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Divya Naradasu, Waheed Miran, Mitsuo Sakamoto, [Akihiro Okamoto](https://orcid.org/0000-0002-8102-4316)

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[Isolation and Characterization of Human Gut Bacteria Capable of Extracellular Electron Transport by Electrochemical Techniques](https://mdr.nims.go.jp/datasets/87d3abd3-88df-4627-9d16-0b3604bc6464)

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Isolation and Characterization of Human Gut Bacteria Capable of Extracellular Electron Transport by Electrochemical Techniquesfmicb-09-03267 January 12, 2019 Time: 17:5 # 1ORIGINAL RESEARCHpublished: 15 January 2019doi: 10.3389/fmicb.2018.03267Edited by:Fanghua Liu,Yantai Institute of Coastal ZoneResearch (CAS), ChinaReviewed by:Suleyman Yildirim,Istanbul Medipol University, TurkeyWolfgang Buckel,University of Marburg, Germany*Correspondence:Akihiro OkamotoOKAMOTO.Akihiro@nims.go.jp†These authors have contributedequally to this workSpecialty section:This article was submitted toMicrobiotechnology, Ecotoxicologyand Bioremediation,a section of the journalFrontiers in MicrobiologyReceived: 18 October 2018Accepted: 17 December 2018Published: 15 January 2019Citation:Naradasu D, Miran W,Sakamoto M and Okamoto A (2019)Isolation and Characterizationof Human Gut Bacteria Capableof Extracellular Electron Transport byElectrochemical Techniques.Front. Microbiol. 9:3267.doi: 10.3389/fmicb.2018.03267Isolation and Characterization ofHuman Gut Bacteria Capable ofExtracellular Electron Transport byElectrochemical TechniquesDivya Naradasu1,2†, Waheed Miran1†, Mitsuo Sakamoto3,4 and Akihiro Okamoto1,4,5*1 International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science, Tsukuba, Japan,2 Department of Advanced Interdisciplinary Studies, Research Center for Advanced Science and Technology, GraduateSchool of Engineering, The University of Tokyo, Tokyo, Japan, 3 Microbe Division/Japan Collection of Microorganisms, RIKENBioResource Research Center, Tsukuba, Japan, 4 PRIME, Japan Agency for Medical Research and Development (AMED),Tsukuba, Japan, 5 Center for Sensor and Actuator Material, National Institute for Materials Science (NIMS), Tsukuba, JapanMicroorganisms are known to exhibit extracellular electron transfer (EET) in a wide varietyof habitats. However, as for the human microbiome which significantly impacts ourhealth, 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 usingan electrochemical enrichment method and examined whether the isolates couple EETwith anaerobic respiration or fermentation. Upon the use of energy-rich or minimummedia (with acetate or lactate) for electrochemical enrichment with the human gutsample 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 subsequentincubation using the δ-MnO2-agar plate with lactate or acetate led to the isolationof two EET-capable microbial strains, Gut-S1 and Gut-S2, having 99% of 16S rRNAgene sequence identity with Enterococcus avium (E. avium) and Klebsiella pneumoniae(K. pneumoniae), respectively. While the enrichment involved anaerobic respiration withacetate and lactate, further electrochemistry with E. avium and K. pneumoniae revealedthat the glucose fermentation was also coupled with EET. These results indicate thatEET couples not only with anaerobic respiration as found in environmental bacteria, butalso with fermentation in the human gut.Keywords: gut microbes, electromicrobiology, fermentative bacteria, electrochemical enrichment, extracellularelectron transferINTRODUCTIONExtracellular electron transfer (EET) mechanisms have evolved in microorganisms as an anaerobicmetabolic strategy that can be coupled to the reduction of extracellular solid materials (Myersand Nealson, 1988; Shi et al., 2016). EET mechanisms are proposed to be mediated by cellsurface transmembrane cytochromes, exogenous or endogenous soluble redox-active compounds,Frontiers in Microbiology | www.frontiersin.org 1 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/journals/microbiology#editorial-boardhttps://www.frontiersin.org/journals/microbiology#editorial-boardhttps://doi.org/10.3389/fmicb.2018.03267http://creativecommons.org/licenses/by/4.0/https://doi.org/10.3389/fmicb.2018.03267http://crossmark.crossref.org/dialog/?doi=10.3389/fmicb.2018.03267&domain=pdf&date_stamp=2019-01-15https://www.frontiersin.org/articles/10.3389/fmicb.2018.03267/fullhttp://loop.frontiersin.org/people/630720/overviewhttp://loop.frontiersin.org/people/329750/overviewhttp://loop.frontiersin.org/people/601679/overviewhttp://loop.frontiersin.org/people/200736/overviewhttps://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 