sustainable energy & fuels · 2017. 7. 6. · the cells were initially grown at 37°c in a 5 ml...
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SustainableEnergy&Fuels
ElectronicSupplementaryInformation
Thisjournalis©TheRoyalSocietyofChemistry2017 SustainableEnergyFuels,2017,XX,1-9|1
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Received28thMay2017,Accepted28thJune2017
DOI:10.1039/c7se00270j
rsc.li/sustainable-energy
InvestigatingExtracellularElectronTransferofRikenellamicrofusus:aRecurringBacteriuminMixed-SpeciesBiofilmsM.Grattieri,aK.Hasan,aR.D.Milton,aS.Abdellaoui,aM.Suvirab,B.Alkotaini,aandS.D.Minteer*a
Materials
R.microfususculture
R. microfusus was obtained from ATCC® (ATCC® 29728TM), strain designation Q-1 [NCTC 11190]. The bacterial cells were
transferredandinoculatedinanaerobicconditionsusinganAtmosbagglovebag(SigmaAldrich)purgedwithArgongasthathad
been scrubbedofO2overaheatedCu catalyst. The cellswere initially grownat37°C ina5mLbrothmediumand following
transferredtoa75mLbrothmedium.Thebrothmediumcomposition(perLiterofde-ionizedwater)was:10gbeefextract,10g
peptone,3gyeastextract,5gglucose,5gsodiumchloride,1gsolublestarch,3gsodiumacetate,0.5gcysteine,and0.5gagar.
All chemicals where from Sigma-Aldrich except for peptone and agar, whichwhere from Becton, Dickinson and Co., sodium
chloridefromVWRandsodiumacetatefromMacronChemicals.Priortoinoculation,thebrothmediumwasautoclavedat121°C
for 15minutes and the Atmosbag glove bag was sterilized wiping with ethanol and by exposure to UV light for at least 30
minutes.Thegrowthcurvewasmonitoredbyopticaldensityanalysisat600nm(OD,ThermoScientific,Evolution260bioUV-Vis
spectrophotometer).WhenthemeasuredODwashigherthan1,thesamplewasdilutedandtheODcalculatedfromthediluted
solution.Theelectrochemicalcellswereinoculatedwithbacterialcellsintheearly-stationaryphaseat40hoursofthebacterial
growth.
Electrodespreparation
Theelectrodesusedforelectrochemicalcharacterizationwerepreparedbyconnectingacarbonclothsheetof1.5x1.5cm(non-
wetproofed,E-TEK)tonickelwire(24BNC,ConsolidatedElectronicWire&Cable).Alloftheelectrodesweresterilizedat121°C
for25minutes.Fortheexperimentsperformedwithpre-acclimatedelectrodes,thecarbonclothelectrodeswereplacedinthe
growthmediumbeforeinoculationandmaintainedinthemediumduringthegrowth.After40hoursofgrowth,theelectrode
was anaerobically transferred to the electrochemical cell. For the experiments performed without pre-acclimation of the
electrodesinthegrowthmedium,theelectrodesweredirectlytransferredtotheelectrochemicalcell.
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Methods
ElectrochemicalCharacterization
Theelectrochemicalexperimentswereperformedwithanumberofbiologicalreplicatesofthree,undercontinuouspurgingof
nitrogengastoensureanaerobicconditions.Athree-electrodesetupwasused,wherethecarbonclothsheetpre-acclimatedin
thebacterialgrowthwasusedas theworkingelectrode,aplatinummeshwas thecounterelectrodeandaAg|AgCl (KCl sat.)
