Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 degrees C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9-14 degrees C at applied organic loading rates (OLRs) of 1.2-3.6 kg chemical oxygen demand (COD)m(-3) d(-1). Replicated treatment performance was observed at 10-14 degrees C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 degrees C at an applied OLR of 2.4 kg COD m(-3) d(-1). Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 degrees C. (C) 2008 Elsevier GmbH. All rights reserved.