Wood-Ljungdahl pathway utilisation during in situ H₂ biomethanation

Biogas production from organic waste is a waste-to-energy technology with the potential to contribute significantly to sustainable energy production. Upgrading of biogas using in situ biomethanation with hydrogen has the potential for surplus electricity storage, and delivery of biogas with a methane content of >90%, allowing for easier integration into the natural gas grid, as well as conversion to other products. Microbial communities in biomethanation reactors undergo changes, however, these changes are largely unexplored. In the present study, metagenome-resolved protein stable isotope probing (Protein-SIP) was applied to laboratory scale batch incubations operating under anaerobic digestion, and (pre-adapted) biomethanation conditions, fed with ¹³C-labelled bicarbonate, in order to gain insight into the microbial activities during CO₂-reduction. The strongest and most microbially diverse isotopic incorporation was observed in the pre-adapted biomethanation incubation. Furthermore, divergent incorporation of ¹³C-labelled bicarbonate was also observed in the Wood-Ljungdahl pathway, with the anaerobic digester incubations primarily showing labelled proteins in the peripheral pathways leading toward production of energy and biomass. The pre-adapted biomethanation incubations consumed H₂ and CO₂, but did not convert it to CH₄, suggesting the production of acetate in these incubations, which was supported by heavy labelling of key enzymes in the Wood-Ljungdahl pathway. Twelve (ten high quality) metagenome-assembled genomes (MAGs) coding for ¹³C-incorporated proteins were extracted from the metagenome, eight of which contained one or more of the key genes in the Wood-Ljungdahl pathway, one of which was affiliated to Methanosarcina. Together, the findings in the present study deepen our knowledge surrounding microbial communities in biomethanation systems, and contribute to the development of better strategies for implementation of biogas upgrading and microbial management.