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Plant root exudates impact the hydrocarbon degradation potential of a weathered-hydrocarbon contaminated soil

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Applied Soil Ecology, 52, 1, pp. 56-64, 2011-11-11

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Plant root exudates impact the hydrocarbon degradation potential of a

weathered-hydrocarbon contaminated soil

Phillips, Lori A.; Greer, Charles W.; Farrell, Richard E.; Germida, James J.

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ContentslistsavailableatSciVerseScienceDirect

Applied

Soil

Ecology

j o u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / a p s o i l

Plant

root

exudates

impact

the

hydrocarbon

degradation

potential

of

a

weathered-hydrocarbon

contaminated

soil

Lori

A.

Phillips

a,∗

,

Charles

W.

Greer

b

,

Richard

E.

Farrell

a

,

James

J.

Germida

a

aDepartmentofSoilScience,UniversityofSaskatchewan,51CampusDrive,SaskatoonSaskatchewan,Canada,S7N5A8 bNationalResearchCouncilCanada,BiotechnologyResearchInstitute,6100RoyalmountAvenue,Montreal,QC,Canada,H4P2R2

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received20July2011

Receivedinrevisedform18October2011 Accepted18October2011 Keywords: Phytoremediation Plant-microbeinteractions Rootexudates Petroleumhydrocarbons QuantitativePCR

a

b

s

t

r

a

c

t

Phytoremediationisapromisinglowcosttechnologyforthecleanupofcontaminatedsites.However, specificplantsmaypromotedegradationunderonesetofconditionsbutnotunderanother,and knowl-edgelimitationssurroundingthemechanismsofphytoremediationhamperattemptsatoptimization. WeaddressedthisissuebyexamininghowexudatesreleasedbyElymusangustus(wildrye)andMedicago sativa(alfalfa),grownunderhydrocarbon-stressedornon-stressedconditions,impactedthedegradation potentialofmicrobialcommunitiesinaweatheredhydrocarbon-contaminatedsoil.Degradation poten-tialwasassessedusingmineralizationassayswith14C-labeledhydrocarbons(hexadecane,naphthalene, phenanthrene)followedbyDGGEofmicrobialcommunitiesandquantitative-PCRofgenesassociated withhydrocarbondegradation.Allrootexudatesrepressedhydrocarbonmineralizationinsoil micro-cosms,withexudatesfromhydrocarbon-stressedwildryehavingtheleastrepressiveimpact.Changesin degradationpotentialwerenotassociatedwithchangesinthedominantmicrobialcommunitystructure orwithsignificantshiftsingeneralmicrobialabundance.Degradationwas,however,associatedwith functionalchangesinmicrobialcommunities.Mineralizationofpolyaromatichydrocarbons(PAH)was highlycorrelatedwithcopynumbersofcatechol2,3dioxygenaseandnaphthalenedioxygenase,two genesinvolvedinPAHdegradation.Bothgenecopynumbersandmineralizationparameterswere sig-nificantlyimpactedbyexudatecomposition,withspecificcompoundsassociatedwitheitherincreased (acetate,alanine)ordecreased(malonate)degradativecapacity.Thesuccessofagiven phytoremedia-tiontreatmentislikelyinfluencedbytherelativeamountoftheseandsimilarcompoundswithinroot exudates.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Phytoremediation, the use of plants and their associated microorganismstoremoveordegradecontaminants,isa promis-ingtechnologyfortheremediationofbothinorganicandorganic soil contaminants (Arthur et al., 2005; Chaudhry et al., 2005). Accumulating phytoremediation studies under both controlled environmentalconditionsandfieldconditionsshowthatthis tech-nologyisafeasibleremediationoptionforpetroleumhydrocarbons (Bankset al.,2003; ListeandPrutz, 2006;Phillips etal., 2006). Thedegradation ofhydrocarbonsis facilitated througha rhizo-sphere effect; plants exude organic compounds through their roots,influencingtheabundance,diversity,oractivityofpotential hydrocarbondegradingmicroorganismsinthezonesurrounding theroots(Andersonetal.,1993).Theactualmechanismsofthis

∗ Correspondingauthor.Presentaddress.DepartmentofPrimaryIndustries,1 ParkDrive,Bundoora,Victoria3083,Australia.

