Background  Coal seams exhibit the extensive distribution of a variety of indigenous functional microorganisms. Among these, some archaea enable methane metabolism using CO₂ as a substrate, providing the potential for the biological CO₂ storage and utilization in coal seams. However, conventional studies focus primarily on the degradation characteristics of coal matrix, leading to a lack of a systematic understanding of the mechanisms governing the adjustment of the methanation process in CO₂-bearing environments.

Methods  This study investigated the bituminous coals with a high volatile content in the Huainan mining area. Using laboratory-scale experiments on methanogenesis through anaerobic fermentation under varying CO₂ concentrations (0‒100%), combined with untargeted metabolomics and microbiomics, this study systematically explored the mechanisms behind the impacts of CO₂ concentration on microbial methanogenesis. Furthermore, CO₂ consumption and methane production were calculated for the quantitative analysis of the conversion relationship between CO₂ and CH₄. Accordingly, the biochemical conversion pathways of CO₂ were established.

Results  The CO₂ concentration threshold was preliminarily observed under an atmosphere with a CO₂∶N₂ ratio of 4∶1 (C80 group). In this case, a peak methane production of 268.98 μmol/g coal was identified at day 45, suggesting a 42.11% increase compared to that of the atmosphere with a CO₂∶N₂ ratio of 0∶5 (189.27 μmol/g coal; CK group). In contrast, the methane production trended downward under atmospheres with CO₂∶N₂ ratios of 9∶1 (C90 group) and 1∶0 (C100 group). Meanwhile, the C80 group exhibited a 51.71% reduction in CO₂ within the headspace of the anaerobic bottle, with 1 mL CO₂ increasing about 0.38 mL CH₄. As the CO₂ concentration increased to 80%, the evolution patterns of the dominant genera of bacteria in the microbial community changed, with the dominant genera shifting from Paraclostridium and Enterococcus to Bacillus and Clostridium. The addition of exogenous CO₂ resulted in significant enrichment of methanogenic archaea, with mixotrophic Methanosarcina in the C80 group showing the most pronounced increase in relative abundance. Data from functional gene analysis also demonstrated that the intervention of exogenous CO₂ significantly increased both the Wood-Ljungdahl pathway of CO₂ fixation in Clostridium and the expression of methanogenesis-related functional genes. The analysis of liquid-phase differential metabolites before and after culture in various experimental groups shows that a high CO₂ concentration promoted the synthesis of metabolites associated with carbon fixation, such as cobamamide a,c-diamide and dihydroxyacetone phosphate (DAHP), while also accelerating the degradation of aromatic components in coals. In combination with results from microbial community composition, PICRUSt2 prediction, and untargeted metabolomics, this study determined the metabolic pathway that coupled the degradation of macromolecular organic matter in coals with CO₂-CH₄ conversion under the synergistic effects of bacteria and archaea.

Conclusion The results of this study reveal that exogenous CO₂ enhance microbial methanogenesis through a triple mechanism, which (1) results in the enrichment of hydrogenotrophic/mixotrophic methanogenic archaea; (2) increases the abundance of functional genes involved in the Wood-Ljungdahl pathway of carbon fixation in Clostridium while also promoting CO₂ reduction; and (3) facilitates both the metabolism induced by the cleavage of aromatic rings and the synthesis of carbon fixation cofactors. The findings that the CO₂ concentration threshold is 80% and that the Wood-Ljungdahl pathway of carbon fixation can be activated by the directional enrichment of Clostridium-Sarcina synergetic microflora provide a theoretical basis and new control strategies for both coalbed methane bioengineering (CGB) and the conversion of geological to biological CO₂ storage.