UC Davis

Extracellular electron transfer (EET) is a bioelectrochemical pathway in which microorganisms reduce electron acceptors in their environment. Through EET, microorganisms can enhance energy conservation and balance intracellular redox homeostasis while simultaneously reducing their extracellular environment. Outside of these direct impacts EET-capable microbial metabolism, the broader effects of EET in conducive environments remain underexplored outside of applications for microbial fuel cells. Chapter 1 of this dissertation focuses on the EET capabilities of bacteria found in diverse plant- and animal-associated niches, including the plant rhizosphere, animal mucosal surfaces, and during the fermentation of plant and animal foods. Ecological impacts of EET metabolism are explored, including the modulation of metal uptake by plants, the growth and proliferation of pathogenic bacteria in mucosal niches, and the increasing environmental acidification and reduction potential during food fermentations. Food fermentations are of particular interest regarding EET due to the impact of lactic acid bacteria (LAB), a diverse group of Gram-positive organisms characterized by their production of lactic acid during fermentation. LAB are found in diverse environmental, plant, mammal, and insect-associated microbiomes, and used in the fermentation of hundreds of foods and beverages. In Chapter 2, the EET is identified and explored in Lactiplantibacillus plantarum, a model organism for studying LAB. We characterized L. plantarum EET through the reduction of extracellular, insoluble ferrihydrite (iron(III) oxyhydroxide) and the generation of current with a graphite anode. The genetic conservation of the L. plantarum flavin-mediated EET (FLEET) locus was identified in other LAB and led to the discovery that LAB EET required Ndh2 (a type-II NADH dehydrogenase) and conditionally required PplA (a membrane-associated flavin-binding reductase) for iron reduction or current generation. The metabolic impacts of EET were also quantified and these data showed that L. plantarum had a shorter lag phase, greater cell abundance and viability, greater intracellular ATP and NAD+/NADH under EET-conducive conditions. Furthermore, L. plantarum EET was demonstrated during the fermentation of kale juice and increased environmental acidification. With the physiological impacts of EET on L. plantarum characterized, we turned towards exploring the role of quinones, an indispensable class of electron shuttles used by all bacteria for EET. In Chapter 3, the specificity of quinones and their physiological and ecological impacts on L. plantarum EET was explored. L. plantarum growth with DHNA led to membrane-associated menaquinone production but was not sufficient to stimulate EET in the absence of an exogenous DHNA. Instead, co-exposure of L. plantarum to DHNA during growth and EET significantly increased iron reduction at both high and low, environmentally relevant concentrations. DHNA and other quinones inhibited L. plantarum growth via hydrogen peroxide production and induced expression of oxidative stress response genes and redox-associated amino acid metabolism and transport. However, co-exposure of DHNA and ferric iron, a terminal electron acceptor, reversed these effects and partially restored L. plantarum growth. Other purified, naphthoquinone-based compounds, as well as spent cell-free growth medium from the LAB Lactococcus lactis and Leuconostoc mesenteroides was also sufficient in stimulated L. plantarum EET. Furthermore, we confirmed quinone cross-feeding in situ by showing that L. plantarum in co-culture with these LAB results in enhanced iron reduction and accelerated environmental acidification. Taken together the results of this dissertation highlight a novel metabolic pathway present in L. plantarum and other LAB and explores both the physiological and ecological impacts of EET on niches inhabited by EET-capable LAB. These findings inform the use of LAB during the fermentation of foods and beverages, during which successful outcomes as well as modulating flavor profiles may be achievable using EET.