1 Overall Introduction

The ubiquitous sulfate-reducing bacteria (SRB) participate in significant biogeological processes such as the cycling of sulfur (Jørgensen 1982; Berner and Raiswell 1983) and carbon and anaerobic oxidation of methane (AOM) (Boetius et al. 2000). Moreover, since hydrogen sulfide (H2S), a metabolite of SRB, is a metal-corroding chemical, SRB induce anaerobic corrosion of metal infrastructure such as underground oil and gas pipelines. This results in significant economic losses amounting to 0.4% of the gross domestic product of an industrial nation (Koch et al. 2001; Beavers and Thompson 2006). SRB are one of the most ubiquitous bacteria in anaerobic environments, especially the marine environments that are abundant with sulfate (~28 mM in seawater) (Christensen 1984; Parkes et al. 1989; Muyzer and Stams 2008; Fichtel et al. 2012). These bacteria reduce sulfate as the electron acceptor to hydrogen sulfide, coupled with the oxidation of diverse organics, hydrocarbon (So and Young 1999), and hydrogen (H2) as the electron donors. In addition to these soluble or gaseous energy sources, recent studies demonstrated that some SRB use solids as electron donor via extracellular electron uptake (EEU) processes (Deng et al. 2015, 2018; Beese-Vasbender et al. 2015; Deng and Okamoto 2018; Deng et al. 2020). Direct electron transport process from metal iron specimens into the cell interior was first proposed in two marine sedimentary SRB strains, Desulfovibrio ferrophilus strain IS5 and Desulfobacterium corrodens strain IS4. This was based on the observation of significantly faster anaerobic corrosion in the absence of organic electron donors and at rates higher than other conventional SRB strains which corrode iron by producing H2S or depleting accumulated H2 on the iron surface (Dinh et al. 2004). The direct EEU process from solids in SRB was shown by excluding the involvement of H2 (generated as a result of proton reduction on the surface of a solid electron donor) as an electron mediator between the solids and SRB cells in whole-cell electrochemical measurements. It has also been proposed that EEU by SRB from solid electron donors is an important mechanism in mediating the interspecies electron transfer process from methane-oxidizing archaea to SRB in the AOM consortia (McGlynn et al. 2015; Wegener et al. 2015; Scheller et al. 2016) as well as the bacterial energy acquisition in energy-scarce subsurface environments (Deng et al. 2018). While the importance and ubiquity of EEU mechanism for the biogeological and biophysical processes and anaerobic iron corrosion have been suggested, the number of microbial species with experimental evidences is still limited.

The present chapter reviews the methodology and background physiochemistry for measuring and calculating the H2-evolution overpotential of an electrode, bioelectrochemical analyses on electrodes poised at potentials in the potential window for avoiding H2 evolution, and the temperature and electrode potential dependency of cell activity analyses for investigating whether H2 is involved in the EEU process. Finally, the limitations of current studies are discussed and critical questions that need to be clarified in future studies are addressed.

2 Experimental Background and Methods to Characterize the EEU Process

Since the oxidation of iron [Fe0 ⇌ Fe2+ + 2e−; E0 = −0.44 V versus standard hydrogen electrode (SHE)] can proceed with proton reduction (2H+ + 2e− ⇌ H2, \( {E}_{\mathrm{pH}\ 7}^0=-0.413\ \mathrm{V} \)), H2 spontaneously forms on the surface of iron under neutral pH condition (Fe0 + 2H+ ⇌ Fe2+ + H2, \( {\Delta G}_{\mathrm{pH}\ 7}^0=-5.2\ \mathrm{kJ}/\mathrm{mol} \)). Therefore, to confirm whether SRB conduct EEU from solids without using H2 as an electron mediator, artificial electrodes with large overpotentials for H2 evolution such as indium-tin doped oxide (ITO) and graphite electrodes have been used instead of iron for tracking EEU processes in three-electrode electrochemical reactors.

