Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO2, biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic- and thermophilic anaerobic cultures were enriched to convert CO2 to CH4 by addition of H2. Enrichment at thermophilic temperature (55 degrees C) resulted in CO2 and H2 bioconversion rate of 320?mL CH4/(gVSS?h), which was more than 60% higher than that under mesophilic temperature (37 degrees C). Different dominant species were found at mesophilic- and thermophilic-enriched cultures, as revealed by PCRDGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH4 content, around 95% at steady-state, at gas (mixture of biogas and hydrogen) injection rate of 6?L/(L?day). The increase of gas injection rate to 12?L/(L?day) resulted in the decrease of CH4 content to around 90%. Further study showed that by decreasing the gasliquid mass transfer by increasing the stirring speed of the mixture the CH4 content was increased to around 95%. Finally, the CH4 content around 90% was achieved in this study with the gas injection rate as high as 24?L/(L?day). Biotechnol. Bioeng. 2012; 109: 27292736. (c) 2012 Wiley Periodicals, Inc.