Culture-independent. molecular techniques were applied to the characterization of microbial communities of an anaerobic granular sludge obtained from a full-scale digester. Procedures were optimised for total DNA recovery and polymerase chain reaction (PCR) amplification of 16S rDNA using archaea- and eubacteria-specific oligonucleotide primers. Cloned PCR products were subsequently screened by amplified rDNA restriction analysis to identify operational taxonomic units (OTUs). Inserts from clones representing each OTU were sequenced and phylogenetic trees were prepared. In addition, the microbial communities were characterised using terminal restriction fragment length polymorphism (T-RFLP). The specific methanogenic activity of the biomass, against various substrates, was also ascertained. Two anaerobic bioreactors were seeded with granular and non-granular (i.e. crushed) aliquots of the characterised sludge, respectively. and used to investigate the treatment of a volatile fatty acid (VFA)-based synthetic wastewater, at a loading rate of 5 kg COD m(-3) day(-1) at low ambient temperatures (18degreesC). DNA was isolated from sludge samples during the test period and shifts in archaeal and eubacterial population structures were elucidated. The start-up period was successful with methane yields and COD removal efficiencies of 60-75% and 65-85%, respectively. Specific methanogenic activities of reactor biomass, obtained at the conclusion of the trial, indicated the development of psychrotolerant biomass during the 90-day experiment. Furthermore, the efficacy of T-RFLP as a molecular tool for use in the surveyance of engineered ecosystems was confirmed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.