To investigate the influence of bicarbonate on the metabolic pathway of methanogenesis, different concentrations of bicarbonate (0-0.2 mol/L) were applied during thermophilic anaerobic digestion of 2.5 and 5 g/L glucose. The stable carbon isotopic results demonstrated that, as the bicarbonate concentration increased, the proportion of total CH4 generated from hydrogenotrophic methanogenesis generally increased. Furthermore, methane production rates and acetate degradation rates were seriously reduced under high levels of bicarbonate (0.15 and 0.2 mol/L). Meanwhile, carbon isotope fractionation was more prominent in treatments with 5 g/L glucose than that of 2.5 g/L glucose. Increased concentrations of bicarbonate altered the dominant methanogens and bacteria and increased the microbial diversity. The inhibitory effects of high concentrations of bicarbonate suggested that bicarbonate should be used cautiously as a buffer salt in anaerobic processes, especially when methanogenetic pathways were studied. (C) 2013 Elsevier Ltd. All rights reserved.