Low-temperature (<20 degrees C) anaerobic digestion is ail emerging, cost-efficient technology, which has been successfully applied to a variety of high-strength hazardous and non-hazardous waste-streams. Operated at low-temperature, expanded granular sludge bed (EGSB) reactors favour the development of a methanogenic consortium adapted to high biodegradation rates. Reactor operating parameters, Such as wastewater characteristics and organic loading rate, affect microbial consortium physiology and therefore Could shift bacterial 16S rRNA ratios, for example by changing growth rates and/or physiological activity. Thus its a screening tool, temporal DGGE-profiling. comparing DNA and cDNA, was employed to highlight the potentially key organisms directly involved in psychrophilic (12 degrees C) toluene methanogenesis in a laboratory-scale EGSB bioreactor. Biomass samples were taken and analysed over a 5-day period, immediately following,in imposed increase in toluene loading rate. Functionally active potential toluene-degraders were Successfully characterized. The results suggested that a Putatively psychrophilic Geobacter-like organism was involved in the process along with hydrogenotropic Methanospirillum-like and acetotrophic Methanosaeta-like Archaea. The presence of additional mesophilic and thermophilic Putative toluene-degraders Suggested, however. that reactor operating temperature may not have been the main factor for the development of this well-established and active microbial methanogenic consortium.