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The substitution of natural gas by renewable biomethane is an interesting option to reduce global carbon footprint. Syngas fermentation has potential in this context, as a diverse range of low-biodegradable materials that can be used. In this study, anaerobic sludge acclimatized to syngas in a multi-orifice baffled bioreactor (MOBB) was used to start enrichments with CO. The main goals were to identify the key players in CO conversion and evaluate potential interspecies metabolic interactions conferring robustness to the process. Anaerobic sludge incubated with 0.7 × 10 5  Pa CO produced methane and acetate. When the antibiotics vancomycin and/or erythromycin were added, no methane was produced, indicating that direct methanogenesis from CO did not occur. Acetobacterium and Sporomusa were the predominant bacterial species in CO-converting enrichments, together with methanogens from the genera Methanobacterium and Methanospirillum. Subsequently, a highly enriched culture mainly composed of a Sporomusa sp. was obtained that could convert up to 1.7 × 10 5  Pa CO to hydrogen and acetate. These results attest the role of Sporomusa species in the enrichment as primary CO utilizers and show their importance for methane production as conveyers of hydrogen to methanogens present in the culture., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)