To determine the onset potential (Eon-set) of H2 evolution for an electrode material under a specific experimental condition, one can apply linear sweep voltammetry (LSV), in which the electrode current is measured as scanning the electrode potential in the negative direction. Because the standard potential of H2 evolution is E = −0.059 pH (V) at 298 K, the H2 evolution can be assigned to the current that exhibits a shift of Eon-set, according to the pH of the electrolyte. A previous study determined that the Eon-set for H2 evolution on an ITO electrode in artificial seawater medium at neutral pH was approximately −0.9 V (Deng et al. 2015). Based on the Eon-set, the potential window suitable for tracking EEU processes for a certain electrode material can be determined, in which the electrode serves as the sole electron donor providing sufficient energy for cell metabolism while inhibiting the production of H2.

Results of LSV measurement also enable the calculation of the H2 evolution current on the electrode at any potential by using the cathodic part of the Butler-Volmer equation as described in Eq. (4.1), provided the H2 evolution current is small enough and the rate-limiting step is not shifted to the H+ diffusion. The anodic part is omitted because oxidation reactions on the negatively poised electrode are negligible.

$$ {j}_{{\mathrm{H}}_2}=-{j}_0\exp \left[-\frac{2{a}_{\mathrm{c}}F\left(E-{E}_{\mathrm{eq}}\right)}{RT}\right] $$
(4.1)

where \( {j}_{{\mathrm{H}}_2} \) is the H2-evolution current density on the electrode, j0 is the exchange current, ac is cathodic charge transfer coefficient, F is Faraday constant, E is electrode potential, Eeq is the equilibrium potential of H2 evolution, R is the universal gas constant, and T is the temperature. ac is first calculated based on two current values (j1 and j2), measured at different electrode potentials, E1 and E2, respectively, as described in the following equation:

$$ {a}_{\mathrm{c}}=-\frac{RT}{2F\left({E}_1-{E}_2\right)}\ln \left(\frac{j_1}{j_2}\right) $$
(4.2)

j0 is then calculated by inserting j1 and E1 (or j2 and E2) into Eq. (4.1). With the calculated values of ac and j0, the \( {j}_{{\mathrm{H}}_2} \) at any electrode potential E can be determined based on Eq. (4.1), unless H+ diffusion limits the reaction rate. By this method, the \( {j}_{{\mathrm{H}}_2} \) on an ITO electrode in artificial seawater electrolyte with a neutral pH at −0.4 V was found to be ~−0.11 nA/cm2. Chronoamperometry measurement which detects currents on an electrode poised at a constant potential showed that the SRB cells capable of EEU produced −0.2 to 20 μA/cm2 currents (Deng et al. 2015, 2018; Beese-Vasbender et al. 2015; Deng and Okamoto 2018), which are 103 to 105-fold larger than the \( {j}_{{\mathrm{H}}_2} \), on electrodes poised at −0.4 V, strongly suggest in the EEU capabilities of SRB cells.

Furthermore, because Eq. (4.1) also describes the relationship between j and T, the theoretical value of H2-evolution current density at temperature T1, \( {j}_{{\mathrm{H}}_2,\mathrm{theoretical},{T}_1} \), can be approximately calculated by using ac and j0 values obtained at a known temperature (different from T1). If the calculated \( {j}_{{\mathrm{H}}_2,\mathrm{theoretical},{T}_1} \) value is consistent with the measured value \( {j}_{{\mathrm{H}}_2,{T}_1} \), it can be considered that the observed current is caused by abiotic reactions (e.g., H2 production). In contrast, if the \( {j}_{{\mathrm{H}}_2,\mathrm{theoretical},{T}_1} \) deviates largely from \( {j}_{{\mathrm{H}}_2,{T}_1} \), it strongly suggests that the observed current is directly associated with microbial metabolic activities, because the cell activity is largely affected by temperature and can be severely hampered at a suboptimal temperature only slightly different from the growth temperature. For example, when the reaction temperature decreases from 30 to 4 °C, in theory, the \( {j}_{{\mathrm{H}}_2,\mathrm{theoretical},4{}^{\circ}\mathrm{C}} \) would be 95% of the \( {j}_{{\mathrm{H}}_2,30{}^{\circ}\mathrm{C}} \); however, the measured current at 4 °C with SRB cells was less than 20% of that at 30 °C, indicating that the current was attributable to the hampered metabolic activity of SRB cells at suboptimal temperature (Pietzsch et al. 1999) rather than to the inorganic H2 formation.

