The Electron Transport Chain ofShewanella oneidensisMR-1 can Operate Bidirectionally to Enable Microbial Electrosynthesis

Abstract

Extracellular electron transfer (EET) is a process by which bacterial cells can exchange electrons with a redox active material located outside of the cell. InShewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive microbial electrosynthesis of 2,3-butanediol (2,3-BDO) inS. oneidensisexogenously expressing butanediol dehydrogenase (Bdh). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via Bdh. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and O2reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical systems (BES), and the translocation of protons across the membrane during O2reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.

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

“…9 Past work with S. oneidensis is also consistent with IMF-dependent EEU to the cytoplasm. 25,29,67,83,84 Tefft and TerAvest (2019) reported EEU in S. oneidensis + Bdh + PR (proteorhodopsin), where PR, a light-driven proton pump, provided PMF. 67 On a cathode, S. oneidensis + Bdh + PR, rapidly responded to changes in light, indicating that EEU might be PMF-limited.…”

“…83,84 Ford and TerAvest (2023) demonstrated that PMF from terminal oxidases was also su cient to drive EEU to the cytoplasm in place of PR in S. oneidensis + Bdh. 29 Tefft et al (2022) found that CCCP interfered with EEU to the cytoplasm, but not to fumarate reductase (FccA) in the periplasm. 25 As illustrated by Ross et al (2011), 85% of EEU to FccA must go through the quinone pool.…”

“…Future work could optimize expression of both H + -pumping oxidases and dissolved oxygen concentration to provide su cient PMF for EEU, without reaching harmful concentrations of H 2 O 2 from oxygen reduced on the cathode. 29 Under optimized conditions, it is possible that IMF would no longer be limiting for EEU. If this occurs, and discrepancies between anodic and cathodic current density magnitudes persist, future work could consider enzymatic engineering.…”