2Naradasu et al. Extracellular Electron Transport in Gutor electrically conductive nanowires (Shi et al., 2016). Thesignificance of EET during anaerobic respiration has provideda reasonable explanation for not only microbial energyconservation and physiology, but also for their interactionwith the environment. While EET has been well characterizedin terms of its mechanistic basis, mainly in two modelbacterial strains, Geobacter sulfurreducens PCA and Shewanellaoneidensis MR-1 (Lovley and Phillips, 1988; Myers and Nealson,1988), electrochemical enrichment studies, combined with16S rRNA-based assessments in a variety of environments,advocate that more physiologically and phylogenetically diversemicroorganisms may be capable of using exterior surfacesas electron acceptors. However, it is vital to mention thatthe survival or enrichment of a microbe on an electrodeis not the ultimate evidence for their EET ability, andhence, further electrochemical characterization is essential toprobe the EET processes after the isolation of the microbes.A number of enrichments have resulted in the isolationof pure cultures that can accomplish EET with electrodes,indicating that EET may be advantageous in a wide varietyof habitats (Zuo et al., 2008; Fedorovich et al., 2009; Logan,2009; Rowe et al., 2017). Moreover, bacteria that utilizefermentation as their main metabolic pathway have alsobeen isolated and characterized for their EET capabilities(Khan et al., 2012; Zhou et al., 2016; Kumar P. et al.,2018).In anaerobic environments with substantially reductivecondition such as the human gut (Edwards et al., 1985),fermentation is the primary mechanism of microbial metabolism,in which the redox cycling of biological electron carriers, suchas nicotinamide adenine dinucleotide (NADH), drives theintracellular oxidation and reduction of organic substrates.As fermentation does not require extracellular electronacceptors for the termination of metabolism, the energygain under such conditions is potentially lower than that ofrespiratory metabolism; therefore, the possibility for EET toincrease the rate of NAD+ regeneration and fermentativemetabolism may be important for these microbes to increasetheir net energy gain and compete with other respiratorybacteria (Okamoto et al., 2017). In fact, a few studies haveshown that fermentative gut microbes are capable of EETusing soluble electron carrier molecules (Khan et al., 2012;Keogh et al., 2018; Light et al., 2018; Pankratova et al., 2018).However, by simply studying isolated bacterial cultures,it is impossible to examine which bacteria primarily relyon EET-coupled metabolism in the human gut and tostudy the ecophysiological importance of EET coupledwith fermentation, compared to anaerobic respiration,which is also abundant in the gut environment (Rey et al.,2013). Here, we examined the growth competition betweenfermentative and respiratory bacteria on an electrode surfacethat enriches for EET-capable bacteria. Specifically, weperformed electrochemical enrichment, which was initiatedusing a diluted gut microbial community, by employingtwo different medium conditions that biased for eitherfermentation or anaerobic respiration. The isolated bacterialstrains were characterized by electrochemical assays for theirmetabolism associated with current production and the EETmechanism.MATERIALS AND METHODSElectrochemical Cell Operation andMedium CompositionElectrochemical measurements were performed in single-chamber and three-electrode reactors. Tin-doped In2O3 (ITO)grown on a glass substrate by spray pyrolysis depositionwas used as the working electrode (WE) having a surfacearea of 3.1 cm2, and thickness 1.1 mm. The WEs wereplaced at the bottom of the reactor with sealing gaskets toavoid any leakage. A platinum wire (approximate diameter of0.1 mm) and Ag/AgCl (sat. KCl) were used as counter andreference electrodes, respectively. Electrochemical experimentswere conducted in a COY anaerobic chamber filled with 100%N2. Electrochemical analysis techniques such as single-potentialamperometry (SA) and differential pulse voltammetry (DPV)were measured with an automatic polarization system (VMP3,Bio-Logic Science Instruments). DPV was measured under thefollowing conditions: pulse increment, 5.0 mV; pulse amplitude,50 mV; pulse width, 300 ms; and pulse period, 5.0 s. Theelectrochemical cell was maintained at 37◦C throughout theexperiment and the WE