Syngas (H2, CO2, CO) produced thermochemically from lignocellulosic biomass is an underexploited source for resource recovery and valorisation through its biological conversion for the production of a wide range of chemicals and fuels. Syngas biomethanation is one such promising bioconversion pathway, displaying interesting features such as high conversion efficiency and product selectivity, as methane is the sole product of the process. The biological conversion of syngas to high purity biomethane is still typically associated with a number of challenges related to the syngas composition, CO toxicity, and gas–liquid mass transfer limitations. In this work, the syngas biomethanation process carried out in trickle bed reactors was investigated at its boundary conditions to explore the limits of the process, focusing on syngas composition and mass-transfer conditions potentially limiting process performance. The process was found to be robust when exposed to CO excess, but highly sensitive to H2 excess, which caused severe inhibition even under small amounts of excess H2. This implies that biomethane purity comparable to natural gas can be achieved by addition of renewable H2, but this requires precise control to avoid process failure. Modulating the liquid recirculation rate and gas residence time allowed for a maximum methane productivity of 9.8 ± 0.5 mmol CH4 h−1 Lreactor−1 with full conversion of H2 and CO at a gas residence time of 1 h. Nevertheless, increasing the gas–liquid mass transfer with increasing liquid recirculation rate did not lead to increased methane productivity, which suggested additional rate-limiting bottlenecks in the process. Careful investigation of other factors potentially limiting the process led to the conclusion that diffusive transport of syngas components in the biofilm was the main bottleneck of the process. This diffusive limitation leads to a scenario of severe substrate scarcity in the biofilm phase that conditions the thermodynamic feasibility of the different biochemical reactions involved in syngas biomethanation. In turn, these thermodynamic constraints were found to drive the stratification of microbial groups and sequential consumption of syngas components along the height of the reactor.
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