E-mailaddress:lori.phillips@dpi.vic.gov.au(L.A.Phillips).

synergisticrelationshipremainill-definedandposeoneofthemore intriguingchallengesinphytoremediationresearchtoday.

Plantspeciesvarygreatlyintheirabilitytoincreasethe degrada-tioncapacityofsoilmicrobialcommunities(Chiapusioetal.,2007; ListeandPrutz,2006;Parrishetal.,2004;Sicilianoetal.,2003). Differencesindegradationpotentialarenothowever,limitedto differentplantspecies.Studiesonthephytoremediationpotential ofspecificplantspeciesoftenprovidecontrastingresults.For exam-ple,onerecentstudy(Rezeketal.,2008)foundlittleinfluenceof Loliumperenne(perennialryegrass)onpolyaromatichydrocarbon (PAH)degradation,whileanother(Binetetal.,2000)reporteda significantrhizosphereeffect.Differencesindegradationpotential arealsoobservedbetweenspecificcultivarsofaplant.Genotypic clonesofMedicagosativacvRiley(alfalfa)havebeenfoundto pro-motedifferentamountsofcrude oildegradation(Schwabetal., 2006;Wiltseetal.,1998).Somegenotypesincreaseddegradation whileothersdecreaseddegradationrelativetoanun-planted con-trol.Suchdifferencesindegradationpotentialmayberelatedto thecompositionofrootexudates,asithasbeenshownthatthe exudatesofrelatedcultivars differentiallyimpactbacterialgene

0929-1393/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2011.10.009

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expression. Mark et al. (2005) foundthat theexudates of two Betavulgaris(beetroot)cultivarspromotedsubstantiallydifferent patternsofgeneexpressioninPseudomonasaeruginosaPA01.For example,ageneencodingprotocatechuate3,4dioxygenase,an aro-matic ring-cleavingdioxygenase,wasup-regulatedby exudates fromonecultivarbutexhibitednochangewithexudatesfromthe othercultivar.Suchfundamentaldifferencesevenwithinaplant specieshighlighttheimportanceofspecificexudationpatternson soilmicrobialcommunityfunctioning.

Severalrecentstudieshaveattemptedtoelucidatetheimpactof exudatesonmicrobialcommunities.Artificialrootexudateshave beenshowntoincrease bacterialdensities,shiftmetabolic pro-files(Baudoinetal.,2003), andstimulatehydrocarbondegrader populations(Joneretal.,2002).Similarly,rootextractsand exu-dateshavebeenshowntostimulatebothgeneral(DaSilvaetal., 2006;MiyaandFirestone,2001)andgroup-specific(Yoshitomiand Shann,2001)increasesinPAHdegraderpopulations.Otherstudies however,have implicated exudates in therepressionof hydro-carbondegradationpotential.Corgiéetal.(2004,2006)observed distance-dependentrepressiveeffectsonPAHdegradationby exu-datesreleasedbyL.perenne.Rentzetal.(2004)foundthatexudates froma varietyof plants repressedthephenanthrene degrading activityofaP.putidastrain,inpartduetotherepressiveeffectsof exudatesonageneinvolvedinnaphthalenedioxygenase transcrip-tion(Kamathetal.,2004).Individualcompoundscommonlyfound inrootexudatescausedeitherrepression(glucose,glutamate, lac-tateandotherorganicacids)orstimulation(salicylicacid)ofgene expression.Rhizospheremicrobialcommunitiesareinfluencedbya myriadofchemical,physical,andbiologicalfactorsandareunlikely torespondtorootexudateinputsinamannercomparableto bac-terialisolates.However,thereis littledoubtthat thesuccessof agivenphytoremediationtreatmentisinextricablylinkedtothe impactthatthecomplexmixtureofrootexudateshasonmicrobial metabolicactivity.Untilwedevelopabetterunderstandingofthis relationshipandthedeterminantcausesofincreaseddegradation, thefullpotentialofphytoremediationwillnotberealized.

