Microorganisms are known to exhibit extracellular electron transfer (EET) in a wide variety of habitats. However, as for the human microbiome which significantly impacts our health, the role and importance of EET has not been widely investigated. In this study, we enriched and isolated the EET-capable bacteria from human gut microbes using an electrochemical enrichment method and examined whether the isolates couple EET with anaerobic respiration or fermentation. Upon the use of energy-rich or minimum media (with acetate or lactate) for electrochemical enrichment with the human gut sample at an electrode potential of +0.4 V [vs. the standard hydrogen electrode (SHE)], both culture conditions showed significant current production. However, EET-capable pure strains were enriched specifically with minimum media, and subsequent incubation using the δ-MnO2-agar plate with lactate or acetate led to the isolation of two EET-capable microbial strains, Gut-S1 and Gut-S2, having 99% of 16S rRNA gene sequence identity with Enterococcus avium (E. avium) and Klebsiella pneumoniae (K. pneumoniae), respectively. While the enrichment involved anaerobic respiration with acetate and lactate, further electrochemistry with E. avium and K. pneumoniae revealed that the glucose fermentation was also coupled with EET. These results indicate that EET couples not only with anaerobic respiration as found in environmental bacteria, but also with fermentation in the human gut.

Continental subsurface environments can present significant energetic challenges to the resident microorganisms. While these environments are geologically diverse, potentially allowing energy harvesting by microorganisms that catalyze redox reactions, many of the abundant electron donors and acceptors are insoluble and therefore not directly bioavailable. Extracellular electron transfer (EET) is a metabolic strategy that microorganisms can deploy to meet the challenges of interacting with redox-active surfaces. Though mechanistically characterized in a few metal-reducing bacteria, the role, extent, and diversity of EET in subsurface ecosystems remains unclear. Since this process can be mimicked on electrode surfaces, it opens the door to electrochemical techniques to enrich for and quantify the activities of environmental microorganisms in situ. Here, we report the electrochemical enrichment of microorganisms from a deep fractured-rock aquifer in Death Valley, CA, USA. In experiments performed in mesocosms containing a synthetic medium based on aquifer chemistry, four working electrodes (WEs) were poised at different redox potentials (272, 373, 472, 572 mV vs. SHE) to serve as electron acceptors, resulting in anodic currents coupled to the oxidation of acetate during enrichment. The anodes were dominated by Betaproteobacteria from the families Comamonadaceae and Rhodocyclaceae. A representative of each dominant family was subsequently isolated from electrode-associated biomass. The EET abilities of the isolated Delftia strain (designated WE1-13) and Azonexus strain (designated WE2-4) were confirmed in electrochemical reactors using WEs poised at 522 mV vs. SHE. The rise in anodic current upon inoculation was correlated with a modest increase in total protein content. Both genera have been previously observed in mixed communities of microbial fuel cell enrichments, but this is the first direct measurement of their electrochemical activity. While alternate metabolisms (e.g., nitrate reduction) by these organisms were previously known, our observations suggest that additional 'hidden' interactions with external electron acceptors are also possible. Electrochemical approaches are well positioned to dissect such extracellular interactions that may be prevalent in the subsurface.

The diversity of microbially mediated redox processes that occur in marine sediments is likely underestimated, especially with respect to the metabolisms that involve solid substrate electron donors or acceptors. Though electrochemical studies that utilize poised potential electrodes as a surrogate for solid substrate or mineral interactions have shed some much needed light on these areas, these studies have traditionally been limited to one redox potential or metabolic condition. This work seeks to uncover the diversity of microbes capable of accepting cathodic electrons from a marine sediment utilizing a range of redox potentials, by coupling electrochemical enrichment approaches to microbial cultivation and isolation techniques. Five lab-scale three-electrode electrochemical systems were constructed, using electrodes that were initially incubated in marine sediment at cathodic or electron-donating voltages (five redox potentials between -400 and -750 mV versus Ag/AgCl) as energy sources for enrichment. Electron uptake was monitored in the laboratory bioreactors and linked to the reduction of supplied terminal electron acceptors (nitrate or sulfate). Enriched communities exhibited differences in community structure dependent on poised redox potential and terminal electron acceptor used. Further cultivation of microbes was conducted using media with reduced iron (Fe0, FeCl2) and sulfur (S0) compounds as electron donors, resulting in the isolation of six electrochemically active strains. The isolates belong to the genera Vallitalea of the Clostridia, Arcobacter of the Epsilonproteobacteria, Desulfovibrio of the Deltaproteobacteria, and Vibrio and Marinobacter of the Gammaproteobacteria. Electrochemical characterization of the isolates with cyclic voltammetry yielded a wide range of midpoint potentials (99.20 to -389.1 mV versus Ag/AgCl), indicating diverse metabolic pathways likely support the observed electron uptake. Our work demonstrates culturing under various electrochemical and geochemical regimes allows for enhanced cultivation of diverse cathode-oxidizing microbes from one environmental system. Understanding the mechanisms of solid substrate oxidation from environmental microbes will further elucidation of the ecological relevance of these electron transfer interactions with implications for microbe-electrode technologies.

