exploring the extracellular electron transfer potential of ... · 1 exploring the extracellular...
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Exploringtheextracellularelectrontransferpotentialofbiocharwithsoilmicrobes
SamuelBarnettDepartmentofMicrobiology,CornellUniversity
MicrobialDiversityCourse,MarineBiologicalLaboratorySummer2017
Abstract Biocharisanagriculturesoilamendmentthatimprovescropyieldsandsoilhealth.Onefunctionthatbiocharmayhaveisasamediatorofextracellularelectrontransfer,couplingmicrobialmetabolismwithdistantelectronacceptorreduction.Thisstudyaimedtoenrichforsoilbacteriacapableoftransferringelectronstobiocharandtoexaminedifferenceinoxygenprofilesinbiocharamendedsoils.Usingacetateasanelectronsourceinamicrobialfuelcell,Ienrichedforacurrentlyunidentifiedbacterium,howevernostrongelectricalcurrentwasgeneratedbytheseenrichmentcultures.Sphericalstructuresofpossiblebacterialoriginwerealsofoundhighlyenrichedonbiocharfromamicrobialfuelcellusingsuccinateaselectronsource.Nodifferencesweremeasuredinoxygenprofilesduetobiocharinamendedsoilswitheithersuccinateoracetateasaddedelectronsources.Whilethisstudyisafirststep,morethoroughexperimentsareneededtoconfirmanddescribeextracellularelectrontransfercapabilityofbiocharwithsoilmicrobes.Introduction
Biocharisasoilamendmentthat,whileusedbyindigenouspeopleofSouthAmericaandAfricaforhundredsofyears,hasonlyrecentlybeenintroducedintomodernagriculture.Biocharisproducedbytheslowanoxicpyrolysisoforganicmattersuchaswoodchipsoragriculturewaste.Theresultingmaterialishighlyenrichedinpurecarbon.Applicationofbiochartofieldsincreasesyieldsofvariouscropsespeciallywhenusedinconjunctionwithfertilizerandnutrientamendments.Thegrowthpromotingpropertiesofbiochararenotfullyunderstood,butevidencepointstoitsimmensewaterholdingandcationexchangecapacities,aswellasitsabilitytoadsorbcompoundsfromthesoilsuchasmetals,organicmatter,andnutrients.Biocharcanalsobeusedasacarbonsequestrationmechanismasitcannotbeeasilybrokendownbyorganismsandremainsinsoilsforhundredsofyears[1].Thepurecarbonthroughoutbiocharalsomakesthematerialabletoconductelectricitysimilartographiteusedinbasicmicrobialfuelcells. Microbialfuelcells(MFC)harnessmicrobialmetabolismtogenerateanelectriccurrent.InabasicMFC,bacteriametabolizeagivensubstrateusingtheanode,oftengraphiteorcarbonpaper,astheelectronacceptor.Theelectronsfromthisprocessthenpasstoacathodewhereafinalelectronsink,suchasoxygen,isreduced.Thecurrentproducedbytheelectronmovementbetweenanodeandcathodecanbeusedforvariousapplicationssuchaspoweringenvironmentalmonitoringequipment.ElectrontransferinanMFCrequireseitherdirectelectrontransferbetweenthebacteriaandtheelectrodeorbymeansofmediatorssuchashumicacids,quinones,orFe(II)/Fe(III)[2].Thisextracellularelectrontransfer(EET)isalsoimportantinenvironmentalsystemswhereeitherelectrondonorandacceptormaynotbeeasilyaccessibletoorganisms.
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MicrobescapableofEETcanbefoundinmanyenvironments,allofwhicharegoodinoculumsourcesforMFCs.SoilhasbeenshowntoharbormanyEETcapablemicrobessuchasmembersofthegenresGeobacter[3]andRhizobiales[4]andClostridium[5].InsituEETisbelievedtoutilizeelectronacceptorssuchasironoxides[5]andhumicacids[6].Duetoitspurecarbonchemicalmakeup,biocharhasalsobeensuggestedasapossibleelectronconduitinamendedsoils[7].PreviousstudieshavefoundthatitcanbeusedeffectivelyasananodeinMFCsinoculatedwithwastewatersludge[8].OthershaveusedbiochartoconnectShewanellaoneidensismetabolism,aknownEETcapablemicrobe,withFe(III)reduction[9].
