Aims:The objective of this work was to provide functional evidence of key metabolic pathways important for anaerobic digestion processes through the identification of highly expressed proteins in a mixed anaerobic microbial consortium.Methods and Results:The microbial communities from an anaerobic industrial-like wastewater treatment bioreactor were characterized using phylogenetic analyses and metaproteomics. Clone libraries indicated that the bacterial community in the bioreactor was diverse while the archaeal population was mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three hundred and eighty-eight reproducible protein spots were obtained on 2-D gels, of which 70 were excised and 33 were identified. The putative functions of the proteins detected in the anaerobic bioreactor were related to cellular processes, including methanogenesis from CO(2) and acetate, glycolysis and the pentose phosphate pathway. Metaproteomics also indicated, by protein assignment, the presence of specific micro-organisms in the bioreactor. However, only a limited overlap was observed between the phylogenetic and metaproteomic analyses.Conclusions:This study provides some direct evidence of the microbial activities taking place during anaerobic digestion.Significance and Impact of Study:This study demonstrates metaproteomics as a useful tool to uncover key biochemical pathways underpinning specific anaerobic bioprocesses.