Iron-oxidizing bacteria (IOB) and iron-reducing bacteria (IRB) can easily adhere onto carbon steel surface to form biofilm and affect corrosion processes. However, the mechanism of mixed consortium induced carbon steel corrosion is relatively underexplored. In this paper, the adsorptions of IOB (Mariprofundus ferrooxydans, M. f.), IRB (Thalassospira sp., T. sp.) and mixed consortium (M. f. and T. sp.) on surface of Q235 carbon steel and their effects on corrosion in seawater were investigated through surface analysis techniques and electrochemical methods. Results showed that local adhesion is a typical characteristic for biofilm on surface of Q235 carbon steel in M. f. and mixed consortium media, which induces localized corrosion of Q235 carbon steel. Corrosion rates of Q235 carbon steel in different culture media decrease in the order: r M.f. > r mixed consortium > r T. sp. > r sterile. The evolution of corrosion rate along with time decreases in M. f. medium, and increases then keeps table in both T. sp. and mixed consortium media. Corrosion mechanism of Q235 carbon steel in mixed consortium medium is discussed through analysis of surface morphology and composition, environmental parameter, and electrochemical behavior.

The extracellular electron transfer (EET) that connects the intracellular metabolism of electroactive microorganisms to external electron donors/acceptors, is the foundation to develop diverse microbial electrochemical technologies. For a particular microbial electrochemical device, the surface chemical property of an employed electrode material plays a crucial role in the EET process owing to the direct and intimate biotic-abiotic interaction. The functional modification of an electrode surface with redox mediators has been proposed as an effectual approach to promote EET, but the underlying mechanism remains unclear. In this work, we investigated the enhancement of electrochemically polymerized riboflavin interface on the bidirectional EET of Shewanella putrefaciens CN32 for boosting bioelectrocatalytic ability. An optimal polyriboflavin functionalized carbon cloth electrode achieved about 4.3-fold output power density (∼707 mW/m2) in microbial fuel cells and 3.7-fold cathodic current density (∼0.78 A/m2) for fumarate reduction in three-electrode cells compared to the control, showing great increases in both outward and inward EET rates. Likewise, the improvement was observed for polyriboflavin-functionalized graphene electrodes. Through comparison between wild-type strain and outer-membrane cytochrome (MtrC/UndA) mutant, the significant improvements were suggested to be attributed to the fast interfacial electron exchange between the polyriboflavin interface with flexible electrochemical activity and good biocompatibility and the outer-membrane cytochromes of the Shewanella strain. This work not only provides an effective approach to boost microbial electrocatalysis for energy conversion, but also offers a new demonstration of broadening the applications of riboflavin-functionalized interface since the widespread contribution of riboflavin in various microbial EET pathways together with the facile electropolymerization approach.

Pneumocystis jirovecii pneumonia (PcP) is a major human immunodeficiency virus (HIV)-related illness, rising among immunocompromised non-HIV patients and in developing countries. Presently, the diagnosis requires respiratory specimens obtained through invasive and costly techniques that are difficult to perform in all patients or implement in all economic settings. Therefore, the development of a faster, cost-effective, non-invasive and field-friendly test to diagnose PcP would be a significant advance. In this study, recombinant synthetic antigens (RSA) of P. jirovecii's major surface glycoprotein (Msg) and kexin-like serine protease (Kex1) were produced and purified. These RSA were applied as antigenic tools in immunoenzymatic assays for detection of specific anti-P. jirovecii antibodies (IgG and IgM) in sera of patients with (n = 48) and without (n = 28) PcP. Results showed that only IgM anti-P. jirovecii levels were significantly increased in patients with PcP compared with patients without P. jirovecii infection (p ≤ 0.001 with both RSA). Thus, two strip lateral flow immunoassays (LFIA), based on the detection of specific IgM anti-P. jirovecii antibodies in human sera samples, were developed using the innovative association of P. jirovecii's RSA with spherical gold nanoparticles (AuNPs). For that, alkanethiol-functionalized spherical AuNPs with ca. ~40 nm in diameter were synthetized and conjugated with the two RSA (Msg or Kex1) produced. These AuNP-RSA conjugates were characterized by agarose gel electrophoresis (AGE) and optimized to improve their ability to interact specifically with serum IgM anti-P. jirovecii antibodies. Finally, two LFIA prototypes were developed and tested with pools of sera from patients with (positive sample) and without (negative sample) PcP. Both LFIA had the expected performance, namely, the presence of a test and control red colored lines with the positive sample, and only a control red colored line with the negative sample. These results provide valuable insights into the possibility of PcP serodiagnosis at point-of-care. The optimization, validation and implementation of this strip-based approach may help to reduce the high cost of medical diagnosis and subsequent treatment of PcP both in industrialized and low-income regions, helping to manage the disease all around the world.

