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INTRODUCTION
Humicsubstancesareubiquitousandheterogeneousorganiccompoundsthatcomprise
upto80%oftheorganicmatterpoolinsoil,sedimentandaquaticenvironments(Mulholland
2003).Untilrecently,humicsubstances(HS)wereconsideredchemicallyunreactivepoolsof
dissolvedorganicmatter.However,ithasbeenshownthatHSformcomplexeswithmetals,
andthiscansignificantlyinfluencethemobility,bioavailabilityandspeciationofmetals(Tipping
1981;SpositoandWeber1986).HSalsoparticipateinreductionandoxidation(redox)reactions
withmetalspeciessuchasiron,otherorganiccompoundsandchlorinatedsolvents(Lovley,et
al.1996).TheredoxreactivityofHSisprimarilyattributedtoquinonemoieties(Scott,etal.
1998).
Phylogeneticallydiversemicroorganismsusethereductionandoxidationofhumic
substancestogenerateenergyand/ortosupportgrowth(Lovely,etal.1996).Manyhumics‐
reducingmicroorganismscoupletheoxidationofacetatetothereductionofquinones(Coates,
etal.1998).Whenpairedtotheoxidationofacetate,thereductionofanthraquinone‐2,6‐
disulfonicacid,amodelquinone‐likecompoundwhichisoftenusedasafunctionalhumic
substanceanalog,yields91.46kJofenergy(Equation1;Straub,etal.2005).Amoreselect
groupofmicroorganismsusehydrogenasanelectrondonorinHSreduction(Cervantes,etal.
2002).
Equation1.
Humics‐reducingmicroorganismshavebeenisolatedfromavarietyofanoxicfreshwater
andmarinesediments(Coates,etal.1998).Evidencesuggeststhathumics‐reducing
microorganismscouldbeasabundantasiron‐reducingandfermentingmicroorganismsin
anaerobicfreshwatersediments(Kappler,etal.2004).Initialinvestigationsfoundthatthe
majorityofhumics‐reducingmicroorganismswereDeltaproteobacteriaintheGeobacteraceae
family(Coates,etal.1998).MorerecentstudieshaveshownthatGammaproteobacteriainthe
Shewanellagenus,aswellashalorespiring,sulphate‐reducingandmethanogenic
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microorganismscanalsoreducehumicsubstances(NewmanandKolter2000;Cervantes,etal.
2002).Whileadiversityoforganismscanreducequinonemoieties,growththroughhumics‐
reductionhasnotbeenconfirmedformanyorganismsoutsideoftheGeobacteraceaefamily.
Theprimarygoalofthisstudywastoisolateacetate‐oxidizinghumics‐reducing
microorganismsfromsedimentsamplescollectedinfreshwaterandsaltwaterenvironments
nearWoodsHole,Massachusetts.Anothergoalofthestudywastomonitortherelative
abundanceofDeltaproteobacteriawithintheenrichmentstotestifDeltaproteobacteriasuchas
thoseintheGeobacteraceaewerethedominantacetate‐oxidizinghumics‐reducing
microorganismsatthesamplesites.Resultsfromthisstudysuggestthatmicroorganismsthat
canpairacetateoxidationtoquinonereductionarepresentintheanoxicsedimentsofCedar
Swamp,SchoolStreetMarch,LittleSippewissettSaltMarshandoffthecoastofNaushonIsland
inMassachusetts(Figure1).
MATERIALSANDMETHODS
SedimentSampleCollection
Freshwaterandsaltwatersedimentswereusedasinoculaforeachofthehumics‐
reducingenrichments.AnoxicfreshwatersedimentswerecollectedfromSchoolStreetMarsh
(onecore)andCedarSwamp(twocores)onJuly8,2009(Figure1).Sedimentfromamicrobial
matintheLittleSippewissettSaltMarsh(onecore)wascollectedonJuly9,2009atlowtide.
SedimentcollectedfromaneelgrassbednearNaushonIslandwascollectedonJuly10,2009.
SedimentsamplesfromSchoolStreetMarsh,CedarSwampandLittleSippewissettSaltMarsh
werestoredinananoxicgas‐packjarthatwasflushedwithN2‐CO2(80:20)gasatfourdegrees
Celsiusuntilthetimeofinoculation.TheNaushonIslandsedimentwascollectedhourspriorto
inoculation,andstoredatfourdegreesCelsiusuntiltheinoculation.