was poised at +0.2 V [vs. Ag/AgCl (sat.KCl)] reference electrode for SA.Gifu Anaerobic Medium (GAM Broth), which is knownfor providing reducing conditions and adequate anaerobiosis,was used as an energy rich medium for the enrichment ofgut microbes. Defined medium 1 (DM1), used as a minimummedium for EET strains enrichment and initial electrochemicalcharacterization experiments had the following composition(L−1): NH4Cl: 1 g; MgCl2 6H2O: 0.8 g; CaCl2.2H2O: 0.1 g;KH2PO4: 0.5 g; yeast extract: 1 g; NaHCO3: 1 g; trace mineral mix:10 mL [with the following composition (L−1): Nitrilotriaceticacid: 1.5 g, MgSO4 7H2O: 3 g, MnSO4 H2O: 0.5 g, NaCl:1 g, FeSO4 7H2O: 0.1 g, CoSO4 7H2O: 0.18 g, CaCl2 2H2O:0.1 g, ZnSO4 7H2O: 0.18 g, CuSO4 5H2O: 0.01 g, KAl (SO4)212H3BO3: 0.02 g, Na2MoO4 2H2O: 0.01 g, NiCl2 6H2O: 0.03 g,Na2SeO3 5H2O: 0.3 mg, Na2WO4 2H2O: 0.4 mg; first dissolvedthe nitrilotriacetic acid and adjusted the pH to 6.5 with KOH,then added the minerals. Finally, pH was adjusted to 7.0 withKOH] and trace vitamin mix: 10 mL [with the followingcomposition (L−1): Biotin: 2 mg, Folic acid: 2 mg, Pyridoxine-HCl: 10 mg, Thiamine-HCl: 5 mg, Riboflavin: 5 mg, Nicotinicacid: 5 mg, D-Ca-pantothenate: 5 mg, Vitamin B12: 0.1 mg,p-Aminobenzoic acid: 5 mg, Lipoic acid: 5 mg]. Acetate (30 mM)or lactate (30 mM) was used as electron donors. DM1 exclusiveof NaHCO3, trace mineral, and trace vitamin was autoclavedfirst, and pH was adjusted to 7.2 after adding the remainingcomponents. The final medium was filtered using 0.22-µm-pore-size filters and deaerated by purging it with 100% N2for 15 min prior to use for experiments. A fine powder ofδ-MnO2 was synthesized as previously reported (Burdige andNealson, 1985). Briefly, Mn2+ was oxidized by permanganateunder basic conditions, and the product was washed andFrontiers in Microbiology | www.frontiersin.org 2 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 3Naradasu et al. Extracellular Electron Transport in Gutresuspended in distilled water. The solid was freeze-dried forstorage, and resuspended in sterilized solution before use in agarplate.Defined minimum medium 2 (DM2), used as an electrolyte forEET experiments had the following composition (L−1): NH4Cl:1 g; MgCl2.6H2O: 0.2 g; CaCl2.2H2O: 0.08 g; yeast extract: 0.5 g;NaHCO3: 2.5 g; NaCl: 10 g; and HEPES buffer: 7.2 g. Acetate(10 mM) or lactate (10 mM) was used as electron donors forEET experiments with DM2. This medium was also autoclavedand deaerated prior to the electrochemical experiments. A totalof 5 mL of medium (including strain culture) was used inthe electrochemical reactor for all experiments. During theelectrochemical measurements, the reactor was operated at 37◦Cthroughout the experiment without any agitation.Electrochemical Enrichment of HumanGut SampleThis study was approved by the RIKEN Ethics Committee. A fecalsample was obtained from a healthy volunteer (45–50 years old).A written informed consent agreement signed by the volunteerwas obtained before the experiment. A 0.5 g of fecal samplewas suspended in 4.5 mL of pre-reduced phosphate buffer saline(PBS) and then serially diluted in 10-fold steps. The sample wasdiluted to a concentration of 10−7 (v/v) and 0.1 mL of this dilutedmicrobial consortium was then added to the electrochemicalreactor with 4.9 mL of GAM medium or DM1 in which theelectrolyte temperature was maintained at 37◦C, and the WE waspoised at+0.2 V throughout the enrichment.At the end of electrochemical enrichment with GAM, orDM1 having acetate or lactate, the WE surface was washed twicewith PBS, and subsequently the electrode-attached biomass fromeach WE was streaked on separate agar plates which were madewith GAM or DM1 having acetate and lactate, and contained50–60 mM δ-MnO2. The plates were incubated at 37◦C underan H2/CO2/N2 (1:1:8 v/v) gas mixture. Given the microbialreduction of δ-MnO2 generate transparent spot in the darkbrown agar plate, this visual clue was used to identify the colonyof EET-capable bacteria. The schematics of isolation procedureare shown in Supplementary Figure S1. After 2–4 days ofincubation, the colonies that formed the transparent spots weresub-cultured on an Eggerth Gagnon agar (Merck) supplementedwith 5% horse blood at 37◦C under the same conditions asmentioned above, and single colonies were picked up for furtheranalysis by sequencing. The strains isolated from agar plateshaving acetate and lactate were named as Gut-S1 and Gut-S2,respectively.Cell Cultures HarvestingThe isolated strains Gut-S1 and Gut-S2 were pre-cultivated