The reproducibility and stability of low-temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular Sludge bed-based bioreactors were used to degrade a volatile fatty acid and glucose-based wastewater under sub-ambient (15 degrees C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient get electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations. Biotechnol. Bioeng. 2010;105:79-87. (C) 2009 Wiley Periodicals, Inc.