Addressing the climate crisis requires not a single breakthrough, but a suite of well-understood, adaptable solutions spanning technology, policy, and practice. To limit warming to no more than 1.5°C above pre-industrial levels, we must approach net zero carbon dioxide (CO2) emissions around the mid-twenty first century. However, achieving this will require a coordinated effort across multiple disciplines, with combinations of biology and technology playing a crucial role in supporting these initiatives. One promising biotechnology, microbial electrosynthesis (MES), has the potential to significantly reduce net CO2 emissions if implemented on an industrial scale. In future MES systems, microbial species capable of extracellular electron transfer (ET) and carbon fixing reactions could recycle CO2 from industrial emissions directly into useful organic molecules. While MES and its key components (the bacteria-electrode interface and ET), could become valuable tools in the broader effort to lower net CO2 emissions, fundamental questions remain for even the most well-understood extracellular ET pathway, the Mtr pathway. The Mtr pathway is the metal reducing pathway from Shewanella oneidensis, a bacterium that can use extracellular electron acceptors when the available oxygen is insufficient for respiration (outward ET). The Mtr pathway is also bidirectional, an important feature for a model organism used to study MES. Because MES requires a robust bacteria-electrode interface for electron transfer into the bacterium (inward ET), the bidirectionality of the Mtr pathway provides an excellent vehicle for studying the mechanisms and bottlenecks that constrain inward ET in S. oneidensis or comparable systems. Despite the established bidirectionality of the Mtr pathway, there is a persistent asymmetry between outward and inward ET, with outward electron transfer being consistently higher in magnitude. Tefft and TerAvest (2019) developed an S. oneidensis strain expressing butanediol dehydrogenase (Bdh), a non-native NADH-dependent enzyme. The enzymatic reaction Bdh catalyzes, acetoin reduction to 2,3-butanediol, can act as an indicator of electron transfer to cytoplasmic carriers via NADH dehydrogenases. However, this direction is the opposite of the respiratory direction and is thermodynamically limited for inward ET. In Chapter 2, I use a thermodynamic model to compare inward ET through S. oneidensis NADH dehydrogenases under three energetic coupling scenarios. I also use a complementary experimental approach to assess qualitative changes in membrane potential at the single cell level for electrode-attached S. oneidensis. In Chapter 3, I compare the extracellular component of inward and outward ET by using two thermodynamically favorable ET paths. Under conditions with and without supplemental flavin (a known ET mediator for S. oneidensis), I use both chronoamperometry and cyclic voltammetry to conclude that inward and outward ET occur through different mechanisms for anaerobic S. oneidensis. In Chapter 4, I follow up on an unpublished observation that growth medium impacts inward ET performance in bioelectrochemical systems (BESs) with minimal medium lacking a carbon source. Specifically, when S. oneidensis was pre-cultured in minimal medium rather than rich medium, inward ET ability increased. I performed differential protein analysis and found that pre-culture in minimal medium appears to prime S. oneidensis for inward ET more effectively than pre-culture in rich medium. Growth in minimal medium made proteins in energy conserving pathways more abundant, and proteins involved in translational processes less abundant. Together, Chapters 2, 3, and 4 describe bottlenecks along the inward ET pathway that, if alleviated, could lessen or eliminate the discrepancy between inward and outward ET rates in S. oneidensis.
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