This study investigated the impact of root exudates of M. sativa and Elymus angustus (Altai wildrye), two commonplant speciesusedinphytoremediationstudies,onmicrobial commu-nitiesindigenoustoaweatheredhydrocarboncontaminatedsoil. Boththeplantsandthesoilusedinthecurrentstudyhavebeen investigatedinpreviousphytoremediationstudiesbyourgroup, underbothcontrolled(Phillipsetal.,2006)andfield(Phillipsetal., 2009)conditions.Theprimaryobjectiveofthisstudywasto deter-mine how differences in exudationpatterns,stimulated bythe presenceofhydrocarbons,impactthedegradationpotentialofsoil microbialcommunities.

2. Materialsandmethods

2.1. Plantgrowthandexudatecollection

E.angustus Trin. (Altai wildrye)and M. sativaL. (alfalfavar. Rambler)weregrownundersterileconditionsinmodifiedLeonard jars.Seedsweresurfacedisinfectedbywashingfor1minwith95% ethanol,followedbya5min(alfalfa)or10min(wildrye)washwith 5.25%sodiumhypochlorite,followedbyaminimumof5rinseswith sterilewater.Seedswerethenincubatedon1/10thstrengthtryptic soyagar(TSA)platesforaminimumof2daysandonlythoseseeds whichshowednobacterialorfungalgrowthwereused.

EachmodifiedLeonardjarconsistedofa quartz-sand(400g) filled330ml straight-bodiedglassfunnelinvertedintoa950ml wide-mouth bottle containing 600ml 0.5 strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950). A fiberglass wick (SpecialtyGaskets, Mississauga Ont.)connected theliquid

reservoirtothesolidgrowthsubstrate.Allcomponentswere ster-ilizedbyautoclavingbothpre-andpost-assembly.Thesandinhalf ofallsystemswassubsequentlyspikedwith200mgkg−1ofboth

pyreneandphenanthrene(98%pure;SigmaAldrich,Mississauga, Ont.)inacetone(40mgml−l),andtheacetonewasallowedto

evap-orateofffor24h.PAH-spikedandnon-spikedsystemswerethen plantedwithsurfacesterilizedseeds(PAH-spiked15seedseach, non-spiked10seedseach),orleftun-plantedforcontrols,and cov-eredbyasterileSunbagwitha0.02␮mgasexchangefilter(Sigma Aldrich,Mississauga,Ont.).Sixtreatmentswereestablishedwith fourreplicateseach;Altaiwildrye,alfalfa,andanon-planted con-trol,inPAH-contaminatedandnon-contaminatedsand.

Plantsweregrowninagrowthchamberwitha16h/25◦Cday

(1500␮molm−2)and8h/15Cnightcyclefor6weeks.Exudates

andcontroleluateswerecollectedbyflushingthesandwith300ml steriledeionizedwater.Exudateandeluatesterilitywasassessed byplating100␮laliquotson1/10thstrengthTSAplates.An addi-tional1mlaliquot,boiledtoreleaseDNA,wasassessedbyPCRusing theeubacterialprimersoutlinedinthefollowingsections.All exu-datesandeluateswerethenfilteredthrougha0.45␮mfilterand frozenuntiluse.Replicateswerenotpooledandweremaintained asseparateunitsthroughoutthestudy.Plantrootsandshootswere separated,driedat60◦Cfor1week,andweighed.

Anybacteriafoundinthegrowthsystemspost-harvestwere iso-latedandassessedbyFAMEanalysis(HewlettPackard5890SeriesII GC),accordingtopreviouslyoutlinedprocedures(deFreitasetal., 1997).IsolateswereidentifiedbylibrarycomparisonusingMIDI MicrobialIdentificationSoftware(SherlockTSBALibraryversion 3.80,MicrobialID,Inc.,Newark,DE,USA).

2.2. TOCanalysis

A5mlsub-sampleofeachexudateorcontroleluate,acidifiedto apHoflessthan2.0usingHCl,wasassessedfortotalorganiccarbon (TOC)usingaShimadzuTOC-5050ATOCAnalyzer.TOC concentra-tionwasdeterminedbycomparisonwithapotassiumhydrogen phthalatestandardcurve.

