Toward complete carbon utilization: Improved methane yield from formate and hydrogen co-feeding through constitutive formate dehydrogenase-gene expression in Methanothermobacter thermautotrophicus ΔH

Formate is emerging as a relevant intermediate in carbon capture and utilization technologies. However, its low energy density limits its value as an energy carrier. Some hydrogenotrophic methanogens can reduce formate to the established energy carrier methane. The stoichiometric limitation of formate disproportionation is that 75 % of the carbon is released as carbon dioxide, and achieving a complete carbon utilization requires co-feeding hydrogen. However, hydrogen-dependent genetic regulation of formate metabolism inhibits simultaneous formate and hydrogen utilization in hydrogenotrophic methanogens. Here, we compared the catalytic performance of the genetically modified strain Methanothermobacter thermautotrophicus ΔH pMVS1111A:PhmtB-fdh Z-245 (pFdh) with M. thermautotrophicus Z-245 using continuous cultivation at different hydrogen concentrations . While M. thermautotrophicus Z-245 is natively formatotrophic, M. thermautotrophicus ΔH (pFdh) was engineered to enable formate utilization via plasmid-borne expression of a formate dehydrogenase-gene cassette. We found that M. thermautotrophicus ΔH (pFdh) can simultaneously utilize formate and hydrogen. It continuously consumed formate at a dissolved hydrogen concentration of 0.069 ± 0.004 mM, enabling a 76.6 % ± 0.9 % carbon utilization. M. thermautotrophicus Z-245 showed declining formate consumption as the dissolved hydrogen concentration increased toward approximately 0.02 mM and reached a maximum stable carbon utilization of 36.2 % ± 0.2 %. These results suggest that M. thermautotrophicus ΔH (pFdh) largely bypasses hydrogen-dependent transcriptional control of formate metabolism; however, it still faces redox-related metabolic limitations at dissolved hydrogen concentrations above 0.32 mM. Overall, the findings reveal a potential strategy to circumvent hydrogen-induced regulation of formate metabolism and identify M. thermautotrophicus ΔH (pFdh) as a promising biocatalyst for formate-to-methane conversion.