EnrichmentCulturePreparation
EnrichmentcultureswerepreparedonJuly10,2009asdescribedinCoates,etal.1998
andStraub,etal.2005.Adetaileddescriptionoftheconcentrationsofconstituentswithinthe
bicarbonate‐bufferedbasalfreshwatermediacanbefoundinLovleyandPhillips1988whereas
therecipeforthebasalmarinemediacanbefoundinCoates,etal.1995.Anaerobicculturing
techniqueswereusedthroughouttheenrichmentculturepreparation(MillerandWolin1974).
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BasalliquidmediawasautoclavedandthencooledunderN2‐CO2(80:20)foratleast20
minutes.Oncecooled,2mMacetateand5mMAQDSwasaddedtothebasalfreshwatermedia.
Next,20mlofmediawasaddedtoBalchtubesandonegramofsedimentwasaddedtoeach
tubewhilegassingtheheadspacewithN2‐CO2(80:20).Uninoculatedfreshwaterandmarine
controlswerealsopreparedusingthesamemethods.Moredetailsaboutthesampleinocula
arelistedinTable1.Freshwaterenrichmentswereincubatedinthedarkat30degreesCelsius
whilemarineenrichmentswereincubatedatroomtemperatureinthedark.
ThereductionofAQDSwasinitiallymonitoredbyobservanceofacolorchangefrom
pink‐ishtoadarkorange.Freshwaterenrichmentculturesweretransferredthreedaysafter
preparationbyadding1mlofprimaryenrichmentcultureto19mloffreshmedia,using
anaerobictechniques.MarineandfreshwaterenrichmentsweretransferredagainonJuly21,
2009toadilutionseries;thedilutionsusedwere10‐2,10‐4,10‐6and10‐8.Thedilutionseries
wereusedtoinoculate1.3%agaroseplateswiththesamechemicalcompositionastheliquid
media.Theseplateswereincubatedanaerobicallyat30degreesCelsius.Growthwasobserved
fortheplateinoculatedwiththeCS2sampleafterfivedays.
CARD‐FISH
ThesedimentsusedtoinoculateenrichmentcultureswerealsousedforCARD‐FISH
analysis.0.5mlofadditionalsedimentwaspreservedatthetimeofinoculationthrough
fixation,usingProtocolv2.0availableathttp://www.arb‐silva.de/fish‐probes/fish‐protocols/.
Fixedsedimentsweresonicatedusingthefollowingmethod:sonicateonlowspeedfor30
seconds,waitfor30seconds,seventimes.Sedimentswerestoredat‐20degreesCelsiusuntil
furtherCARD‐FISHpreparation.Primaryandsecondaryenrichmentculturesampleswerealso
fixedandstoredaccordingtothepreviouslymentionedprotocolattimepointsthroughoutthe
experiment.50μloffixedandsonicatedsamplewasaddedto10mlof1xPBSandvacuum‐
filteredonaMilliporeGTTPmembranefilterwith0.2μmporesize.Filterswerethenrinsed
with20mlofsterileMilliQwater.Next,filterswerepreparedaccordingtotheCARD‐FISH
protocolforsedimentsamplesavailableathttp://www.arb‐
silva.de/fileadmin/graphics_fish/SILVA_FISH_protocols_card_080702.pdf(Ishii,etal.2004).
FilterswerehybridizedwiththeEubacteria338,Archaea915,DeltaproteobacteriaandNonsense
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probes;moredetailsareavailableathttp://www.microbial‐
ecology.net/probebase/list.asp?list=insitu‐probes.
ChemicalAnalysis
AcetateandAQDSconcentrationsweremonitoredthroughouttheenrichmentculture
experiment.Samplesforacetateanalysiswerepreparedbyextracting1mlofenrichment
cultureandcentrifugingthesampleonhighspeedfor5minutes,oruntilapelletformed.Then,
500μlofthesupernatantwastransferredintoa1.5mlsamplevial.Acetateconcentrations
weremeasuredonaShimadzuLC‐2010C‐HTHighPerformanceLiquidChromatography
instrument.TherearefewprecisemethodsformeasuringconcentrationsofAQDSinwater
samples.SincethereductionofAQDStoanthrahydroquinone‐2,6‐disulfonicacid(AHDS)results
inacolorchangetheprocesscanbemonitoredthroughobservations.Inaddition,the
absorbanceofasampleat450nmcanprovideanestimateoftheamountofAQDSreduction
thathasoccurred.AnothermethodformeasuringAQDSreductionindirectlyincludesreacting
avolumeoffilteredsamplewithanequalvolumeofferriciron,andthenmeasuringthe
concentrationofferrousironproducedbythetransferofelectronsfromAHDStoferriciron
(Straub,etal.2005).Ferrousironconcentrationscanbedeterminedbytheferrozinemethod
(Stookey1970).EachofthesemethodswasusedtoestimateAQDSreductioninthe
incubations.
