sustainable energy & fuels · 2017. 7. 6. · the cells were initially grown at 37°c in a 5 ml...

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ElectronicSupplementaryInformation

Thisjournalis©TheRoyalSocietyofChemistry2017 SustainableEnergyFuels,2017,XX,1-9|1

Pleasedonotadjustmargins


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)





FigureS2showstheCVsforthethreereplicatestestswherecarbonclothelectrodespreviouslyexposedtobacterialcellsfor40hourswere

utilizedfortheanalysis.

FigureS2-CyclicvoltammogramsforthreereplicatetestswithadditionofexogenousRB.Scanrate:1mVs-1.





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.





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.





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.





FromFigureS7,thetypicalrodshapeofR.microfususcellcanberecognizedonthecarbonfibers.

FigureS7-SEMimagesofR.microfususcellsattachedtothecarbonfibercloth.Scalebarof10and20mmforAandBrespectively.





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

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