Scope: Salmonellosis is a prevalent food-borne illness that causes diarrhea in over 130 million humans yearly and can lead to death. There is an urgent need to find alternatives to antibiotics as many salmonellae are now multidrug resistant. As such, specific beneficial bacteria and dietary fibers can be an alternative as they may prevent Salmonella Typhimurium (STM) infection and spreading by strengthening intestinal barrier function. Methods and Results: We tested whether immune active long-chain inulin-type fructans and/or L. acidophilus W37, L. brevis W63, and L. casei W56 can strengthen barrier integrity of intestinal Caco-2 cells in the presence and absence of a STM. Effects of the ingredients on intestinal barrier function were first evaluated by quantifying trans-epithelial electric resistance (TEER) and regulation of gene expression by microarray. Only L. acidophilus had effects on TEER and modulated a group of 26 genes related to tight-junctions. Inulin-type fructans, L. brevis W63 and L. casei W56 regulated other genes, unrelated to tight-junctions. L. acidophilus also had unique effects on a group of six genes regulating epithelial phenotype toward follicle-associated epithelium. L. acidophilus W37 was therefore selected for a challenge with STM and prevented STM-induced barrier disruption and decreased secretion of IL-8. Conclusion:L. acidophilus W37 increases TEER and can protect against STM induced disruption of gut epithelial cells integrity in vitro. Our results suggest that selection of specific bacterial strains for enforcing barrier function may be a promising strategy to reduce or prevent STM infections.

Biophotovoltaic devices utilize photosynthetic organisms such as the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) to generate current for power or hydrogen production from light. These devices have been improved by both architecture engineering and genetic engineering of the phototrophic organism. However, genetic approaches are limited by lack of understanding of cellular mechanisms of electron transfer from internal metabolism to the cell exterior. Type IV pili have been implicated in extracellular electron transfer (EET) in some species of heterotrophic bacteria. Furthermore, conductive cell surface filaments have been reported for cyanobacteria, including Synechocystis. However, it remains unclear whether these filaments are type IV pili and whether they are involved in EET. Herein, a mediatorless electrochemical setup is used to compare the electrogenic output of wild-type Synechocystis to that of a ΔpilD mutant that cannot produce type IV pili. No differences in photocurrent, i.e., current in response to illumination, are detectable. Furthermore, measurements of individual pili using conductive atomic force microscopy indicate these structures are not conductive. These results suggest that pili are not required for EET by Synechocystis, supporting a role for shuttling of electrons via soluble redox mediators or direct interactions between the cell surface and extracellular substrates.

Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems (BES). We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 μg/mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 μA/cm(2) anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains.

According to the literature, pyrite (FeS2) oxidation has been previously determined to involve thiosulfate as the first aqueous intermediate sulfur product, which is further oxidized to sulfate. In the present study, pyrite oxidation by Acidithiobacillus ferrooxidans was studied using electrochemical and metabolic approaches in an effort to extend existing knowledge on the oxidation mechanism. Due to the small surface area, the reaction rate of a compact pyrite electrode in the form of polycrystalline pyrite aggregate in A. ferrooxidans suspension was very slow at a spontaneously formed high redox potential. The slow rate made it possible to investigate the oxidation process in detail over a term of 100 days. Using electrochemical parameters from polarization curves and levels of released iron, the number of exchanged electrons per pyrite molecule was estimated. The values close to 14 and 2 electrons were determined for the oxidation with and without bacteria, respectively. These results indicated that sulfate was the dominant first aqueous sulfur species formed in the presence of bacteria and elemental sulfur was predominantly formed without bacteria. The stoichiometric calculations are consistent with high iron-oxidizing activities of bacteria that continually keep the released iron in the ferric form, resulting in a high redox potential. The sulfur entity of pyrite was oxidized to sulfate by Fe3+ without intermediate thiosulfate under these conditions. Cell attachment on the corroded pyrite electrode surface was documented although pyrite surface corrosion by Fe3+ was evident without bacterial participation. Attached cells may be important in initiating the oxidation of the pyrite surface to release iron from the mineral. During the active phase of oxidation of a pyrite concentrate sample, the ATP levels in attached and planktonic bacteria were consistent with previously established ATP content of iron-oxidizing cells. No significant upregulation of three essential genes involved in energy metabolism of sulfur compounds was observed in the planktonic cells, which represented the dominant biomass in the pyrite culture. The study demonstrated the formation of sulfate as the first dissolved sulfur species with iron-oxidizing bacteria under high redox potential conditions. Minor aqueous sulfur intermediates may be formed but as a result of side reactions.

