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Highlights • A Methanoperedenceae-dominated methanotrophic consortium was able to respirate to electrodes. • PHB was identified as energy source for the extracellular respiration of the consortium. • Acetate was a likely intermediate from degradation of PHB and EPSs in the consortium. • Methanoperedenceae interacted with Geobacter in the consortia via the diffusion of acetate. Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH4) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by ‘Candidatus Methanoperedens nitroreducens’ in a CH4-fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH4 directly. In the absence of CH4, current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of ‘Ca. M. nitroreducens’ to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems.

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modelling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modelling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilisation with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.