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.

Fermented dairy products have emerged as promising functional foods for improving gastrointestinal (GI) health, yet the mechanisms underlying the benefits of fermented dairy products are not yet known. This dissertation aimed to review the current evidence on fermented dairy and GI health (Chapter 1) and investigate the capacity of probiotic-fermented milk to mitigate inflammation and improve barrier function using a mouse model for inflammatory bowel disease (Chapter 2) and in vitro using the Caco-2 cell line (Chapter 3). The review of human clinical trials assessed the effects of fermented dairy consumption on gut symptoms and biomarkers of gut health, which included inflammatory markers, barrier integrity, short-chain fatty acid levels, and microbiota composition (Chapter 1). In general, fermented dairy intake resulted in modest improvements in some GI symptoms, with fermented milk containing probiotic strains demonstrating the most promise in improving gut health. However, there was no consistent trend in how fermented dairy products altered the gut microbiota composition. The outcomes varied substantially depending on the fermented dairy product and probiotic strain. Since many products contained added probiotics, this highlighted the need to delineate the dairy matrix versus probiotic effects. To investigate this in my dissertation, I used a mouse model of dextran sodium sulfate (DSS)-induced colitis (Chapter 2). My findings showed sex-dependent differences in DSS-induced colitis progression and recovery, with worse outcomes in females, and these effects may have been due to differences in their intestinal microbiota. Feeding mice milk fermented by Lacticaseibacillus casei BL23 improved weight recovery, specifically among females. Milk supported better weight retention among males, while BL23-milk enhanced colonic ion transport. To determine how milk and probiotics can directly modulate the intestinal epithelium, the capacity of fermented milk to mitigate the effects of the inflammatory cytokine, IFNγ, on intestinal epithelial cells (IECs) was assessed (Chapter 3). A cell-free preparation of milk fermented by L. casei BL23 or L. casei ATCC334, but not regular milk, mitigated inflammatory disruption of the intestinal epithelial barrier. It was shown that this occurred via epidermal growth factor receptor activation in intestinal epithelial cells. Transcriptomic analysis of the IECs revealed that milk fermented by either L. casei BL23 or ATCC334 increased the expression of the gene encoding for the tight junction protein claudin-2. Both fermented milks also reversed inflammation-induced suppression of sterol metabolism, potentially through the master regulator of cholesterol synthesis and uptake known as sterol regulatory element-binding protein 2 (SREBP2). Higher sterol gene expression correlated with milder colitis in DSS-treated mice, implicating sterol regulation in L. casei-mediated barrier effects. In summary, this dissertation significantly expands the understanding of the benefits of fermented dairy and probiotic consumption on gut health. The findings also underscore the intricate interactions between diet, microbiota, host factors, and intracellular pathways governing GI responses to inflammation. This work further provides molecular insights into the impact of probiotic-fermented dairy on gut health.

Lacto-fermented fruits and vegetables (FFVs) have gained popularity for their positive impact on health, however, insights of the mechanistic details underpinning the benefits of FFVs are limited. This dissertation aimed to first examine the current evidence on the effects of whole FFVs consumption and the health-promoting potentials of the constituents in FFVs (Chapter 1). Next, the capacity of fermented cabbage to ameliorate inflammation-induced intestinal barrier disruption was investigated using an in vitro Caco-2 monolayer model (Chapter 2). Furthermore, this dissertation focused on Plantaricin EF (PlnEF), a bacteriocin peptide produced by Lactiplantibacillus plantarum, a member of FFV microbiomes, as a case study for assessing the feasibility of encapsulation for intestinal delivery of bioactive peptides (Chapter 3) and elucidating the dual functionality of PlnEF as an effector molecule with intestinal barrier protective property (Chapter 4) and antimicrobial activity (Chapter 5). Consumption of whole FFVs was associated with improvement of metabolic and immune functions and reduction of oxidative stress, which overlaps with the bioactivity of fermentation derived metabolites found in FFVs (Chapter 1). Despite the enticing support for the health benefits of whole FFVs and their bioactive constituents, more mechanistic studies focusing on the influences of individual bioactive components in the context of the whole FFV food are needed. To help bridge this gap, the capability of fermented cabbage and fermentation-derived metabolites to mitigate cytokine-induced intestinal barrier permeabilization in Caco-2 monolayers was examined (Chapter 2). My findings revealed that fermented cabbage, but not raw cabbage in brine, prevented cytokine-induced barrier disruption, and this activity that was attributed to the distinct metabolomes and specific metabolites after fermentation. To focus in on specific bioactive compounds expected to be present in FFV, this dissertation evaluated the effectiveness of encapsulation in protecting the peptides, PlnEF, during simulated gastrointestinal (GI) transit (Chapter 3). The results revealed that although the cross-linked calcium alginate microcapsules (CLAMs) were not able to protect PlnEF from gastric pepsin degradation, mixing CLAMs-encapsulated PlnEF in food (peanut butter) was successful in retaining active PlnEF through simulated GI digestion. This dissertation also showed that PlnEF exhibited a putative prophylactic effect in preventing disruptions to intestinal barrier permeability in Caco-2 cells, likely attributed to the modulation of tight junction proteins and other PlnEF-receptor mediated pathways (Chapter 4). Lastly, the antibacterial activity of PlnEF was shown by the increased the lag phase times of a PlnEF sensitive L. plantarum strain in a dose-dependent manner (Chapter 5). A spontaneous, PlnEF-resistant mutant of L. plantarum with a mutation in the Mg2+ efflux protein, CorC, contained higher intracellular Mg2+ levels regardless of the presence of PlnEF, pointing to the roles of intracellular Mg2+ in the heterogenous cellular response against the bacteriocin. In summary, this dissertation markedly contributes to the understanding of the effects of bioactive compounds produced by FFVs-associated LAB on intestinal barrier integrity. The findings showed that FFVs can serve as a feasible dietary source of bioactive constituents with beneficial impact on intestinal barrier function. This work also underlines the complexities of these microbe-host and microbe-microbe interactions mediated through microbial metabolites.