Another electrochemical measurement method, differential pulse voltammetry (DPV), is used to examine whether H2 is involved in the EEU process by microbes. In DPV, a small potential pulse (pulse height ΔE = 10–100 mV, pulse width Pw = ~50 ms level) is repetitively applied to the working electrode (with an interval time Δt = 0.5–5 s), while the potential of the working electrode is slightly changed for several mV after each pulse toward the negative (reduction) direction or the positive (oxidative) direction. Currents at timings right before (before faradic reaction, I1) and near the end of the pulse (in the middle of faradic reaction; I2) are measured. By recording the difference of I1 and I2 as ΔI, the charging current can be largely eliminated. Therefore, DPV detects the redox potential and relative amount of active species on the electrode surface with a high sensitivity (as low as 10−8 M). The DPV analysis of D. ferrophilus IS5 cells on the ITO electrode surface revealed that its OMCs had a redox potential of ~−0.46 V and a half width of ~130 mV (Deng et al. 2018). Because the redox potential of OMCs is much more positive compared to the H2 evolution potential, the result indicates that OMCs solely mediate EEU in IS5 cells without the involvement with H2. Additionally, the exclusion of H2 in the EEU process of SRB was also achieved by observing the unchanged current by introducing H2-metabolizing SRB (e.g., Desulfobacterium vacuolatum and Desulfovibrio sp. strain HS3) on the electrode surface (Beese-Vasbender et al. 2015; Deng et al. 2018).

3 EEU Pathways Across the Cellular Outer Membrane of SRB

Mechanisms of EEU in SRB have been studied using electrochemical, microbiological, and bioinformatic methods. Outer-membrane cytochromes (OMCs), biosynthesized iron sulfide nanoparticles (FeS NPs), and soluble redox mediators are proposed to mediate EEU in cells and solid surfaces (Fig. 4.1). These mechanisms are similar to those identified in iron-reducing bacteria (IRB) that export metabolically generated electrons to the extracellular solid electron acceptors.

Fig. 4.1
figure 1

Schematic illustration of identified mechanisms of extracellular electron uptake from solid electron donors coupled with sulfate reduction in sulfate-reducing bacteria (SRB). Electrons (e−) were transferred across the outer membrane (OM) via (a) outer-membrane cytochromes (OMCs) and nanowire structure that is an extension of the OM, (b) electrically conductive iron sulfide nanoparticles (FeS NPs) biosynthesized on cell surface, and (c) diffusive redox mediators (Mox/Mred). Periplasmic cytochromes (c in red circles) and/or periplasmic FeS NPs may facilitate the extracted electrons to be further transported to the cellular electron transport chain. IM inner membrane, TMC transmembrane complex

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3.1 Outer-Membrane Cytochromes (OMCs) and Nanowires

OMCs are cytochrome proteins located on the cell outer membrane (OM) and were first identified in Shewanella oneidensis MR-1 (Myers and Myers 1992), an iron-reducing bacterium which respires oxide minerals (e.g., MnO2 and Fe2O3) as electron acceptors when other soluble electron acceptors (e.g., oxygen) become scarce (Myers and Nealson 1988). The OMCs of S. oneidensis MR-1 consist of extracellularly localized decaheme cytochromes OmcA and MtrC, periplasmic decaheme MtrA, and an OM porin MtrB. The extracellular OmcA and MtrC interact with the periplasmic MtrA through MtrB (Myers and Myers 1992). These heme-porin complexes enable extracellular electron transport (EET) in Shewanella cells (Bretschger et al. 2007; Bucking et al. 2010; Jensen et al. 2010). Protein crystal structures (Clarke et al. 2011; Edwards et al. 2012, 2014, 2017), binding cofactors (Okamoto et al. 2013, 2014a; Hong and Pachter 2016; Tokunou et al. 2016), and electron transport mechanisms (Breuer et al. 2014) of OMCs have been studied extensively using Shewanella OMCs. Meanwhile, similar heme-porin complexes have been just recently identified in D. ferrophilus IS5 by gene and protein identification methods (Deng et al. 2018).