RESULTS
Observationsandchemicalanalysisofenrichmentculturesidentifieddifferences
betweenfreshwaterandmarineenrichmentcultures.Theculturemediaintheprimary
freshwaterenrichmentschangedtoadarkorangecolor,asignofAQDSreduction,afteronly3
daysofincubationat30degreesCelsius.Incontrast,thesamemagnitudeofcolorchangewas
notobservedinthemarineenrichmentsevenafter17daysofincubationatroomtemperature.
TheNaushonIslandeelgrassbedenrichmentshowedthelargestamountofcolorchangeduring
theenrichmentexperiment,andtheenrichmentinoculatedwithshallowsedimentformthe
LittleSippewissettSaltMarshshowedamoderateamountofcolorchangeduringthesame
periodoftime.Thesecondaryfreshwaterenrichmentswereslowertoshowacolorchange
thantheprimaryenrichments;8daysforthesecondaryenrichmentsasopposedtothreedays
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fortheprimaryenrichments.Additionally,onlytheCS2enrichmentshowedanyAQDS
reductionthroughacolorchangewithinsevendaysofthethirdtransfer,thedilutionseries
(Table1).
Acetatemeasurementsovertheexperimentalperioddemonstratethatacetatewas
consumedintheenrichmentcultures(Figures2‐4).Asstatedpreviously,2mMacetatewas
addedineachoftheenrichmentcultures.Atthetimeofthefirstacetatemeasurement,three
daysafterinoculation,thefreshwaterenrichmentsfromCedarSwampshowedsome
consumptionofacetatecomparedtothecontrolenrichment.Bytheendoftheprimary
enrichmentexperiment,13daysafterinoculation,alloftheaddedacetatehadbeenconsumed
inthefreshwaterenrichments(Figure2).Acetatemeasurementsofthesecondaryfreshwater
enrichmentsalsoindictedthatacetateconsumptionoccurredovertheenrichmentperiod,and
thegreaterconsumptionoccurredinthesamplesfromCedarSwamp(Figure4).Incontrast,
acetateconcentrationsdecreasedverylittle,andevenincreasedinthemarineenrichments
fromtheLittleSippewissettSaltMarsh(Figure3).
ResultsfromAQDSreductionestimateswereinconsistentwithacetateconsumption
(Figure7).Inaddition,AHDSconcentrationswerehigherinthemarineuninoculatedcontrol
enrichmentthanintheinoculatedenrichments(Figure5).TheseresultsindicatethattheAHDS
measurementsmaynotbeanaccuratemeasureofbiologicalAQDSreduction.However,there
wasastrongrelationshipbetweenresultsfromthetwomethodsusedtomeasureAQDS
reduction(Figure6).
TheCARD‐FISHhybridizationwasnotverysuccessfulbecauseitwasdifficulttofindthe
dilutionfactorforwhichtherewasanappropriateproportionofcellstobackground
particulates.AsshowninFigures8‐10,theparticulatebackgroundincreasedbetweenthe
sedimentsamplestotheenrichmentsprobablybecauseofhighconcentrationsofAQDSinthe
enrichments.ForLittleSippewissett1thepercentageofDeltaproteobacteriatototalDAPI
countsdecreasedfrom20%to6%betweentheinoculationsedimenttotheprimary
enrichment.CARD‐FISHhybridizationresultsfortheCedarSwamp1primaryenrichment
sampleshowedthatEubacteriacomprised50%ofthetotalDAPIcounts.
