A hydrogen-adapted microbial community enhances in situ biomethanation robustness under increasing organic loading...

In situ biomethanation upgrades biogas by converting CO₂ and externally supplied H₂ into CH₄ directly within anaerobic digesters. However, elevated H₂ partial pressure can disrupt syntrophic interactions and impair anaerobic digestion (AD). Microbial adaptation, particularly within archaeal communities, may mitigate these effects. This study assessed how prior H₂ exposure influences organic matter conversion under increasing organic loading rate (OLR). Two 3-L mesophilic continuous stirred-tank reactors were operated either in in situ biomethanation mode (i.e., H₂-supplied AD), with H₂ supplied at 2 NmL·min^-1, or as AD without H₂ addition. Using an H₂-adapted inoculum, the OLR was increased stepwise from 2.5 to 6.0 gVS·L^-1·d^-1, while maintaining the hydraulic retention time (∼30 days). The maximum CH₄ production rate at the highest OLR applied (6.0gVS·L^-1·d^-1) increased by 351 NmL CH₄·L^-1·d^-1 in AD, while it was doubled in the in situ biomethanation reactor (733 NmL CH₄·L^-1·d^-1). Similarly, AD and in situ biomethanation produced 57 % and 76 % of the theoretical CH₄ expected from the additional organic matter added when increasing the OLR, and therefore a higher accumulation of volatile fatty acids was observed in AD. Despite the increasing OLR, H₂ conversion remained above 98 % throughout the experiment under in situ biomethanation. H₂ addition led to a more balanced archaeal community, with similar abundances between hydrogenotrophic methanogens and Methanosarcina sp., while in AD, Methanosarcina sp. largely dominated the archaeal community with the increasing OLR. Whereas bacterial community trajectories remained broadly similar between reactors. Overall, H₂-adapted in situ biomethanation enhanced CH₄-oriented carbon conversion while impeding metabolic imbalance.