Aims:Anaerobic sludge granules underpin high-rate waste-to-energy bioreactors. Granulation is a microbiological phenomenon involving the self-immobilization of several trophic groups. Low-temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low-temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15 degrees C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules.Methods and Results:One mesophilic (R1; 37 degrees C) and two low-temperature (R2 and R3, 15 degrees C) laboratory-scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter < 0 center dot 4 mm) granules and were fed a glucose-based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low-temperature (LT) bioreactors.Conclusions:Granulation was observed at 15 degrees C in anaerobic bioreactors and was associated with H-2/CO2-mediated methanogenesis and distinct community structure development.Significance and Impact of the Study:Granulation underpins high-rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation < 20 degrees C was not previously documented.