electrodewasthereferenceelectrode(RE).AllpotentialsusedinthispaperareversusthisRE.Theplatinummeshwassterilized
innitricacid;thereferenceelectrodewassterilizedwithethanol.Theelectrochemicalcellsandrubberstoppersweresterilized
at121°Cfor25minutes.Theelectrolytefortheelectrochemicalexperimentswas34mLof0.1Mphosphatebuffersolution(PBS)
+0.1MKCl(pH7)including4mLofcellinoculumgrownattheearlystationaryphase(40h).ThePBSwaspreviouslysterilizedat
121°Cfor25minutes.Dilutionofthecellgrowthwasperformedtoensureoptimalsolutionconductivityandavoidnoiseinthe
electrochemical response due to the complex growth medium. The use of PBS solution in the study of bioelectrochemical
systems is beneficial since the increase in solution conductivitymight facilitatean increase in current generation,particularly
whenthesolutionresistanceplaysasignificantrole,andithasbeenreportedfordifferentmicrobialfuelcellsetups.1-3Inorder
to investigate the capability of R. microfusus to utilize a redox mediator to facilitate the EET, one set of experiments was
performedbyadding0.5mLofa500µMRB5’-monophosphatesodiumsalthydrate(Sigma)solutionintotheelectrolyte.Cyclic
voltammetrictests(CVs)wereperformedusingaVSPBio-LogicInstrumentspotentiostat,atasweeprateof1mVs-1.FiveCVs
wereperformedforeachmeasurement,toensureastableelectrochemicalresponse.Thelastcyclewasusedforrepresentation
throughoutthemanuscript.Chronoamperometricexperiments(CA,VSPBio-LogicInstruments)wereperformedaftertheCVby
continuouslypolarizingtheWEatapotentialof+0.2Vvs.Ag|AgCl(KClsat.) for70hours.Additionsof2mLofa1Mglucose
stocksolutionwereperformedtoprovidethesubstrateforthebacterialcells(finalconcentration50mM).Theelectrochemical
potentialforCAswasselectedfromtheresultsobtainedbyCVs,ensuringasufficientoverpotential(η)fortheanodicreaction
(η= 0.28V). Control (negative) experiments, to confirm the role of R. microfusus in the bioelectrocatalytic process, were
performedutilizingasterilizedmediumandaworkingelectrodepre-acclimatedinthesterilebrothmediawithoutbacterialcells.
At the end of the electrochemical tests, the electrodeswere dried in sterilized petri dishes at room temperature (20 ± 2°C)
beforeperformingscanningelectronmicroscopy(SEM).TheSEMmicrographswereobtainedusingaFEIQuanta600FEG.
GelElectrophoresisanalysis
Thebacterialcellsintheearly-stationaryphaseat40hoursofgrowthwerecentrifugedusingAmiconUltra10Kfilterunitsfor20
min at 15000 rpm (Allegra X-15R, Beckman Coulter). The supernatant containing proteins above 10KDa was collected and
centrifuged again at 13000 rpm for 5min (Eppendorf™ 5424R). Gel electrophoresis (Thermo Scientific Owl® EC3000XL) was
performedonapre-castgel(Tris-GlycineNN4-20%,NuSep)usingaTris-Glycinesodiumdodecylsulfate(SDS)buffer(25mMTris,
0.19MGlycine,0.1%SDS).Thesamplewaspreparedbyadding2µLof50%glyceroland6µLofPierce™lanemarkerreducing
samplebuffer(ThermoScientific)to22µLofthesupernatantpreviouslyobtained.Proteomicsmassspectrometryanalysiswas
performedattheMassSpectrometryandProteomicsCoreFacilityattheUniversityofUtah.Massspectrometryequipmentwas
obtainedthroughNCRRSharedInstrumentationGrant#1S10RR020883-01and1S10RR025532-01A1.Theproteinsequences
wereanalyzedbyMascot™database.
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SupplementaryFigures,ResultsandDiscussion
FigureS1showsthebacterialgrowthcurve.Theearly-stationaryphasewasreachedafter40hoursofgrowth.Thistimewasselectedtoharvestthecellsforelectrochemicalexperiments.
FigureS1–ThegrowthcurveofR.microfusus(OD=600nm)
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FigureS2showstheCVsforthethreereplicatestestswherecarbonclothelectrodespreviouslyexposedtobacterialcellsfor40hourswere
utilizedfortheanalysis.
FigureS2-CyclicvoltammogramsforthreereplicatetestswithadditionofexogenousRB.Scanrate:1mVs-1.
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Figure S3 reports the chronoamperometric traces, performed at +0.2 V (vs. Ag|AgCl (KCl sat.)) after the cyclic voltammetric analysis
reportedinFigure1.Duringtheinitial45hoursofpolarization,thecurrentresponseinthepresence(red)andabsence(blue)ofexogenous
RBwascomparable.After45hours,2mLofa1Mglucosesolutionwasadded(toafinalconcentrationof50mM)andasignificantincrease
ofthecatalyticcurrentoutputwasobtainedonlyfortheexperimentsthatcontainedadditionalexogenousRB.Thehighercurrentoutput
lasteduntil52hours.Interestingly,theadditionofglucosetoR.microfususthatdidnotcontainadditionalRBonlyyieldansmallincreasein
theoxidativecurrentupto47hours,whenthebaselinecurrentwasrestored.
