Development of a mixed microbial culture for robust high-throughput ex-situ thermophilic biomethanation

Ex-situ biomethanation using mixed microbial cultures is a promising approach for carbon capture and utilisation, converting CO₂ and renewable H₂ into grid-compatible methane. However, achieving high H₂/CO₂ throughputs at ambient pressure remains constrained by microbial competition and process instability. This study presents a novel operational strategy integrating autoclave pretreatment, targeted microbial conditioning, and intermittent washouts to achieve stable, high throughput ex-situ biomethanation using autoclaved, microbe rich sludge. Autoclave pretreatment of seed sludge reduced total bacteria by ~92.5%, while key methanogens, Methanobacteria, Methanosarcina, and Methanosaeta, were declined by 91.5, 85, and 77%, respectively, but not fully eliminated. This non-specific biomass reduction enabled controlled management of a resilient mixed consortium, promoting rapid adaptation and enhanced methanogenic performance under high H₂/CO₂ throughputs. At 37 °C and H₂/CO₂ throughput of 450 L/L/d, methane content remained below 60%. In contrast, conditioning the microbial community at 50 °C under a stoichiometric H₂: CO₂ ratio of 4:1 significantly enhanced performance, increasing methane content to 82%. Further increasing the throughput to 500 L/L/d raised methane content to 86%, with potential to exceed 90%. This represents the highest reported throughput and methane quality in continuously stirred tank reactors operating under atmospheric pressure and 50 °C. The optimised system developed a stable consortium dominated by Euryarchaeota (78%), alongside Firmicutes (16%) and Coprothermobacterota (4%). Intermittent washouts effectively regulated metabolic intermediates, stabilising volatile fatty acids at ~3.5 g/L, primarily acetate (73%). Overall, this study demonstrates a robust strategy for high-throughput ex-situ biomethanation, advancing CO₂ valorisation and supporting energy storage and circular carbon management.