Energetic constraints of metal-reducing bacteria as biocatalysts for microbial electrosynthesis

Background As outlined by the Intergovernmental Panel on Climate Change, we need to approach global net zero CO ₂ emissions by approximately 2050 to prevent warming beyond 1.5°C and the associated environmental tipping points. Future MES systems could decrease net CO ₂ emissions by capturing it from industrial sources. Microbial electrosynthesis (MES) is a process where electroactive microorganisms convert the carbon from CO ₂ and reduction power from a cathode into reduced organic compounds. However, no MES system has attained an efficiency compatible with a financially feasible scale-up. To improve MES efficiency, we need to consider the energetic constraints of extracellular electron uptake (EEU) from an electrode to cytoplasmic electron carriers like NAD ⁺ . In many microbes, EEU to the cytoplasm must pass through the respiratory quinone pool (Q-pool). However, electron transfer from the Q-pool to cytoplasmic NAD ⁺ is thermodynamically unfavorable. Here, we model the thermodynamic barrier for Q-pool dependent EEU using the well-characterized bidirectional electron transfer pathway of Shewanella oneidensis , which has NADH dehydrogenases that are energetically coupled to proton-motive force (PMF), sodium-motive force (SMF), or uncoupled. We also tested our hypothesis that Q-pool dependent EEU to NAD ⁺ is ion-motive force (IMF)-limited in S. oneidensis expressing butanediol dehydrogenase (Bdh), a heterologous NADH-dependent enzyme. We assessed membrane potential changes in S. oneidensis  + Bdh on a cathode at the single-cell level pre to post injection with acetoin, the substrate of Bdh. Results We modeled the Gibbs free energy change for electron transfer from respiratory quinones to NADH under conditions reflecting changes in membrane potential, pH, reactant to product ratio, and energetically coupled IMF. Of the 40 conditions modeled for each method of energetic coupling (PMF, SMF, and uncoupled), none were thermodynamically favorable without PMF or SMF. We also found that membrane potential decreased upon initiation of EEU to NAD ⁺ for S. oneidensis on a cathode. Conclusions Our results suggest that Q-pool-dependent EEU is both IMF-dependent and is IMF-limited in a proof-of-concept system. Because microbes that rely on Q-pool-dependent EEU are among the most genetically tractable and metabolically flexible options for MES systems, it is important that we account for this thermodynamic bottleneck in future MES platform designs.