The IS5 genome harbors 26 genes encoding cytochromes with four or more heme-binding motifs [CXnCH (n=2–5)], 7 of which are predicted to be localized in the OM region (Table 4.1). Furthermore, some of the protein products encoded by the potential OMCs gene clusters were detected on the isolated OM. The presence of OMCs in IS5 cells was confirmed by spectroscopic absorption measurement of the isolated OM fraction and was visualized by transmission electron microscopic (TEM) of cell cross-sections stained by a cytochrome-reactive diaminobenzidine (DAB)-H2O2 method (McGlynn et al. 2015; Graham and Karnovsky 1966). Additionally, it was found that IS5 not only expressed OMCs on the cell surface but also the segmented nanowires with diameters ranging from 30 to 50 nm, which were most likely the extensions of OM and periplasmic space in a form of aligned OM vesicles (Fig. 4.1a) (Deng et al. 2018). Similar nanowire structures were also observed in Shewanella cells (Deng et al. 2018; Pirbadian et al. 2014; Subramanian et al. 2018). The capability of OMCs in mediating EEU was confirmed by IS5 cells upon controlling the expression of OMCs. The redox potential of IS5 OMCs was approximately −0.46 V (Deng et al. 2018), which is slightly more negative compared to that of the OMCs in Shewanellacells (−0.39 V) under EEU conditions. However, much needs to be explored about the IS5 OMCs, for instance, their protein crystal structure, potential binding cofactors, and whether they participate in mediation of the EET process.

Table 4.1 Potential gene clusters encoding OMCs complex in the genome of D. ferrophilus IS5
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The identified genes encoding OMCs in IS5 are widely found in the genomes of bacteria belonging to the phyla Aquificae, Thermodesulfobacteria, and Proteobacteria, which respire various sulfur compounds (e.g., thiosulfate, polysulfide, and sulfite) and primarily inhabit sedimentary environments (Deng et al. 2018). In contrast, the genes encoding OMCs in IRB strains, Shewanella and Geobacter, were found in the genomes of a few highly similar IRB strains living in oxic-anoxic water column and surface sediments. Because abundant minerals (e.g., iron-copper-sulfides) with adequately negative redox potentials can serve as potential electron donors for microbes, EEU mediated by OMCs may be potential energy acquisition strategy for a wide range of bacteria in these environments to surpass the competition for organic and gaseous electron donors (Deng and Okamoto 2017).

3.2 Conductive FeS Nanoparticle (NPs)-Mediated EEU

Electrically conductive NPs, such as palladium precipitated on the cell surface, were reported to function similarly to OMCs by mediating the transportation of respiratory electrons to solid electron acceptors in some Gram-negative bacteria (Wu et al. 2011). The model of biosynthesized FeS NPs-mediated EEU in SRB was recently verified by a study using Desulfovibrio vulgaris Hildenborough, because many SRB like D. vulgaris lack a known EEU pathway, including OMCs and secretion of redox mediators (Heidelberg et al. 2004), but produce FeS.

D. vulgaris cells, cultivated in its growth medium supplemented with 2 mM Fe2+, biosynthesized conductive FeS NPs (e.g., mackinawite) extracellularly, intracellularly, and on the cell surface. Electrochemical studies revealed that cells with biosynthesized FeS NPs became capable of direct EEU on the surface of −0.4 V-poised ITO electrodes, serving as the sole electron donor (Deng et al. 2020). By controlling the cell activity on the electrodes (e.g., by adding antibiotic, removing/re-supplementing sulfate or by maintaining a suboptimal growth temperature), it was revealed that the EEU of D. vulgaris was associated with its active cell metabolism. Notably, the electron uptake rate of FeS NPs-mediated EEU was approximately sevenfold faster in D. vulgaris than that mediated by OMCs in D. ferrophilus IS5, indicating that biosynthesized FeS NPs function as an efficient EEU conduit in SRB.

