Appropriate enrichment of anaerobic microorganism's consortium is crucial for accurate biochemical methane potential (BMP) assays. An alternative method to produce and maintain a mesophilic methanogenic inoculum was demonstrated. Three sources of inoculum were mixed and acclimated for 857 days in order to reach steady conditions (pH = 7.90 +/- 0.46; VS/TS > 50%; VFA/alkalinity = 0.16 +/- 0.04 g(Acetic Acid)/g(CaCO3)). Biogas yield > 80% was obtained after 70 days of inoculum acclimation in comparison to standard cellulose (>600 mL(N)/g(VS)). Methanogen community analysis based on 16S rDNA of the inoculum revealed Archaea concentration of 3 x 10(12) gene copies/g (Methanobacteriales 8 x 10(10); Methanomicrobiales 8 x 10(10); and Methanosarcinales 4 x 10(11) gene copies/g). The proposed method for development and maintenance of microorganism enrichment inoculum demonstrates consistent BMP data which is a requirement for dependable prediction of biogas production at field scale operations. (C) 2016 Elsevier Ltd. All rights reserved.