Extracellular electron transfer (EET) plays a fundamental role in microbial reduction/oxidation of minerals. Extracellular polymeric substances (EPS) surrounding the cells constitute a matrix that separates the cell's outer membrane from insoluble minerals and environmental fluid. This study investigated the effects of EPS on EET processes during microbial reduction of hematite by the iron-reducing strain Shewanella oneidensis MR-1 (MR-1). Electrochemical characterization techniques were employed to determine the influence of EPS components on the redox ability of MR-1. Cells with removed EPS exhibited approximately 30% higher hematite reduction than regular MR-1 cells, and produced a current density of 56 μA cm-2, corresponding to 3-4 fold that of regular MR-1. The superior EET of EPS-deprived cells could be attributed to direct contact between outer membrane proteins and hematite surface, as indicated by more redox peaks being detected by cyclic voltammetry and differential pulse voltammetry. The significantly reduced current density of MR-1 cells treated with proteinase K and deoxyribonuclease suggests that the electron transfer capacity across the EPS layer depends mainly on the spatial distribution of specific proteins and electron shuttles. Exopolysaccharides in EPS tend to inhibit electron transfer, however they also favor the attachment of cells onto hematite surfaces. Consistently, the charge transfer resistance of cells lacking EPS was only 116.3 Ω, approximately 44 times lower than that of regular cells (5,139.1 Ω). These findings point to a negative influence of EPS on EET processes for microbial reduction/oxidation of minerals.

The increase of industrial discharges is the first cause of the contamination of water bodies. The bacterial survival strategies contribute to the equilibrium restoration of ecosystems being useful tools for the development of innovative environmental biotechnologies. The aim of this work was to study the Cu(II) and Cd(II) biosensing, removal and recovery, mediated by whole cells, exopolymeric substances (EPS) and biosurfactants of the indigenous and non-pathogenic Pseudomonas veronii 2E to be applied in the development of wastewater biotreatments. An electrochemical biosensor was developed using P. veronii 2E biosorption mechanism mediated by the cell surface associated to bound exopolymeric substances. A Carbon Paste Electrode modified with P. veronii 2E (CPEM) was built using mineral oil, pre-washed graphite power and 24 h-dried cells. For Cd(II) quantification the CPEM was immersed in Cd(II) (1-25 μM), detected by Square Wave Voltammetry. A similar procedure was used for 1-50 μM Cu(II). Regarding Cd(II), removal mediated by immobilized EPS was tested in a 50 ml bioreactor with 0.13 mM Cd(II), pH 7.5. A 54% metal retention by EPS was achieved after 7 h of continuous operation, while a 40% was removed by a control resin. In addition, surfactants produced by P. veronii 2E were studied for recovery of Cd(II) adsorbed on diatomite, obtaining a 36% desorption efficiency at pH 6.5. Cu(II) adsorption from a 1 mM solution was tested using P. veronii 2E purified soluble EPS in 50 mL- batch reactors (pH = 5.5, 32°C). An 80% of the initial Cu(II) was retained using 1.04 g immobilized EPS. Focusing on metal recovery, Cu nanoparticles (NPs) biosynthesis by P. veronii 2E was carried out in Cu(II)-PYG Broth at 25°C for 5 days. Extracellular CuNPs were characterized by UV-Vis spectral analysis while both extracellular and intracellular NPs were analyzed by SEM and TEM techniques. Responses of P. veronii 2E and its products as biosurfactants, bound and soluble EPS allowed Cu(II) and Cd(II) removal, recovery and biosensing resulting in a multiple and versatile tool for sustainable wastewater biotreatments.