This finding significantly expands the ubiquity of EEU, which was previously considered to be a specific microbial process limited to those bacteria that possess OMCs, to a wider range of bacteria capable of self-synthesis of FeS NPs or have FeS NPs precipitated on the cell surface. FeS NPs are ubiquitously identified in nature, such as marine sediments, because they are predominantly produced by the ubiquitous SRB (Fortin et al. 1994; Donald and Southam 1999; Watson et al. 2000; Williams et al. 2005; Picard et al. 2016) and also in geological processes, e.g., the eruption of hydrothermal fluids and earthquakes (Findlay et al. 2019). These FeS NPs can be transported via water/ocean flow and precipitated on the cell surface of other bacteria. Based on the Pourbaix diagram, mackinawite and pyrrhotite that have been identified as EEU pathways in SRB are stable at conditions with pH > 4, potentially more negative than −0.2 V, and a wide temperature range. The stable pH and potential ranges for mackinawite and pyrrhotite slightly shift to the acidic and reduced direction in accordance with elevated temperatures (Fig. 4.2) (Ning et al. 2015). This indicates that the EEU mechanism mediated by mackinawite and pyrrhotite can be exploited by a wide range of psychrophilic, mesophilic, and thermophilic bacteria for energy acquisition in slightly acidic, neutral, or alkaline reductive environments. In comparison, the semiconductive pyrite and greigite stay stable in lower pH even at strongly oxidative potentials. To further elucidate the distribution of FeS NPs-mediated EEU process, it would be important to explore whether the semiconductive greigite and pyrite can mediate EEU in SRB because they are also found in acidic environments with pH < 4 (Sen and Johnson 1999; Meier et al. 2012; Sanchez-Andrea et al. 2013).

Fig. 4.2
figure 2

Pourbaix diagrams for H2S–H2O–Fe system showing step changes in temperature up to 250 °C (T = 25–250 °C, [H2S]aq = 9.4 × 10−3 M, [Fe2+] = 10 ppm, [Fe3+] = 10−6 M): (a) mackinawite/greigite and (b) mackinawite/greigite/pyrrhotite/pyrite. (Adapted from J. Ning et al., Materials Science, 2015)

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Given that SRB play a primary role in carbon mineralization in marine sedimentary environments (Jørgensen 1982; Berner and Raiswell 1983), the identified FeS NPs-mediated EEU brings novel insights into the biogeochemical cycles of carbon, iron, and sulfur. In addition, FeS NPs-mediated EEU may also provide a pathway for iron oxidation by SRB. A previous corrosion study has observed persistent corrosion of carbon steel by D. vulgaris after organic electrons depletion for up to 55 days (Chen et al. 2015). Therefore, future identification of the mechanism underlying the synthesis of long, electrically conductive pathways via FeS NPs would contribute to the development of an effective strategy for inhibiting microbial iron corrosion.

3.3 Soluble Redox Electron Shuttles to Mediate EEU

Soluble redox mediators have been demonstrated to mediate the electron transportation process from cell interior to cell exterior in various microbes. So far, endogenous redox mediators, e.g., riboflavin (RF; E°′ = −260 mV), flavin mononucleotide (FMN; E°′ = −205 mV), and phenazine derivatives (e.g., phenazine-1-carboxylic acid, phenazine-1-carboxamide, and procyanin, with E°′ = −275, −150, and −32 mV, respectively), were identified to be secreted by Geobacter sulfurreducens, S. oneidensis MR-1, and Pseudomonas aeruginosa during cell growth (von Canstein et al. 2008; Marsili et al. 2008; Okamoto et al. 2014b). Meanwhile, exogenous redox mediators derived from the degradation of microbial and plant matter, such as humic acids (HA, E°′ = −314 to 430 mV) (Lovley et al. 1996; Jiang and Kappler 2008) artificial mediators [e.g., anthraquinone-2,6-disulfonate (2,6-AQDS; E°′ = −186 mV), and potassium ferricyanide (E°′ = 436 mV at pH 7)], were also identified (Nevin and Lovley 2000). The redox potentials of these membrane-permeable redox mediators are compatible to microbial cell metabolic processes, and some of these mediators, such as RF and FMN, can function as specific binding cofactors for OMCs (Okamoto et al. 2013, 2014b). Therefore, soluble mediators enable or accelerate electron transfer processes between cells and extracellular solids.

