THEENRICHMENTOFHUMIC‐REDUCINGBACTERIA

TerryLegg

UniversityofColorado/INSTAAR

MicrobialDiversity2009

2

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

3

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).

4

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

5

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

6

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.

7

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.

8

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.

9

10

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

11

Figure1.SedimentsamplingsitesnearWoodsHole,Massacusetts.SedimentInoculaforenrichmentcultureswerecollectedatSchoolStreetMarsh,CedarSwamp,LittleSippewissettSaltMarshandoffthecoastofNaushonIsland(courtesyofDr.ColleenCavanaugh).

12

Figure2.Acetateconcentrationsinprimaryenrichmentcultureexperimentsforhumics‐reducingbacteriafromfreshwaterenvironments.

13

Figure 3. Acetate concentrations in primary enrichment culture experiments for humics‐reducingbacteriafrommarineenvironments.

14

Figure 4. Acetate concentrations in secondary enrichment culture experiments for humics‐reducingbacteriafromfreshwaterenvironments.

15

Figure5.AHDSconcentrationestimatesinprimaryenrichmentcultures.AHDSconcentrationswereestimatedbyreacting500μlofsamplewith500μlof5mMferriccitrate for15minutes.Next, I performed the ferrozine method on the mixture to determine the concentration offerrousiron.IestimatedAHDSconcentrationbymultiplyingtheconcentrationofferrousironby2sincethereductionofFe(III)toFe(II) isaoneelectrontransfer,whereastheoxidationofAHDStoAQDSisatwoelectrontransfer.

16

Figure6.Therelationshipbetweentheabsorbanceoffiltered(0.7μmGF/F)enrichmentculturesamplesat450nmandAHDSconcentration.

17

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.

18

Figure8.DAPIimagesofsedimentsusedtoinoculatefreshwaterenrichmentcultures.A.CedarSwamp1;B.CedarSwamp2;C.SchoolSt.Marsh.Sampleswerefilteredusingthesameprocedureandthesamedilutionfactor.

A

B

C

19

Figure9.DAPIimagesofsedimentsusedtoinoculatemarineenrichmentcultures.A.LittleSippewissett1;B.LittleSippewissett2;C.NaushonIslandeelgrassbed.Sampleswerefilteredusingthesameprocedureandthesamedilutionfactor.

A

B

C

20

Figure10.DAPIimagesofCedarSwamp1A.inoculationsediment;B.primaryenrichmentandC.secondaryenrichment.

A

B

C

21

Figure11.CARD‐FISHhybridizationimagesoftheDeltaproteobacteriaprobe.Imagesareofinoculationsediment(A)andprimaryenrichment(B)fromshallowsedimentcollectedatLittleSippewissettSaltMarsh.TheproportionofDeltaproteobacteriatototalDAPIcountsdecreasedfrom20%intheinoculationsedimentto6%intheprimaryenrichment.

A

B

22

Figure12.CARD‐FISHhybridizationimagesoftheEubacteriaprobe.Imagesareoftheprimaryenrichment(A)DAPIand(B)EubacteriaprobefromSchoolStreetMarsh.AccordingtothehydridizationresultsEubacteriacomprised50%ofthetotalDAPIcounts.

A

B