectively hydrolyze cellulosic substrates and degrade the metabolites to hydrogen and volatile fatty acids. The strain Clostridium acetobutylicum X-9 was noted to have the highest hydrogen yield amongst the three isolates in all tests. Co-cultures of any two of the three isolates and with another strain Ethanoigenens harbinense B-49 demonstrated higher biohydrogen yield and cellulose hydrolysis ratio compared with the mono-cultured tests. Bioaugmentation with co-cultures X-9 + B-49 efficiently improved cellulose hydrolysis and subsequent hydrogen production rates from carboxymethyl cellulose. The strain X-9 significantly hydrolyzed corn stalks pretreated with H2SO4, NaOH, and NH3 soaking, and steam explosion in 10-12 h. Hydrogen was yielded in conjunction with the noted cellulose hydrolysis. The steam explosion + hydrolysis/fermentation with X-9 present the most effective method amongst the four tested pretreatments for hydrolyzing cellulose and yielding hydrogen. (C) 2008 international Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.