in40 mL of Lysogeny broth (LB) in butyl-rubber-stoppered vials at37◦C with an anoxic headspace of CO2/N2 (20:80 v/v). Microbialcultures were harvested in the late exponential phase when theOD600 was about 1.0. The cultures were centrifuged at 7800 rpmat 37◦C for 10 min in a 50 mL falcon tubes. The resultant cellpellet was washed twice with defined media (DM1 or DM2)by resuspending and centrifugation. The resuspended cells indefined media were then added into the reactors to a final OD600of 0.1.Metabolites DeterminationSamples for metabolites were collected from the electrochemicalcells at every 8-h time interval for 24-h during the electrochemicaloperations. The collected samples were filtered using 0.22-µm-pore-size filters to remove the cells and stored at −20◦Cuntil further analysis. For metabolite analysis, samples werediluted 100 times with distilled water. Metabolic productswere quantified by using an ion chromatography (IC) system(HIC-20Asuper, Shimadzu Corporation, Japan). Fifty microlitersamples were injected, and the anions were analyzed in non-suppressor mode. Shim-pack IC-A3 and Shim-pack IC-GA3(Shimadzu Corporation, Japan) were used as the analyticalcolumn and guard column, respectively. The mobile phasecontained 8 mM p-hydroxybenzoic acid, 3.2 mM Bis–Tris, and50 mM boric acid, and the flow rate was 1.2 mL/min. The columntemperature was maintained at 40◦C, and the detector (CDD-10ASP) parameters were set according to the manufacturer’sguidelines. Peak area analysis was conducted by Shimadzuanalytical workstation software LabSolutions provided by themanufacturer. The standard curves showed sufficiently highlinearity (R2 = 0.999). Glucose concentrations were measured byusing a glucose assay kit (GAGO-20, Sigma–Aldrich) accordingto the manufacturer’s protocol.DNA Isolation and Phylogenetic TreeThe discrete colonies that were sub-cultured from the transparentspots of δ-MnO2 were analyzed by 16S rRNA gene sequencing.The forward and reverse primers for PCR were 27 F (5′-AGAGTT TGA TCC TGG CTC AG-3′) and 1492 R (5′-GGT TACCTT GTT ACG ACT T-3′). The 16S rRNA gene sequences werecompared with the sequences of closely related strains by usingthe BLAST program in the GenBank database. These sequencedata were deposited in NCBI GenBank under accession numbersMK051424 and MK051423 for Gut-S1 and Gut-S2, respectively.For the construction of a 16S rRNA phylogenetic tree, 16S rRNAsequences were collected from the NCBI nucleotide database,aligned using MUSCLE (Edgar, 2004), and analyzed by theneighbor-joining method (Saitou and Nei, 1987) using MolecularEvolutionary Genetics Analysis package (MEGA, version X.0)(Kumar S. et al., 2018).Scanning Electron MicroscopyFor the scanning electron microscopy, ITO electrodes wereremoved from the reactors after performing the electrochemicalmeasurements. Microbial fixation on electrodes was carried outwith 2.5% glutaraldehyde for 10 min in the dark at roomtemperature. This was followed by washing three times in 0.1 Mphosphate buffer (pH 7.4) for 15 min each. These washed sampleswere then dehydrated in 30, 50, 70, 90, and 100% ethanolgradients (prepared in the 0.1 M buffer) for 15 min each. Ethanolgradient dehydrated samples were exchanged thrice with 100%t-butanol and finally freeze-dried under vacuum. The driedsamples were coated with platinum and then observed using aKeyence VE-9800 microscope.Frontiers in Microbiology | www.frontiersin.org 3 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 4Naradasu et al. Extracellular Electron Transport in GutSupernatant Exchange During CurrentProducing ConditionThe electron transfer mechanism of gut microbes was evaluatedusing a medium-replacement experiment used to test electrontransfer in environmental model microbes S. oneidensis MR1(Marsili et al., 2008). Here, the medium in the electrochemicalcell was removed; the biofilm was rinsed with N2-sparged DMtwice at each replacement; and the headspace was continuouslysparged with N2 during each replacement to avoid the leakageof oxygen into the electrochemical cell. The cell was refilled withN2-sparged sterile medium (10 mM acetate or lactate). DPVmeasurements were performed as previously described to detectthe redox molecules (Okamoto et al., 2009) before and aftersupernatant exchanges.RESULTSElectrochemical Enrichment andIsolation of EET-Capable Human GutMicrobesFor the isolation of fermentative bacteria capable of EET, weinitiated electrochemical enrichment in GAM medium with amicrobial consortium sample collected from the human gutand diluted