Although information about the capability of biosynthesizing and utilizing redox mediators remains very limited in SRB, it was reported that Desulfotomaculum reducens MI-1, a Gram-positive SRB strain, secreted flavins during cell growth and used the flavins to facilitate the reduction of extracellular Fe3+ compounds (e.g., solid-phase hydrous ferric oxide). This suggested that this SRB strain may conduct EET mediated by flavins (Dalla Vecchia et al. 2014). Moreover, it was reported that the addition of redox mediators, RF and flavin adenine dinucleotide (FAD, E°′ = −340 mV), accelerated the corrosion of carbon steel and stainless steel by D. vulgaris, thereby suggesting that redox mediators may accelerate the EEU from metal iron in this SRB strain (Fig. 4.1c) (Li et al. 2015; Zhang et al. 2015). Further examination of the roles and mechanisms of redox mediators in the OMCs by SRB would contribute to our understanding of EEU mechanisms in SRB.

4 Microbial Physiology Coupled with EEU in SRB

4.1 Single Cell Activity Measurement for EEU Process

The surface-based cell activity analysis method, nanoscale secondary ion mass spectrometry (NanoSIMS), can be used to measure cell isotopic ratios (e.g., 13C/Ctotal and 15N/Ntotal) at nanometer scale (Nana et al. 2018). It has been observed that when incubated on electrodes with isotopic C and nitrogen (N) sources (e.g., [1-13C]acetate and 15NH4Cl), the cells which obtain more energy from the electrode will assimilate more C and N, resulting in higher 13C/Ctotal and 15N/Ntotal. NanoSIMS analysis has been commonly applied to analyze the distribution of cell activity on electrodes (Deng and Okamoto 2018; Saito et al. 2017).

NanoSIMS measurement of the cell activity of D. ferrophilus IS5 cells on electrodes at varied potentials (−0.2, −0.3, −0.4, and −0.5 V) demonstrated that cells, which conducted EEU at −0.4 and −0.5 V, obtained energy for C and N assimilation (Deng and Okamoto 2018). In contrast, cells at −0.2 and −0.3 V, which did not conduct EEU, had only 13C/Ctotal and 15N/Ntotal of natural abundances. Additionally, NanoSIMS measurement of cell activity at varied potentials also revealed the Eon-set for EEU was consistent with the LSV measurement result. Therefore, NanoSIMS measurement of the potential dependency of cell activity could be applied to detect slow microbial EEU process and determine the Eon-set of EEU process where the microbial signals are comparable to or smaller than the background signals.

4.2 Cell Growth of SRB During EEU

Electrochemical and NanoSIMS analyses on various SRB species with different EEU pathways, including D. ferrophilus IS5, Desulfobacterium corrodens IS4, and D. vulgaris Hildenborough, showed that their Eon-set was similar and was approximately −0.3 V (Beese-Vasbender et al. 2015; Deng and Okamoto 2018). Therefore, EEU thermodynamically enables the generation of NAD(P)H (E°′ = −0.32 V) by reduction reactions and fuels cell growth (Fig. 4.3). However, in electrochemical studies using single-chamber three-electrode reactors equipped with −0.4 V-poised electrodes as the sole electron donor for incubating D. ferrophilus IS5 cells, cell activity was too slow to allow replication in a time span up to 66 days (Deng et al. Under review). The lack of cell growth on electrode surface in single-chamber reactors was also reported for the IRB strain S. oneidensis MR-1, which couples EEU with the reduction of oxygen or fumarate (Rowe et al. 2018). In contrast, some iron-oxidizing bacteria (IOB), e.g., Acidithiobacillus ferrooxidans, could grow in single-chamber reactors by coupling EEU with oxygen (O2) reduction (Summers et al. 2013; Ishii et al. 2015). Therefore, although factors that differentiate SRB and IRB cells from IOB for growing on electrodes are unclear, EEU was proposed to supply limited energy for cell maintenance but not cell growth.

