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The potent greenhouse gas methane presents a widely accessible resource, being the primary component in natural gas as well as in bio-gas from anaerobic digesters. Given its relatively low heating-value and several issues concerning its storage and transportation, methane upgrading to liquid fuels is of particular interest. Microbial methane conversion/utilization and upgrading is gaining increasing interest due to its high conversion efficiency. In this study we computationally compare aerobic and anaerobic microbial pathways for CH4 oxidation and discuss theoretically achievable biomass yields as well as the possibility for building synthetic biological production platforms for liquid fuels. Specifically, the presented in-silico work investigates the potential of microbial methane upgrading in a metabolic network analysis by means of elementary flux modes. Aerobic fixation of methane via conversion of methane to methanol by a methane monooxygenase (MMO) and different subsequent formaldehyde assimilation pathways (Serine-cycle, RuMP, XMP/DHA-pathway) is compared with anaerobic pathways for oxidation of methane (AOM) by means of reverse-methanogenesis or via a presumed glycyl-radical enzyme, which uses fumarate for activation of methane. The different pathways for aerobic and anaerobic methane oxidation are compared in different central carbon-metabolism envelopes in order to identify highest achievable carbon yields. The capability of efficient CO2 fixation, as well as energy preservation in form of reducing equivalents is identified as crucial to enable high yields, which ranged from 22% to 100%. The potential of the different microbes to grow on these gas streams is assessed by means of the maximum achievable biomass yield and the CO2/CH4 uptake ratio. CO2 co-utilisation, by transferring reducing power between the two co-substrates, is highest, when combining reverse-methanogenesis with the Wood-Ljungdahl pathway, effectively replacing the need for H2 with CH4. Further, the possibility to upgrade methane into liquid (drop-in) bio-fuels is investigated. Established routes to methanol, ethanol, C4-alcoholes and farnesene are evaluated in the most promising substrate-pathway / organism combinations. Stoichiometric, thermodynamic and kinetic limitations are assessed and recommendations regarding potential industrial feasibility are given. The results presented here should guide future research efforts in search for feasible ways of (co)utilizing novel carbon substrates for sustainable production of fuels and chemicals.

Metabolic Network Analysis of Microbial Methane Utilization for Biomass Formation and Upgrading to Bio-Fuels