it to a concentration of 10−7 (v/v) at 37◦C.Current production (Ic) was measured to be approximately 0.3– 0.4 µA cm−2 for each cycle at around 48 h, and it graduallydecreased. After 1 week of incubation, we replaced the spentmedium with fresh GAM medium, and after another week ofelectrochemical incubation, the electrode surface was washedto collect the enriched bacterial cells. Although the subsequentplating resulted in the formation of many colonies, none of thecolonies showed transparent spots on the black δ-MnO2 agarplates, indicating that MnO2 was not reduced, and hence, nocolonies of EET-capable bacteria were isolated. This unsuccessfulresult was probably due to the fact that the GAM medium grewtoo many fermentative bacterial cells that were incapable ofEET in bulk, and the EET-capable bacteria that were potentiallyenriched on the electrode became a minority.Next, we used DM1, containing either 30 mM acetate or30 mM lactate as an electron donor, to enrich for bacteria thatcan couple EET with anaerobic respiration during the sametime frame as the enrichment with the GAM medium. Althoughthe Ic only reached 15–20 nA cm−2 during the DM1 cycles(Supplementary Figures S2a,b), transparent spots were observedon the δ-MnO2 agar plates, after the incubation of the cellscollected from the reactors enriched with either lactate or acetate.All of the colonies with the transparent spots looked identical,and one colony from each plate was analyzed for its 16S rRNAgene sequence. Sequence alignment using NCBI showed thatthe strain isolated from the acetate-fed reactor belongs to thegenus Enterococcus, and the strain isolated from the lactate-fed reactor belongs to the genus Klebsiella. Analysis of the 16SrRNA gene sequences of the strain Gut-S1 using the GenBankdatabase showed that it has more than 99% identity withEnterococcus avium, and analysis of the 16S rRNA gene sequencesof the lactate-enriched strain Gut-S2 showed that it has morethan 99% identity with Klebsiella pneumoniae. The ribosomalRNA gene sequences of these isolated strains were aligned withrepresentative microbial community sequences from human gutmicrobes (Enterococcus faecalis and Faecalibacterium prausnitzii)that had been previously reported to have EET capability. Thephylogenetic analysis is shown in Figure 1. E. avium andK. pneumoniae, which are similar to Gut-S1 and Gut-S2, areGram-positive and Gram-negative strains, respectively, and bothare fermentative under anaerobic conditions.Electrochemical Characterization of theMetabolism in the Isolated StrainsAn SA experiment with the isolated strains was performed usingthe DM1 medium without the addition of acetate or lactate atthe start of the incubation (Figure 2). The anodic current wasobserved to increase once Gut-S1 was added to the reactor withsterile media (Figure 2A), suggesting the microbial capability ofEET for the anode in Gut-S1. However, this medium did notcontain acetate, and hence, the current production was mostlikely due to the oxidation of the yeast extract, as no other organicsource was present. To our surprise, upon addition of acetate(30 mM), the current production immediately decreased by 10%and continued to decrease gradually, followed by a short currentrecovery, suggesting that 30 mM acetate might damage Gut-S1,FIGURE 1 | Ribosomal RNA gene sequences of isolated electrogenicmicrobes (isolated in bold red) were aligned with the representative gutmicrobial sequences previously reported for their EET capability. Alignmentwas carried out using MUSCLE and the neighbor-joining method wasemployed for phylogenetic tree construction.Frontiers in Microbiology | www.frontiersin.org 4 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 5Naradasu et al. Extracellular Electron Transport in GutFIGURE 2 | Representative current production versus time in isolated (A)Gut-S1 and (B) Gut-S2 measured with the ITO electrode poised at 0.4 V vs.SHE, initiated with the sterile DM1 medium containing only yeast extract as acarbon source at time = 0. At indicated times with arrows, microbes, acetateor lactate, and glucose were added.although we had been able to use acetate to enrich for the Gut-S1strain. The current increased to 120 nA cm−2 after the additionof glucose, demonstrating the microbial viability of Gut-S1 andits ability to couple glucose oxidation with current production.These data strongly suggested that Gut-S1 was enriched by thecomplex organic substrates of yeast extract, rather than by theacetate, implying that there was no microbial strain that