Fig. 4.3
figure 3

Energy diagram of the extracellular electron uptake from extracellular solids via OMCs or FeS NPs in SRB. The onset potential for EEU is more negative than −0.3 V, which allows sufficient energy for generating nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and/or reduced menaquinone (MQH2) which drives the reduction of oxidized sulfur (S) compounds (e.g., sulfate, SO42−; sulfite, SO32−) and intermediates, such as adenosine phosphosulfate (APS). If the solid has a redox potential negative enough for the proton (H+) reduction, hydrogen (H2) is formed on the electrode surface serving as an alternative electron donor for SRB. cyt periplasmic cytochrome, OM outer membrane, IM inner membrane, TMX transmembrane complex. Unit, V versus standard hydrogen electrode

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A recent study revealed factors specific to the electrochemical reactors that restricted cell growth on the electrodes by focusing on the generation of oxidative stress in the electrochemical reactors (Deng et al. Under review). By incubating IS5 cells in H-type reactors in which the counter electrode (CE) at which oxidation reactions occur associated with EEU process is separated from cells using a proton-exchange membrane, the first evidence of the growth of D. ferrophilus IS5 cells on −0.4 V-poised electrodes was obtained by NanoSIMS analysis. Moreover, in addition to the exogenous oxidative stress, IS5 cell activity was also likely to be restricted by the endogenous oxidative stress produced by the reduction of trace amount of O2 by the reduced cellular electron transport chain. Therefore, these results demonstrated that the EEU process is intrinsically coupled with the production of exogenous and endogenous oxidative stress and clarified that EEU is an important mechanism supporting cell growth under energy-limited conditions, rather than a mere support for cell maintenance.

However, it is possible that factors specific to the electrochemical systems that inhibit cell growth on the electrode surface. For example, the oxidizing species produced at the counter electrode or an unsuitable working electrode potential may supress cell acitity on the working electrode. These possibilities are currently under examination by Deng et al.

4.3 Gene Expression of D. ferrophilus IS5 During EEU

Transcriptome analysis of IS5 cells which generated currents on electrodes demonstrated upregulated expressions of genes encoding central energy metabolism, including ATP synthesis, sulfate-reducing pathway, and cell division and cell wall synthesis, compared to cells that generated lower currents or cells incubated without an electrode (Deng and Okamoto 2018). The analysis further revealed the significant upregulation of genes encoding OMCs but not those encoding periplasmic hydrogenases required for H2 utilization in cells that produced higher currents. This in turn strongly suggested that OMCs are the pathway mediating the EEU process without the requirement of oxidizing H2 as an electron mediator. Moreover, the upregulated levels of antioxidative genes were also observed in cells that produced higher current on an electrode poised at −0.5 V, compared to cells that produced lower currents at −0.4 V. This implied that cells that obtained more energy during EEU had to deal with more antioxidative stress, most likely originating endogenously from the reduction of trace O2 in the system via the electron transport chain (Deng et al. Under review).

5 Future Perspective of EEU by SRB

5.1 Exploration of New Genes for EEU Mechanism in SRB

OMCs, FeS NPs, and redox mediators have been identified as different pathways mediating EEU in SRB. However, there is a possibility of the presence of new pathways. Therefore, the EEU potential should be explored in SRB living in various environments. For example, a different EET pathway was recently identified in the Gram-positive bacterium Listeria monocytogenes, whereby a novel membrane-attached cytoplasmic NADH dehydrogenase mediated the transportation of respiratory electrons to membrane-localized quinone pool that was further transported to extracellular flavoprotein and/or flavin shuttles to reach extracellular electron acceptors (Light et al. 2018).

5.2 Proof of EEU Process in AOM and Iron Corrosion Processes

Although the capability of EEU from electrode has been identified in SRB (Dinh et al. 2004), electron extraction by cells from iron still remains unverified, largely due to the fact that H2 spontaneously generates on the iron surface and serves as a potential electron donor. To exclusively identify EEU process in iron corrosion by SRB, approaches such as comparison of corrosion rates in wild-type, OMC-deficient, and hydrogenase-deficient strains would be useful. In addition, comparative gene expression analyses of OMCs and hydrogenases of SRB in the presence of iron and H2 as the sole electron donor could provide insights into the corrosion mechanism.

