Biodiesel production from cotton-seed cake (CSC) and the pretreatment of the remaining biomass for dark fermentative hydrogen production was investigated. The direct conversion to biodiesel with alkali free fatty acids neutralization pretreatment and alkali transesterification resulted in a biodiesel with high esters content and physicochemical properties fulfilling the EN-standards. Blends of cotton-seed oil methyl esters (CME) and diesel showed an improvement in lubricity and cetane number. Moreover, CME showed good compatibility with commercial biodiesel additives. On the basis of conversion of the remaining CSC to sugars fermentable towards hydrogen, the optimal conditions included removal of the oil of CSC and pretreatment at 10% NaOH (w/w dry matter). The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production, 84-112% of the control, from NaOH-pretreated CSC and low hydrogen production, 15-20% of the control, from the oil-rich and not chemically pretreated CSC, and from Ca(OH)(2)-pretreated CSC. (C) 2013 Elsevier Ltd. All rights reserved.