coulduse the abundant acetate to outcompete the bacteria that couldutilize the yeast extract.In contrast, a gradual current increase of 15 nA cm−2was observed upon lactate addition in the case of Gut-S2(Figure 2B), suggesting that lactate oxidation contributed tothe current production. As glucose addition resulted in animmediate current increase in Gut-S1 (Figure 2A) and Gut-S2(Figure 2B), the current production may be limited by the rateof the metabolic reaction in both strains. These results indicatedFIGURE 3 | Metabolites concentrations measured at every 8-h time intervalduring the current production of isolated strains in the presence of acetate orlactate. (A) Acetate production in the presence of Gut-S1 using DM2containing 10 mM acetate and 0.5 g/L yeast extract. (B) Lactate consumptionand acetate production in case of Gut-S2 using DM2 containing 10 mMlactate and 0.5 g/L yeast extract. Similar tendency was observed in more thantwo individual experiments.that the lactate-oxidation metabolism takes the lead role for theenrichment of the Gut-S2 cells on the electrode surface during theelectrochemical enrichment.We further characterized the metabolism of the two strainsand the EET mechanisms in these strains by electrochemistry anda metabolite assay in a different minimal medium DM2, whichwe usually use to characterize the current production capabilityand EET mechanism of S. oneidensis MR-1 (Okamoto et al.,2013; Saito et al., 2016; Rowe et al., 2017). This medium hasa lower yeast extract concentration than DM1 and lacks traceminerals and vitamin solutions. Additionally, the acetate andlactate concentrations were reduced to 10 mM to eliminate thepossibility of toxicity to the bacterial cells from the high organicconcentrations. No significant change was observed in thecurrent generation with and without acetate in the yeast extract-containing media forGut-S1, signifying that EET was not coupledFrontiers in Microbiology | www.frontiersin.org 5 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 6Naradasu et al. Extracellular Electron Transport in GutFIGURE 4 | The metabolism of Gut-S1 and Gut-S2 during the current production associated with glucose fermentation. (A) Time course of glucose consumptionand its metabolites concentration with (B) Gut-S1 and (C) Gut-S2 during current production using DM2 containing 10 mM glucose. The data shown are the meanvalues ± standard deviations of two replicate experiments.with acetate oxidation in the case of Gut-S1 (SupplementaryFigure S3). Furthermore, metabolite quantification for Gut-S1 in IC showed that the acetate concentration increased andthat it was not consumed, which was most likely due to theproduction of acetate from the oxidation of the yeast extract(Figure 3A). In the case of Gut-S2, we observed lactate oxidationand acetate production associated with current production(Figure 3B), indicating that lactate was not fermented butanaerobically respired. A slightly lower consumption of lactate,compared to the production of acetate, indicated the oxidationof the yeast extract to produce acetate in Gut-S2. These resultsdemonstrated that Gut-S2 has an EET capability associated withlactate oxidation, which is similar to the anaerobic respiration inthe EET model microbial strain, S. oneidensis MR-1.To examine their ability to couple EET with fermentation,the metabolites were further explored using glucose in theelectrochemical system. The consumption and production ratesfor glucose and the metabolites, respectively, were identifiedusing Gut-S1 and Gut-S2 in DM2. Ten millimolars of glucosewas completely consumed by both strains in 24 h, which is 50–80% faster than the consumption rate of lactate in S. oneidensisMR-1, indicating considerable microbial activity (Figure 4A).Gut-S1 produced lactate and acetate (Figure 4B), while acetateand formate were the main metabolites for Gut-S2 (Figure 4C).Lactate and formate are known as common end products forbacterial fermentation, and formate can be further oxidized toCO2 and H2 under anoxic conditions (Lim et al., 2014). Thehydrogen produced in electrochemical cells may be oxidized atthe ITO electrode surface and may contribute to the currentgeneration by Gut-S2. A very low coulombic efficiency, i.e., lessthan 0.02%, was observed with both strains, based on the glucoseconsumption and coulombs generated. Given that this value ismuch lower than that of environmental bacteria, like S. oneidensisMR-1 (Bretschger et al., 2007), which was also observed in thesimilar electrochemical set up that