FigureS3-ChronoamperometricanalysisofR.microfususwith(red)andwithout(blue)exogenousRB.Control experimentcomprisingsterilizedelectrodeandelectrolyte(black).Glucoseaddedafter45hoursof continuous operation (final concentration 50mM).Appliedpotential,EAPP = +0.2 Vvs.Ag|AgCl (KCl sat.). Current densities are calculated based on the projected surface of the carbonclothanodes.
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FigureS4showstheCVsforthepre-acclimatedelectrodesafterthechronoamperometrictestsofFigureS3(CA,70hourswithanappliedpotentialof+0.2Vvs.Ag|AgCl(KClsat.)).AftertheCAsthemaximumcurrentachievedwasdecreasedto20and16µAwithandwithoutRB, respectively. However, the onset for the oxidation of glucose is shifted tomore negative potentials, at approximately -0.21 V (vs.Ag|AgCl (KCl sat.)). The shift could indicate an enhanced communication between bacterial cells and electrodes,whichmay be due toincreasedconcentrationofredoxmediatorswithamorenegativeredoxpotentialsecretedbythemicroorganisms.
FigureS4 -Cyclic voltammogramswith(red), andwithout(blue)additionofexogenousRB conductedafterthechronoamperometrictestsofFigureS3.Controlexperimentswerecarriedoutwithsterileelectrodesandelectrolyte(black)(50mMglucose).Scanrate:1mVs-1.
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Scanningelectronmicroscopy(SEM)
FigureS5showstheSEManalysisforcontrol(negative)experiments,wherenobacterialcellswherepresentintheelectrolyte(sterileelectrolyte).
FigureS6showsthewidespreadcolonizationofthecarbonclothelectrodeobtainedwhenexogenousriboflavinwasaddedtotheelectrolyte.
FigureS5-Controlexperiment.SEMimagesofcarbonclothelectrodeinsterileelectrolyte(absenceofR.microfusus).
Figure S6 - SEM images of possible extracellular polymeric substancessecreted from R. microfusus with addition of 6.25 mM exogenousriboflavin.
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FromFigureS7,thetypicalrodshapeofR.microfususcellcanberecognizedonthecarbonfibers.
FigureS7-SEMimagesofR.microfususcellsattachedtothecarbonfibercloth.Scalebarof10and20mmforAandBrespectively.
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TableS1reportsthecompleteresultsoftheproteomicanalysis,includingpeptidessequencesmatchingproteinswithalowscore.
TableS1.ProteomicdataanalysisofpeptidessequencesincludingproteinswithlowscorefromgelelectrophoresisofR.microfususgrowthmedium.
References
1. D.Sun,D.Call,A.Wang,S.ChengandB.E.Logan,Environ.Microbiol.Rep.,2014,6,723-729.2. C.Santoro,S.Babanova,P.Atanassov,B.Li,I.IeropoulosandP.Cristiani,J.Electrochem.Soc.,2013,160,H720-H726.3. A.Kumlanghan,J.Liu,P.Thavarungkul,P.KanatharanaandB.Mattiasson,Biosens.Bioelectron.,2007,22,2939-2944.
NCBIAccessionno. Mass Score Protein DetectedPeptidessequences
gi|488469612 45773 71 Phosphopyruvatehydratase
K.IQIVGDDLFVTNPK.RR.AAVPSGASTGQFEAVELR.DR.IEEELGDSAEYAGASAFPR.FK.VNQIGSLSETIDAVELAHR.NR.AAVPSGASTGQFEAVELR.D
gi|85681833 48565 161 ProteaseIV
R.GGLYGGPSYCGAPTSQR.NR.AAPLAPKPGTPLQVGVGLK.T
K.YSQGNVSAVGVTYDGHTALTR.VK.KYSQGNVSAVGVTYDGHTALTR.V
gi|50840727 32154 33 LysozymeM1precursorK.FATNETATPR.H
K.ATEGTSYQNPFYASQYNGSQSAGLIR.G
gi|282582005 37331 24 Ser/Thrphosphatasefamilyprotein R.IATGANLFTPVVEAR.N