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Background  Commercial biogas upgrading facilities are expensive and consume energy. Biological biogas upgrading may serve as a low-cost approach because it can be easily integrated with existing facilities at biogas plants. The microbial communities found in anaerobic digesters typically contain hydrogenotrophic methanogens, which can use hydrogen (H 2 ) as a reducing agent for conversion of carbon dioxide (CO 2 ) into methane (CH 4 ). Thus, biological biogas upgrading through the exogenous addition of H 2  into biogas digesters for the conversion of CO 2  into CH 4  can increase CH 4  yield and lower CO 2  emission.    Results  The addition of 4 mol of H 2  per mol of CO 2  was optimal for batch biogas reactors and increased the CH 4  content of the biogas from 67 to 94%. The CO 2  content of the biogas was reduced from 33 to 3% and the average residual H 2  content was 3%. At molar H 2 :CO 2  ratios > 4:1, all CO 2  was converted into CH 4 , but the pH increased above 8 due to depletion of CO 2 , which negatively influenced the process stability. Additionally, high residual H 2  content in these reactors was unfavourable, causing volatile fatty acid accumulation and reduced CH 4  yields. The reactor microbial communities shifted in composition over time, which corresponded to changes in the reactor variables. Numerous taxa responded to the H 2  inputs, and in particular the hydrogenotrophic methanogen  Methanobacterium  increased in abundance with addition of H 2 . In addition, the apparent rapid response of hydrogenotrophic methanogens to intermittent H 2  feeding indicates the suitability of biological methanation for variable H 2  inputs, aligning well with fluctuations in renewable electricity production that may be used to produce H 2 .    Conclusions  Our research demonstrates that the H 2 :CO 2  ratio has a significant effect on reactor performance during in situ biological methanation. Consequently, the H 2 :CO 2  molar ratio should be kept at 4:1 to avoid process instability. A shift toward hydrogenotrophic methanogenesis was indicated by an increase in the abundance of the obligate hydrogenotrophic methanogen  Methanobacterium .    Electronic supplementary material  The online version of this article (10.1186/s13068-019-1443-6) contains supplementary material, which is available to authorized users.

Effects of H2:CO2 ratio and H2 supply fluctuation on methane content and microbial community composition during in-situ biological biogas upgrading