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DISCUSSION
Resultsfromvisualobservationsandchemicalanalysissuggestthattheenrichmentof
humics‐reducingmicroorganismsfromfreshwaterenvironmentsassuccessful.Themorerapid
colorchangeinprimaryenrichmentsthaninsecondaryenrichmentscouldbeattributedto
abioticredoxreactionsbetweenAQDSinthemediaandchemicalspeciesintheinoculation
sediment,suchasferrousiron.Inaddition,thesurprisingacetateandAHDSconcentrationsin
themarineenrichmentscouldbetheresultofredoxreactionsbetweenthesediment,and
acetateandAQDSinthemedia.Inaddition,acetateincreasesintheLittleSippewissett1
primaryenrichmentcouldhavebeenproducedbyacetogenicmicroorganismsinthe
inoculationsediment.MethodsformeasuringthereductionofAQDSintheenrichment
culturesshouldbeoptimizedsothattheresultsfromthesemethodsarestronglycorrelated
withoneanother,andacetateconsumptionovertheexperimentalperiod.
ThegoaloftheCARD‐FISHexperimentwastomonitorchangesinthemicrobial
communityintheenrichmentculturesovertime.However,duetoproblemsinthe
optimizationofsampledilutionfactorsitwasnotpossibletogetaccuratecountsofthe
microbialtargetgroups.IntheoneenrichmentseriesforwhichDeltaproteobacteriacounts
werepossible,thepercentageofDeltaproteobacteriatototalDAPIcountsdecreased.This
resultwasindirectcontrasttothefindingthatthemajorityofacetate‐oxidizinghumics‐
reducingbacteriaareDeltaproteobacteria.
CONCLUSIONS
Futureexperimentsaimedatenrichingforandisolatinghumics‐reducingbacteria
shouldusesmalleramountsofsedimentinoculum,andshoulddevelopmoreaccuratemethods
forestimatingAQDSreduction.CARD‐FISHisaveryeffectivemethodfortrackingthe
developmentofenrichmentcultureshowever,humics‐reducingenrichmentswillbemore
difficulttotrackduetothebackgroundinterferencefromhumicsubstances.Also,itis
importanttonotethathumics‐reducingorganismsgrowrelativelyslowlyandtherefore,itmay
notbepossibletoisolatethesemicroorganismswithinashortperiodoftime.
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REFERENCES
Cervantes,F.J.,F.A.M.deBok,T.Duong‐Dac,A.J.M.Stams,G.LettingaandJ.A.Field.2002.Reductionofhumicsubstancesbyhalorespiring,sulphate‐reducingandmethanogenicmicroorganisms,EnvironmentalMicrobiology4(1):51‐57.
Coates,J.D.,D.J.Lonergan,andD.R.Lovley.1995.Desulfuromonaspalmitatissp.nov.,along‐chainfattyacidoxidizingFe(III)reducerfrommarinesediments,ArchivesofMicrobiology164(6):406‐413.
Ishii,K.,M.Mußmann,B.J.MacGregor,andR.Amann.2004.Animprovedfluorescenceinsituhybridizationprotocolfortheidentificationofbacteriaandarchaeainmarinesediments,FEMSMicrobiologyEcology50:203‐212.
Kappler,A.,M.Benz,B.Schink,andA.Brune.2004.Electronshuttlingviahumicacidsinmicrobialiron(III)reductioninafreshwatersediment,FEMSMicrobiologyEcology47:85‐92.
Lovley,D.R.andE.J.P.Phillips.1988.Novelmodeofmicrobialenergymetabolism:organiccarbonoxidationcoupledtodissimilatoryreductionofironormanganese,AppliedandEnvironmentalMicrobiology54(6):1472‐1480.
Lovley,D.R.,J.D.Coates,E.L.Blunt‐Harris,E.J.P.Phillips,andJ.C.Woodward.1996.Humicsubstancesaselectronacceptorsformicrobialrespiration,Nature382:445‐448.
Miller,T.L.andM.J.Wolin.1974.AserumbottlemodificationoftheHungatetechniqueforculturingobligateanaerobes,AppliedMicrobiology27(5):985‐987.
Mulholland,P.J.2003.Large‐scalepatternsindissolvedorganiccarbonconcentration,fluxandsources.InInteractivityofDissolvedOrganicMatter;Findlay,S.E.G.,Sinsabaugh,R.L.,Eds.;AcademicPress:SanDiego,CA:139‐159.
Newman,D.K.andR.Kolter.2000.Aroleforexcretedquinonesinextracellularelectrontransfer,Nature405:94‐97.
