Microbial community dynamics during the anaerobic co-digestion of pig manure, pasteurized slaughterhouse waste and glycerin were studied in a lab-scale CSTR. The feed composition was optimized through progressive co-substrate additions for enhanced methane production and organic matter removal without accumulation of intermediate compounds. Microbial community structure of biomass samples was studied by means of qPCR and DGGE profiling of 16S rRNA genes (Bacteria and Archaea), and genus-specific qPCR of the methyl coenzyme M reductase gene (mcrA), which encodes for an enzyme universally involved in methanogenesis. The composition of the dominant bacterial populations remained relatively stable, when compared to those in the influent, but the highest changes were observed upon the introduction of glycerin. Biodiversity of archaea was restricted to a few representatives of the genera Methanosaeta and Methanosarcina, but Methanospirillum sp. was detected only when glycerin was introduced in the feeding. Glycerin supplementation coincided with the strongest increase in methane yield (from 0.22 to 0.64 m3CH4 m-3 d-1).