Modelling microbial and metabolic shifts in trickle bed reactor biomethanation at decreasing gas retention times

Biological methanation harnesses archaeal and bacterial consortia to convert H₂ and CO₂ into renewable CH₄, yet process efficiency diminishes when residence time is shortened. We combined genome-resolved metagenomics with condition-specific community genome-scale metabolic models to follow a thermophilic trickle-bed reactor subjected to stepwise reductions in gas-retention time. Abrupt shortening provoked a reproducible community transition: putative homoacetogens displaced minor bacteria, acetate accumulated, and CH₄ purity fell from >98 % to <93 %. Despite the upheaval, two Methanothermobacter species dominated throughout; only M. thermoautotrophicus sustained exponential growth, whereas M. marburgensis remained the principal CH₄ producer. Model-driven flux balance analysis exposed a shift from hydrogenotrophic to acetate-centred bacterial metabolism and identified Pseudothermotoga and Saccharicenans species as syntrophic acetate oxidisers that benefit most from cross-feeding with M. thermoautotrophicus. In silico bioaugmentation predicted that introducing the methylotrophic methanogen Methanosuranticola petrocarbonis or supplementing the medium with S-adenosyl-L-methionine and selected amino acids can restore high CH₄ yields at short retention times. Our integrative analysis pinpoints metabolic bottlenecks and offers actionable strategies to stabilise high-rate biological methanation.