Scott,D.T.,D.M.McKnight,E.L.Blunt‐Harris,S.E.Kolesar,andD.R.Lovley.1998.Quinonemoietiesactaselectronacceptorsinthereductionofhumicsubstancesbyhumics‐reducingmicroorganisms,EnvironmentalScienceandTechnology32:2984‐2989.
Sposito,G.andJ.H.Weber.1986.Sorptionoftracemetalsbyhumicmaterialsinsoilsandnaturalwaters,CriticalReviewsinEnvironmentalScienceandTechnology16(2):193‐229.
Straub,K.L.,A.Kappler,andB.Schink.2005.Enrichmentandisolationofferric‐iron‐andhumic‐acid‐reducingbacteria,EnvironmentalMicrobiology397:58‐77.
Stookey,L.L.1970.Ferrozine:anewspectrophotometricreagentforiron,AnalyticalChemistry42:779‐781.
Tipping,E.1981.Theadsorptionofaquatichumicsubstancesbyironoxides,GeochimicaetCosmochimicaActa45(2):191‐199.
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Table1.Detailsonthesedimentsamplesusedtoinoculateenrichmentcultures.
SampleCollectionSite SampleID SedimentDepth(cm) Comments
CedarSwamp CS1 7.5 Organic‐rich
CedarSwamp CS2 7.5 Sandysediment
SchoolStreetMarsh SS 14
LittleSippewissett LS1 1.25 Organic‐rich
LittleSippewissett LS2 10.15 Sandysediment
NaushonIsland
Eelgrassbed
EelG 35(approximately) Black,organic‐and
sulfide‐rich
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Figure1.SedimentsamplingsitesnearWoodsHole,Massacusetts.SedimentInoculaforenrichmentcultureswerecollectedatSchoolStreetMarsh,CedarSwamp,LittleSippewissettSaltMarshandoffthecoastofNaushonIsland(courtesyofDr.ColleenCavanaugh).
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Figure2.Acetateconcentrationsinprimaryenrichmentcultureexperimentsforhumics‐reducingbacteriafromfreshwaterenvironments.
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Figure 3. Acetate concentrations in primary enrichment culture experiments for humics‐reducingbacteriafrommarineenvironments.
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Figure 4. Acetate concentrations in secondary enrichment culture experiments for humics‐reducingbacteriafromfreshwaterenvironments.
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Figure5.AHDSconcentrationestimatesinprimaryenrichmentcultures.AHDSconcentrationswereestimatedbyreacting500μlofsamplewith500μlof5mMferriccitrate for15minutes.Next, I performed the ferrozine method on the mixture to determine the concentration offerrousiron.IestimatedAHDSconcentrationbymultiplyingtheconcentrationofferrousironby2sincethereductionofFe(III)toFe(II) isaoneelectrontransfer,whereastheoxidationofAHDStoAQDSisatwoelectrontransfer.
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Figure6.Therelationshipbetweentheabsorbanceoffiltered(0.7μmGF/F)enrichmentculturesamplesat450nmandAHDSconcentration.
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Figure 7. The relationship between acetate and AHDS concentrations in primary enrichmentcultures. The R2 of the linear relationship between acetate andAHDS concentrations is lessthan0.1.
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Figure8.DAPIimagesofsedimentsusedtoinoculatefreshwaterenrichmentcultures.A.CedarSwamp1;B.CedarSwamp2;C.SchoolSt.Marsh.Sampleswerefilteredusingthesameprocedureandthesamedilutionfactor.
A
B
C
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Figure9.DAPIimagesofsedimentsusedtoinoculatemarineenrichmentcultures.A.LittleSippewissett1;B.LittleSippewissett2;C.NaushonIslandeelgrassbed.Sampleswerefilteredusingthesameprocedureandthesamedilutionfactor.
A
B
C
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Figure10.DAPIimagesofCedarSwamp1A.inoculationsediment;B.primaryenrichmentandC.secondaryenrichment.
A
B
C
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Figure11.CARD‐FISHhybridizationimagesoftheDeltaproteobacteriaprobe.Imagesareofinoculationsediment(A)andprimaryenrichment(B)fromshallowsedimentcollectedatLittleSippewissettSaltMarsh.TheproportionofDeltaproteobacteriatototalDAPIcountsdecreasedfrom20%intheinoculationsedimentto6%intheprimaryenrichment.
A
B