was used in our current study(Okamoto et al., 2013), the role of EET in fermentation maybe distinct from that of well-studied EET microbes, which hasassociated EET with anaerobic respiration.The EET Mechanism in the Two IsolatesShewanella oneidensis MR-1 has two potential EET mechanisms:direct electron transfer and indirect electron transport,mediated by a cell-surface enzyme and soluble electroncarriers, respectively (Okamoto et al., 2013). To distinguish thetwo mechanisms for current production, medium exchangeexperiments have been performed to elucidate the contributionof the soluble electron carriers to the net current production(Marsili et al., 2008). After replacing the spent medium withfresh medium, there was a 20% decrease in the current for ashort period of time. It recovered to the current level beforethe medium exchange in the case of Gut-S1 (Figure 5A),suggesting a low contribution from the soluble electron carriersFrontiers in Microbiology | www.frontiersin.org 6 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 7Naradasu et al. Extracellular Electron Transport in GutFIGURE 5 | Medium exchange experiments for examining the contribution of soluble electron carrier to the current production, and DP voltammograms before andafter medium exchange in reactors with Gut-S1 (A and B) and Gut-S2 (C and D), the similar tendency was observed in more than two experiments.to the current production. Accordingly, the differential pulse(DP) voltammogram measured before and after the exchangeof the electrolyte did not show a significant change, with theoxidative current peaks observed at around −0.35 and 0.0 V[vs. the standard hydrogen electrode (SHE)] (Figure 5B). Incontrast, the current production decreased by approximately50% and did not recover to the level from before the mediumexchange for Gut-S2 (Figure 5C); the oxidative peak potentialalso shifted significantly to the positive region in the DPvoltammogram (Figure 5D), indicating a larger contributionfrom the soluble electron shuttle for the current production.Given that the Gut-S2 cells most likely generate hydrogen, weobserved the reduction in the formate concentration duringcurrent production (Figure 4C), which is a precursor forhydrogen generation. These data suggest that the Gut-S2 currentproduction could be assigned to the oxidation of fermentativelygenerated hydrogen. However, because a clear peak wasstill observed at −0.05 V (vs. SHE), even after the mediumexchange, Gut-S2 may also have a cell surface redox enzyme.Accordingly, for both Gut-S1 and Gut-S2, cellular attachment onthe electrode surface was confirmed by SEM analysis after thecurrent production in the presence of glucose (SupplementaryFigure S4). Taken together, the different peak positions andintensities of the oxidative peaks in the DP voltammogramssuggested that different redox proteins are involved in these twostrains.DISCUSSIONIn this study, the EET ability of isolates from a human gut samplewas investigated, and the metabolic pathways were studied.Our data provide evidence that the physiological role of EETcoupled with fermentation is somehow distinct from that ofanaerobic microbial respiration. The magnitude of the anodiccurrents reported here, detected from these isolated strains, issignificantly lower than those typically reported for the metal-reducing microbes usually investigated as EET model systems,and thus, these currents can be easily missed by traditionalcultivation strategies. For example, S. oneidensis MR-1 producestwo orders of magnitude more current than Gut-S1 and Gut-S2(Xu et al., 2016). The low currents observed here also point to thelower ability of these isolated strains to gain cellular energy fromexternal redox active surfaces. The physiological role of this modeof energy acquisition from external substrates should be furtherinvestigated in detail.The phylogenetic analysis showed that Gut-S1 and Gut-S2were similar to E. avium and K. pneumoniae, respectively. BothFrontiers in Microbiology | www.frontiersin.org 7 January 2019 | Volume 9 | Article 3267https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articlesfmicb-09-03267 January 12, 2019 Time: 17:5 # 8Naradasu et al. Extracellular Electron Transport in Gutstrains are facultative anaerobes, but not strict anaerobes, like themajority in the human gut. E. avium is a Gram-positive bacteriaand is known as a rare human pathogen, and only a few caseseries exist (Lee et al., 2004). While the EET capability of thisspecific strain has not been reported, K. pneumoniae