2.3. Identificationandquantificationofrootexudatecomponents Organicacids,aminoacids,andphenoliccompoundsoftheplant rootexudateswereisolatedbysolidphaseextraction(SPE)ofbulk exudates usingcation andanion exchangemembranes(organic acids, amino acids)or resins (phenolics) and quantified by gas chromatography–massspectrometry(GC–MS)analysisof deriva-tizedsub-samples.Organicacidandaminoacidrecoverymethods were modified from previously developed protocols (Gillespie, 2003).SPEandGC–MSwereusedtodetermineifresidualPAHs werepresentinexudates fromPAHspikedLeonardjars.Unless otherwise stated, all standards and derivatizing reagents were obtainedfromSigma–Aldrich(Mississauga,Ont.)andallsolvents werehighpurityGCgrade(OmniSolv).

IonexchangemembraneswereobtainedfromBio-Rad Labora-tories,Inc.(Hercules,CA).Cationandanionexchangemembranes werepreparedbywashing4timeswith1.0MNaOHor1.0MHCl, respectively,followedbyaregenerationstepofwashing4times with1.0MHClor1.0MNaOH,respectively.Exudatesandcontrol eluateswerethawed,transferredtoasterileErlenmeyerflask con-tainingananionandacationmembrane,andshakeninthedarkon arotaryshakerfor12h.Allmembraneswerethenremovedfrom solution,rinsedwithdeionizedwater,placed inseparatesterile 6mlvialscontainingeither5mlof0.5MHCl(anionmembranes) or5mlof2.0MNH4OH(cationmembranes),andshakeninthedark

for8h.

Fororganicacidanalysis,a1mlaliquotofanionexchange elu-atewastransferredtoa4mlvialcontaining20␮lofglutaricacid

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(100␮gml−1,internalstandard).Theeluatewasshakenfor10s

with2mldiethyl ether(DEE),thephases wereallowedto sep-arate, and the non-aqueous phase was transferred to a 1.8ml autosamplervialcontaining40␮ltriethylamine(EMScience)and evaporatedunderN2atroomtemperature.TheDEEextraction

pro-cesswasrepeatedforatotalof3extractions.Organicacidswere derivatizedtotert-butyldimethylsilyl(t-bdms)esterderivativesby dissolvingthefinalDEEresidue in75␮l isooctaneand25␮l N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide(MTBSTFA), cappingthevial,andheatingat70◦Cfor45min.

Foraminoacidanalysis,a1mlaliquotofcationexchange elu-atewastransferredtoa1.8mlautosamplervialcontaining20␮lof norleucine(100␮gml−1,internalstandard)andevaporatedto

dry-nessunderN2atroomtemperature.Aminoacidswerederivatized

tot-bdmsesterderivativesbydissolvingthedriedresiduein100␮l N-N-dimethylformamide, and 100␮l MTBSTFA (Sigma–Aldrich, Mississauga, Ont.), capping the vial, and heating at 70◦C

for30min.

Phenoliccompoundswereextracted usingDiscoveryDPA-6S SPEcolumns(Supelco,Bellefonte,PA).Columnswereconditioned with5mlethylacetate,andrinsedwith5mlmethanolfollowedby 3×5-mldeionizedwater.Samples(50ml)wereacidifiedtopH3.0, runthroughatarateof3mlmin−1,andthenthecolumnwasrinsed

with10mldeionizedwater.Columnswereallowedtodryfor1h undervacuumandphenoliccompoundswereelutedwith10ml ethylacetate.Theeluted phenolicsweretransferredtoa1.8ml autosampler vial containing 50␮l of naringenin (100␮gml−1,

internalstandard)anddriedunderN2atroomtemperature.

Pheno-licswerederivatizedbydissolvingtheethylacetateresiduein75␮l acetonitrile and 25␮l N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA;Supelco,Bellefonte,PA),cappingthevial,andheatingat 70◦Cfor40min.