Thegoalofthisstudyistofurtherexaminetheroleofbiocharasanelectronconduitinsoils.TodothisItookbothanMFCandmicrocosmapproachtoenrichformicrobescapableofusingbiocharassoleelectronacceptorandmeasureprofilesofoxygen,themainenvironmentalelectronacceptor,inbiocharamendedsoils.MethodsSoilandbiochar: SoilwascollectedfromafieldatCoonamessettFarminEastFalmouth,MA(41.6762˚N,70.5767˚W).ThefieldsitehadnotbeenplantedsincetheFallofthepreviousyearandonlycontainedasparsecoveringofvariousweeds.Soilwastakenfromthetop10cmandconsistedprimarilyoftheorganichorizon.Soilwaspassedthrougha2mmsieveandincubatedatroomtemperaturewithadepthof3cmfor5dayspriortosuccinatefuelcells,6dayspriortosuccinatemicrocosms,8dayspriortoacetatefuelcells,and11dayspriortoacetatemicrocosms. Twotypesofbiochar,CCandSC,wereusedforthisexperiment,bothofwhichwereobtainedfromBrianvonHerzenoftheClimateFoundation.CCbiocharisofcellulosicoriginwhichunderwentpyrolysisataround500˚C.SCbiocharisfromsawdustfeedstockpyrolyzedatamaximumof480˚CandoriginallyfromZHAWinWadenswil,Switzerland.MicrobialFuelCells: Biocharanodesweremadebyfillinga1mlpipettetipwithgranulatedbiochar(Figure1a).Holesweremadeinthesideofthetiptoallowbetterentryofanolytemedia.Abarecopperwirewasinsertedintowideendofthepipettetipintobiocharandthensealedwithsiliconerubber. SuccinateMFCswerebuiltaspicturedinFigure1b.Theanaerobicandaerobicvesselsconsistedofa250mLand500mLPyrexbottlerespectively.Eachchamberwassealedwitharubberstoppercontainingholesforelectrodesandsaltbridge.Succinateanolytemediacontainedperlitersolution:2.06gsodiumsuccinatedibasichexahydrate,0.31gammoniumchloride,0.13gpotassiumchloride,2.93gsodiumphosphatemonobasicmonohydrate,4.09gsodiumphosphatedibasicanhydrous,1mLtraceelementsolution,5mLMESbufferatpH6.15,and1mLvitaminsolution[8].A1%agarosein3Msodiumchloridesaltbridgespanningtheanaerobicandaerobicchamberswasmadebyinjectingthemoltensolutionintoa3mmtubeandsolidifying.Thecounterelectrodewasagraphiterod(AlphaAesar#14738)insertedintotheaerobicchamber.TheAg/AgClreferenceelectrode(BASiMF-2052)wasinsertedintotheanaerobicchamber.TheexperimentalMFCusedabiocharanodeastheworkingelectrodewhilethecontrolMFCusedanindiumtinoxide(ITO)coatedslide(SigmaAldrich#703184)
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attachedtoanalligatorclipthatwasnotsubmergedintheanolytemedia.Aconstantnitrogengasflowthroughtheanaerobicchambersmaintainedananoxicenvironment.BothMFCswereinoculatedwith5gsoil.Chamberswerefilledwithenoughanolytemediatopartiallycoverelectrodesbutmaintainasmallheadspaceforthenitrogengas. Acetatebasedfuelcellsweresinglechambersconsistingof125mLPyrexbottlesasshowninFigure1c.Chambersweresealedwithrubberstopperswithholesforworking,counterandreferenceelectrodes.Acetateanolytemediacontainedperlitersolution2.5gsodiumacetateanhydrous,0.31gammoniumchloride,0.13gpotassiumchloride,2.93gsodiumphosphatemonobasicmonohydrate,4.09gsodiumphosphatedibasicanhydrous,1mLtraceelementsolution,5mLMESbufferatpH6.15,and1mLvitaminsolution[8].BothMFCsusedagraphitecounterelectrodeandAg/AgClreferenceelectrode.Aconstantnitrogengasflowthroughtheanaerobicchambersmaintainedananoxicenvironment.TheexperimentalMFCusedabiocharanodeastheworkingelectrodewhilethecontrolMFCusedasecondgraphiterod.BothMFCswereinoculatedwith5gsoil.Chamberswerefilledwithenoughanolytemediatopartiallycoverelectrodesbutmaintainasmallheadspaceforthenitrogengas.Acetateanolytemediawasremovedandreplacedbyfreshmediaafter4days.