EEU in SRB was also reported to be important for the activity of syntrophic consortia performing AOM. An interspecies electron transport model has been proposed for AOM consortia. This model states that SRB likely receive electrons directly from the methanotrophic archaea via OMCs and/or conductive pili structures (McGlynn et al. 2015; Wegener et al. 2015; Scheller et al. 2016) rather than using soluble intermediating compounds such as a methane-derived organic carbon compound (Moran et al. 2008). To directly prove this model, electrochemical analysis using archaeal and bacterial isolates that are put in separate anodic and cathodic chambers, respectively, would be required.

5.3 Electrical Incubation to Isolate Uncultivated SRB

It is estimated that more than 99.9% of subsurface microbes are uncultivated by conventional methods using soluble electron donors (Short 1997). Because minerals with sufficiently negative potentials, such as iron-copper-sulfides, and microbes potentially possessing an EEU pathway, are widespread in anoxic subsurface environments, electrical incubation methods using electrochemical reactors may enable the enrichment and isolation of novel uncultivated EEU-capable strains from these environments. Moreover, since EEU is not limited to cell-mineral interactions but also intercellular/interspecies interactions, EEU-capable microbes may also be found in non-mineral environments, such as animal guts. Gut microbial strains, such as Desulfovibrio piger and Faecalibacterium prausnitzii, have been reported to conduct EET (Khan et al. 2012; de Campos Rodrigues and Rosenbaum Miriam 2014). Testing the capability and significance of EEU in these microbes related with host health may contribute to the development of new technologies for treating EEU-capable pathogens. Because previous studies suggested that different EEU-capable microbes may prefer different electrode potentials and electrode physical properties, using different electrode materials with different range of potential windows may enable the isolation of different EEU-capable microbes.

5.4 Proof of Ubiquity of EEU in Environments

Given that EEU-mediating pathways, including OMCs, FeS NPs, and redox mediators, are potentially widespread in sediment bacteria and electric current flow on the mineral surface of sediment hydrothermal vents has been reported (Nakamura et al. 2010), EEU likely plays important role in the energy acquisition by a wide range of bacteria in sediment environments. However, such a model describing EEU-supported subsurface biosphere would require identification of EEU-capable microbes in different sites and the comparison of cell metabolism in situ and ex situ. If EEU capability and the pathways can be identified in abundant microbial lineages, rigorous analysis of gene heritage in the phylogenetic tree would be possible and reveal the origin of EEU biomarker. If the biomarker is conserved vertically in numerous strains living in closely related habitat, it would signify the importance of EEU mediated by the biomarker in the environment. Furthermore, because hydrothermal vent systems like those on Earth are also present in extraterrestrial planets, such as on the icy surface of Europa (Gaidos et al. 1999; McCollom 1999; Chyba 2000; Zolotov and Shock 2003) and on ancient Mars (Michalski et al. 2017), identification of the capability of EEU via non-enzymatic pathways in SRB as well as other microbes with an ancient origin from minerals of hydrothermal vents would provide insights on the origin of life on Earth as well as possible life on extraterrestrial planets.

6 Conclusion

In this chapter, we introduced electrochemical methods and studies for identifying direct EEU process in SRB, the most ubiquitous and ancient bacteria on Earth (Shen and Buick 2004). The evolution of H2 on the surface of solid electron donors has been a major obstacle in the research progress of the EEU mechanisms. By using stable electrodes with large overpotential for H2 evolution and controlling the physicochemical parameters (e.g., temperature) during the EEU process, the possibility of H2 involvement in EEU is finally excluded. So far, the identified EEU pathways in SRB include OMCs, FeS NPs, and possibly electron shuttles and can be ubiquitously found in subsurface environments. EEU might be an ancient energy conservation mechanism for supporting the subsurface ecosystems. These findings contribute to our understanding of SRB physiology and have broad implications in other critical processes associated with SRB, such as anaerobic iron corrosion, AOM, and biogeochemical cycles of iron, sulfur, and carbon. In the future, we anticipate that new EEU pathways in SRB and the related electrosynthetic and biomedical applications will be established and an increasing number of microbes capable of EEU will be identified.