has beenstudied previously for its EET capability. This Gram-negativebacterium was isolated from subterranean forest sediment andinvestigated using glucose and starch as the carbon sources withsuccessful current generation (Zhang et al., 2008). Furthermore,one study has reported an electron shuttling mechanism inK. pneumoniae based-microbial fuel cells (MFCs) (Deng et al.,2010). In our electrochemical analysis, these strains showed aspontaneous increase in current production with the fermentablecarbon source such as glucose, and a less consumption of thenonfermentable carbon sources like acetate/lactate, indicating thefermentation associated EET.In our study, one of the isolated strains was Gram-negativeand the other was Gram-positive, and therefore, they shouldhave different redox proteins for the transfer of the electronsto the electrode surface. Gram-negative bacteria are known torely on cell surface-exposed cytochromes for the oxidation orreduction of extracellular minerals (Stams and Plugge, 2009;Wegener et al., 2015; White et al., 2016). Much less is knownabout the electroactivity of gram-positive bacteria than gram-negative ones. The cell envelope of gram-positive bacteria lacksan outer membrane, and the peptidoglycan layer is thicker (20–35 nm) (Beeby et al., 2013). Gram-positive bacteria are known tobe poor in terms of their current production, but they can donateelectrons to an external electrode (Pankratova and Gorton, 2017)and are frequent members of the microbial community in MFCs(Rabaey et al., 2004). A detailed understanding of this aspect maybe important for devising strategies for pathogenicity control andthe improvement of human health.In addition to identifying new microbial candidates for EET,this study provides information on the role of EET associatedwith fermentation, distinct from that of anaerobic respiration.We believe that this new EET mode, coupled with fermentation,may open new windows for biotechnological applications andpathogenicity control models. 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Microbiol. 74, 3130–3137. doi: 10.1128/AEM.02732-07Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.Copyright © 2019 Naradasu, Miran, Sakamoto and Okamoto. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, providedthe original author(s) and the copyright owner(s) are credited and that the originalpublication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with theseterms.Frontiers in Microbiology | www.frontiersin.org 9 January 2019 | Volume 9 | Article 3267https://doi.org/10.1073/pnas.0710525105https://doi.org/10.1073/pnas.0710525105https://doi.org/10.1126/science.240.4857.1319https://doi.org/10.1073/pnas.1220823110https://doi.org/10.1073/pnas.1220823110https://doi.org/10.1002/cbic.200900422https://doi.org/10.1002/anie.201704241https://doi.org/10.1016/j.coelec.2017.09.013https://doi.org/10.1016/j.coelec.2017.09.013https://doi.org/10.1021/acs.biochem.8b00600https://doi.org/10.1128/AEM.70.9.5373-5382.2004https://doi.org/10.1128/AEM.70.9.5373-5382.2004https://doi.org/10.1073/pnas.1312524110https://doi.org/10.1073/pnas.1312524110https://doi.org/10.1111/1462-2920.13723https://doi.org/10.1016/j.electacta.2016.09.002https://doi.org/10.1038/nrmicro.2016.93https://doi.org/10.1038/nrmicro2166https://doi.org/10.1038/nature15733https://doi.org/10.1016/bs.ampbs.2016.02.002https://doi.org/10.1016/bs.ampbs.2016.02.002https://doi.org/10.1016/j.electacta.2016.03.074https://doi.org/10.1016/j.electacta.2016.03.074https://doi.org/10.1016/j.elecom.2008.08.030https://doi.org/10.1002/elan.201501052https://doi.org/10.1128/AEM.02732-07https://doi.org/10.1128/AEM.02732-07http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/https://www.frontiersin.org/journals/microbiology/https://www.frontiersin.org/https://www.frontiersin.org/journals/microbiology#articles Isolation and Characterization of Human Gut Bacteria Capable of Extracellular Electron Transport by Electrochemical Techniques Introduction Materials and Methods Electrochemical Cell Operation and Medium Composition Electrochemical Enrichment of Human Gut Sample Cell Cultures Harvesting Metabolites Determination DNA Isolation and Phylogenetic Tree Scanning Electron Microscopy Supernatant Exchange During Current Producing Condition Results Electrochemical Enrichment and Isolation of EET-Capable Human Gut Microbes Electrochemical Characterization of the Metabolism in the Isolated Strains The EET Mechanism in the Two Isolates Discussion Author Contributions Funding Supplementary Material References