Figure1:a)biocharanodes.b)succinateMFCwithbiocharanodeusingbothanaerobic(small)andaerobic(big)bottles.c)AcetateMFCwithbiocharanodeinoneanaerobicbottle.
MFCswerehookeduptoseparateGamryInstrumentInterface1000potentiostatsandcontrolledusingGamryInstrumentFrameworkversion6.20software.Duringrun,MFCsweremaintainedusingcontrolledpotentialcoulometryatapotentialof200mV,samplingtimeof1min,andmaximumcurrentof1mA.Priortorunandattheendofeachrunweperformedcyclicvoltammetrywithapotentialrangefrom-500to400mV,scanrateof100mV/s,stepsizeof2mV,maxcurrentof10mA,andpowerfactorcorrectionof50ohm.AcetatedMFCswererestarted6timesinthefirst4daysduetocomputercutoffs.CyclicvoltammetrywasperformedatthebeginningandendofeachcontrolledpotentialcoulometryrunofallMFCsandbeforeandafterreplacementofanolytemediaintheacetateMFCs.
a b c
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Microcosmsetup: Allmicrocosmsweresetupin20x150mmHungatetubesandsealedwithParafilm.Therewereeightmicrocosmstomeasuresuccinateusebysoilmicroorganisms.Eachsuccinatemicrocosmcontained10mLlowersoilslurrytoppedby20mLuppersoilslurry.Succinatemicrocosmsetupisdescribedintable1.Succinatesolutioncontained1.02gsodiumsuccinatedibasichexahydratein500mLwater.Fourmicrocosmsweresetuptomeasureacetateuse.Acetatemicrocosmsetupisdescribedintable2.Acetatesolutioncontained1.25gsodiumacetateanhydrousin250mLwater.Forbothmicrocosms,after24hoursofincubation,excessliquidonsurfaceofsoilwasremovedpriortomicroelectrodemeasurements.
Table1:Succinatemicrocosmdesigns.
Table2:Acetatemicrocosmdesigns.
ScanningElectronMicroscopy: Biocharanode,copperwirefrombiocharanode,andITOslidefromsuccinateMFCswereimagedusingaZeissSUPRA40VPscanningelectronmicroscope.Sampleswerefixedin4%paraformaldehydeatroomtemperaturefor4hoursfollowedby4˚Cfor2hours.Theywerethenwashedthreetimesin1xPBS.Allsamplesweredehydratedby25minutesubmersionsof25%,50%,75%and100%ethanol.Sampleswerestoredin100%ethanoluntilcriticalpointdryingandplatinumcoating.HPLC:
LevelsofacetateinsamplesofanolytemediafromacetateMFCsandextractsfromtheacetatemicrocosmsweremeasuredusingUV/visdetectiononanHPLC(ShimadzuLC-2010C-HTwithBio-RadAminexHPX-87Hcolumn).Initialanolytesolutionwassampledafter3daysandagainfromreplenishedanolytesolutionafter7daysrunningintotal.Freshunusedanolytesolutionwasmeasuredforbaselineacetateconcentration.Soilextractsweremadeattheconclusionofthemicrocosmexperiment,day3.25mLpurewaterwasaddedtoeachmicrocosmandtheslurrywashomogenizedbyshakingandvortexingtubes.10mLofslurrywasthencentrifugedat4100Gfor10minutes.Soilextractwaspurifiedbyfiltering3mLofslurrysupernatantwasfilteredthrough0.2µmmembranefilter.Fluorescenceinsituhybridization: AnolytesolutionwithsuspendedcellaggregateswassampledfrominitialacetateMFCsafter4days.Samplewasfixedwith2%paraformaldehydeat4˚Cfor24hours.Afterfixing,
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samplewaswashed3timesin1xPBSandpassedthrough0.2µmfiltertocapturecells.Twosubsampleswerehybridizedfor2hoursat46˚Cin30%formamidewith2setsofprobes,onesettargetingGammaproteobacteria(mixofDelta495a-CY3,Delta495b-CY3,andDelta495c-CY3)andtheothertargetingDeltaproteobacteria(GAM42a-CY3).Bothprobesetsalsoincludedagenericeubacterialprobe(EUB338-FITC).Afterhybridization,sampleswerewashedinstandardwashbuffer,PBSandmilliQwaterfollowedbydehydrationin96%ethanolandstainingwithDAPI.SampleswereimagedusingaZeissAxio-A2fluorescentmicroscope.Microelectrodeoxygenmeasurements: Oxygenprofilesweremeasuredinallmicrocosmsusinga1.1x40mmneedlesensoroxygenprobe(UnisenseOX-N)coupledwithaUnisensefieldmicrosensormultimeter.SensorswerecontrolledanddataanalyzedusingUnisenseSensorTracesoftwareversion2.8.200.21688.Probesmeasuredtoadepthof1cmwithastepsizeof100µm.Oncealldatawascollected,gradientsfromasinglesamplingdaywerenormalizedtoaccountforvaryingstartingdepths.ResultsMFCbasedenrichments: NopeakincurrentwasobservedinanyMFCovertheentiretimecourse.Therewasaconstantcurrentat6.25µAinthesuccinatebiocharMFC,5.08µAinthesuccinateITOMFC,4.71µAintheacetatebiocharMFC,and4.53µAintheacetategraphiteMFC.CyclicvoltammogramsforbothsuccinateMFCsdidnotchangebetweenthestartandendofrun.PlotsforthebiocharanodeinthesuccinateMFCandfirstmeasurementoftheacetateMFCweremostlylinearwithnodistinguishingpeaks.TheplotforthebiocharanodeintheacetateMFCchangedbetweenstartofrunandthepre-andpost-replacementofanolytemediawithpeaksappearingataround175mVagainsttheAg/AgClreference(Figure2a).Thispeakwentawayinthefinalcyclicvoltammogramwhenexperimentwasfinishedandtheplotbecamerelativelylinear.ThecyclicvoltammogramforthegraphiteelectrodeintheacetateMFCalsochangedbetweentimepointswithapeakat260mVinitially,-200mVpre-replacementofanolytemedia,and50mVinbothpost-replacementofanolytemediaandfinalreading,allagainstAg/AgClreference(Figure2b).SamplesoftheanolytemediaweretakenfromtheacetateMFCspriortoreplacementofmediaandattheendofthestudyformeasurementbyHPLCbutnostrongdecreaseinacetateconcentrationwasobservedwhencomparedtofreshanolytemedia.
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Figure2:CyclicvoltammograminacetateMFCsforstart,pre-replacementofanolytemedia,post-replacementofanolytemedia,andendofexperimentwiththea)biocharanodeandb)graphiteanode.
WhilenoclearbacteriawereobservedthroughSEMonthebiocharanodefromthesuccinatebiocharMFC,spheresoffinefilamentswereseenthroughoutthebiocharandcoatingthecopperwireconnector(Figures3a-c).NumerousbacteriawerefoundontheITOslideanodefromthesuccinateITOMFCthemajorityofwhichwererodshapedbacteriawithspiralmarkingsrunningtheirlength(Figure3d).Alsoobservedwerecigar(Figure3e),oval(Figure3f),variousotherrod(Figures3g-h)shapedbacteriaandsomeotherformsthatmayormaynotbebiologicalinorigin.(Figure3i).VariousmorphologiesofbacteriawereobservedintheanolytemediaofbothsuccinateMFCsafterrunsbutnoclearenrichmentwasobserved.
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Figure3:SEMimagesofa)fibroussphereonbiocharanodefragmentfromasuccinateMFC,b)manyfibrousspheresonbiocharanodefragmentfromasuccinateMFC,c)fibrousspheresoncopperwirefrombiocharanodeinasuccinateMFCandbacteriaofvariousmorphologiesfoundontheITOslideanodefromasuccinateMFCincludingd)arodwithspiralpattern,e)cigar,f)oval,g)longerrod,h)roundedirregularrod,aswellasi)otherpossiblebiologicalstructures.
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WhitewispyaggregatesofbacteriawerefoundfloatingandattachedtosurfacesintheacetatebiocharMFCpriortorefreshingofanolytemedia(Figure4a).Theaggregateswereformedbythickrodshapedbacteriacontainingnumerousphasebrightinclusionswithinthecells.Cellswerearrangedinclumpsandtwistedcords(Figure4b).NofluorescencewasobservedwithhybridizationofeitherDeltaproteobacteria(Figure5a)orGammaproteobacteria(Figure5b)FISHprobes,howeverfluorescencewasobservedwiththeeubacteriaprobeandDAPI.Nootherbacteriashowedclearenrichment.Whileaggregatescontinuedtobeseen,theenrichedbacteriawerenotseenaswidelyinanolytesamplesfromtherefreshedmediabiocharMFC.Aggregatesimagedunderphasecontrastmicroscopyappearedtobemadeupofcellulardebrisfromthepreviouslyenrichedbacteria.
Figure4:EnrichedbacteriainthesuccinateMFCwithbiocharanodeobservedasa)whitewispyaggregatesfloatingandattachedtoMFCsurfacesandasb)rodshapedbacteriaformingtwistedcordlikestructuresinphasecontrastmicroscopy
Figure5:FISHimageenrichedbacteriafromsuccinateMFCwithbiocharanodehybridizedwithprobesfora)Deltaproteobacteriaandb)GammaproteobacteriaalongwithEubacteriaandstainedwithDAPI.NotenofluorescencewitheithertheDeltaproteobacteriaorGammaproteobacteriaprobes.
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Microcosmbasedcarbonutilization: Nodifferencewasobservedintheoxygenprofilesofanysuccinatemicrocosmsinanytimepoint(Figure6a-b).Nodifferencewasobservedintheoxygenprofilesoftheacetatemicrocosmsonday1(Figure7a),butthemicrocosmscontainingacetateonday3showedasteeperdecreaseinoxygenwithdepthascomparedtothenoacetatecontrols.Biocharshowednoinfluenceonthemicrocosmscontainingacetatebutseemedtoincreasetheslopeoftheoxygengradientinthenoacetatecontrols(Figure7b).AcetateconcentrationinsoilextractsweredeterminedattheconclusionoftheexperimentusingHPLCresultingin0.077mMand0.733mMinthe-biochar-acetateand+biochar-acetatemicrocosmsrespectivelyand7.758mMand8.084mMacetateinthe-biochar+acetateand+biochar+acetatemicrocosmsrespectively.
Figure6:Oxygenprofilesthroughfirst1cmofsuccinatemicrocosmsoils,a)measuredonday1andb)measuredonday3.
Figure7:Oxygenprofilesthroughfirst1cmofacetatemicrocosmsoils,a)measuredonday1andb)Measuredonday3.
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Discussion Thisstudyaimedtoexaminetheabilityofbiochartoactasanelectronconduitforsoilmicrobes.WhilenoincreaseincurrentwasobservedintheMFCs,theincreasedsignalinthecyclicvoltammogramoverMFCruntimesuggestachangeinelectrontransferkineticsofthebiocharelectrode.Twopossiblecausesforthiscouldbechangeinanolytesolutionorchangetoanodecomposition.Replacementoftheanolytesolutionresultedinnochangetothecyclicvoltammogramwhencomparedtoplotcreatedjustpriortoreplacement.Thissuggeststhatitisamodificationtotheanodeitselfthatresultsintheelectrontransferkinetics.Twopossibleanodemodificationsincludecompletesaturationofanodebyanolytemediaorbuildupofbacterialbiofilmonthesurfacesoftheanodes.Thehighsurfaceareaandporespaceofbiocharcouldallowformoreanolytemediatocomeintocontactwiththeanodesurfaceovertime,increasingtheinteractionbetweenanolyteionsandtheanode[9].ThiscouldalsoexplainthehigherconstantcurrentobservedinthebiocharanodescomparedtographiteorITOslides.WhenreplacingtheanolytemediaintheacetateMFCs,theoldmediawithintheanodewouldlargelytransferwiththeanodeitself,keepingtheincreasedconductivitywhenthepotentialwasrestarted.However,thisshiftincyclicvoltammetrywasnotseeninthesuccinatebiocharMFC,wherethissaturationeffectshouldalsooccur.Biofilmdevelopmentontheanodeisanotherpossibilityfortheshiftinelectrontransferkineticsasenrichedbacteriawasobservedintheanodeduringtherun.Previousstudieshaveshownbiofilmdevelopmentincreasingcyclicvoltammetrysignalwithcarbonpaperanodes[10].Thelossofthepeaksinthecyclicvoltammogramattheendoftheexperimentcouldthenbeduetotheapparentlossoftheenrichedbacteria.Furtherstudiesshouldexaminetheextentofbiofilmdevelopmentonorbacterialinteractionwithbiocharanodestoseeifbacteriaareindeedthecauseoftheshiftcyclicvoltammograms. ThisstudywasabletoenrichforasinglebacterialtypeintheacetatebiocharMFCs.IwasunabletoidentifythebacteriabutnegativeresultsfromtheFISHexperimentsuggestthatitisnotamemberofeitherGammaproteobacteriaorDeltaproteobacteria,whichcontainknownEETcapablebacteriaShewanellaandGeobacterrespectively.Moreexperimentationincludingisolationandsequencingwillbeneededtoidentifythisbacterium.ItisalsounknownifthisbacteriumdirectlyinteractswiththebiocharanodeorconductsEET.Whileclustersofthebacteriawerefoundattachedtotheanode,theywerealsofoundfloatingintheanolytemediaaswellasattachedtothecounterandreferenceelectrodes.Thetwistedcordlikestructureofsomeofthebacterialaggregatesisalsointriguingandshouldbeexaminedinmoredetail.Thepoorhealthofthisenrichmentafterreplacementoftheanolytemediasuggestsacompoundinthesoilitselfwasrequiredforbacterialgrowth.UnderstandingthemicronutrientsinsoilswillbeimportantforfurtherenrichmentofsoilmicroorganismsandindevelopmentofmoreefficientMFCs.SEMimagesofbiocharandthecopperwirefromthesuccinatebiocharMFC,whilenotproducingclearsignsofbacteria,doshowinterestingstructuresthatmaybeofbacterialorigin.Thesefibrousspheresareinhighconcentrationonsomeofthebiocharfragmentsaswellasthecopperwire,suggestingacloserelationshiptotheelectronpotentialofthesesurfacesintheworkingMFC.Properidentificationandassessmentofthesestructuresisneeded. AccordingtoSEMimages,rod-shapedbacteriaalsoseemedtobeenrichedontheITOslideanodeofthesuccinateMFC.Thesebacteriahadaspiralgroovedstructurerunningthe
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lengthofthecells.Itispossiblethatthesestructureswerecausedbythedehydrationandfixingprocesseshoweverfurtherimagingisneededtoexaminethenatureofthiscellmorphology.ContinuedculturingandlongerrunswiththisMFCarealsoneededtoconfirmtheconnectionbetweenthebacteriaandtheanode. Thisstudydidnotfindanymajordifferenceinoxygen-depthprofilesduetobiochar,exceptpossiblyanincreaseinslopeoftheoxygengradientinthebiocharamendedsoiloftheno-acetatemicrocosmascomparedtothenon-amendedsoil.Additionofacetatedoesshowasharpincreaseinoxygengradientsloperegardlessofbiocharaddition.Thisismostlikelyduetoincreasedmicrobialmetabolismfromexcesscarbonsource.Inordertofullytesttheelectrontransfercapabilityofbiocharinsoilitisimportanttomeasureconductivitythroughoutasoilprofilewithbiocharamendmentsandtoremoveorreduceanyothersourceofelectronflowotherthanbiochar.Useofbiocharembeddedmembranesmadeofnon-conductivematerialsuchasNafioncouldaccomplishthis.ExperimentswithopenairMFCssuchasthoseusedinWolińskaetal.[11]orJiangetal.[5]orinsituMFCscouldalsobeuseful.Conclusions Biocharhasthepotentialforbeinganelectronconduitformicroorganismsinanagriculturalsoilenvironment.ItsuseasananodeforMFCssuggeststhatmicrobescanuseitasanelectronacceptorandcoupletheirmetabolismswithreductionreactionsawayfromthecell.Moreworkneedstobedonetoshowthatthisisoccurringinsituanduncoverthemechanismsbehindtheprocess.IfEETisoccurringwithbiochar,thisprocesscouldexplainonebeneficialfunctionthisamendmenthasintreatedsoil.Acknowledgements IwouldliketothankDianneNewmanandJaredLeadbetterfortheirfantasticdirectionofthiscourseaswellasKurtHanselmann,ScottSaunders,BingranCheng,GeorgiaSquyres,andViolaKrukenbergforcloseassistanceandadviceonthisproject.Iwouldalsoliketothankalltheotherfaculty,TAsandlecturerswhohavecontributedtotherunningofthiscourseandthelaboratory.ThisprojectwouldnotbepossiblewithoutassistancefromthestaffatCoonamessettFarmforallowingmetosampletheirsoil,BrianvonHerzenfromtheClimateFoundationforhisdonationofbiochar,Unisenseforlendingusthemicroelectrodesetup,theGirguisLabatHarvardUniversityforlendingustheirpotentiostats,Zeissforlendingusfluorescentmicroscopes,andtheMBLCentralMicroscopyFacilityfortheirhelpwithSEMprepandimaging.ThefundingallowingmetoattendthiscoursecamefromtheSimonsMDScholarshipaswellascontributionfromtheBuckleyLabatCornellUniversity.References
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