ResidualPAHswereextractedfrom10mlsubsamplesofeluate fromeachPAHspikedtreatmentusingSupelcleanENVI-18SSPE tubes(Supelco,Bellefonte,PA).Columns wereconditionedwith 9mlacetonitrilefollowed by12mldeionizedwater:isopropanol (90:10;pH2.5 withHCl) ata flow rateof 3mlmin−1.Samples

amendedwith10%isopropanolwererunthroughcolumnsataflow rateof10mlmin−1,columnswerewashedwith30mldeionized

water:isopropanol(90:10;pH2.5withHCl),driedundervacuum for30min,andthenPAHswereelutedwith2×4-mlaliquotsof hexane:acetone:isopropanol(90:5:5)ataflowrateof1mlmin−1.

Toluene(0.5ml)wasaddedtoeachfinaleluate,whichwasthen driedunderN2toafinalvolumeof0.5ml,dilutedto1.8mlwith

toluene,andassessedbyGC–MS.EluatesfromLeonardjars con-tainingnoPAHsservedascontrols.Serialdilutionsofthetarget compoundswereusedtoestablisha0.004–2.000␮gml−1

calibra-tioncurve.

Samples were analysed on a Varian CP-3800 GC equipped with a Saturn 2200 MS detector and an 8400 autosampler. A Varian FactorFourTM column (VF-Xms) with dimensions of

30m×0.25mmi.d.anda0.25␮mstationary-phasefilmthickness wasusedforanalyticalseparation.Thecarriergaswasheliumwith aconstant flow rateof1mlmin−1.Theinjectionport

tempera-turewas250◦C.A1␮linjectionvolumewasdeliveredwitha5␮l

syringe.TheMSwasoperatedintheEIionizationmodeandwasset toscanfromm/z45to650withascanrateof0.420scanss−1.

Spec-traldatawereacquiredusingalinkedSaturnGC/MSWorkstation version5.5(VarianInc.,WalnutCreek,CA).Fororganicandamino acidanalysis,theinitialcolumnoventemperaturewasmaintained at60◦Cfor1min,andthenrampedat5Cmin−1to300Cwhere

itwasheldfor1minor6min,respectively.Forphenolicanalysis theinitialcolumntemperaturewasmaintainedat80◦Cfor1min,

thenrampedatarateof20◦Cmin−1to250Candheldfor1min,

thenrampedatarateof6◦Cmin−1to300Candheldfor2min,and

thenrampedatarateof20◦Cmin−1to320Candheldfor4min.

Mixedstandardsforeachgroupofcompounds(seesupplementary TableS1)werederivatizedasoutlinedaboveandusedtoidentify andquantifyindividualcompoundswithintheexudates.ForPAH analysis,theinitialcolumnoventemperaturewasmaintainedat 100◦Cfor1minandthenrampedat10Cmin−1to320C,whereit

washeldfor17min.Atargetedm/zscanbetweentherangeofm/z 60and650andascanrateof0.50scanss−1wasperformedfrom

3minonward.

2.4. Hydrocarbonmineralizationpotential

[14C]hydrocarbon mineralization assays wereused to

deter-mine what impact each exudate or control eluate had onthe hydrocarbon degrading activity of soil microbial communities. Thesoil,collectedfromaweathered-hydrocarboncontaminated site insouth-easternSaskatchewan,Canada,had aclaytexture, pHof 8.0,electrical conductivity5.8dSm−1,sodiumabsorption

ratio20.3,cationexchangecapacity18.46cmolkg−1,andNO 3–N,

PandKconcentrationsof1.6,1.0,and332mgkg−1,respectively.

Total hydrocarbon concentration (C16–C50) was 3700mgkg−1;

PAH concentration wasless than 0.50mgkg−1.Soil was sieved

througha4.75mmsievetoensurehomogeneity,storedat4◦Cfor

onemonthuntiluse,and acclimatizedtoroomtemperaturefor 2weekspriortomicrocosmsetup.Moisturecontentwas deter-mined by oven-drying 10g sub-samples of each soil at 100◦C

for 24h. Serum vial microcosms were set up and 14CO 2 was

sampled (at days 1, 3, 5 8, 12, 15, 21 and 28) as outlined in

Phillipsetal.(2006).Exudates/eluateswereaddedtomicrocosms containing6gsoilatarateof10␮gTOCg−1 soil,andfinal

mois-turewas adjusted to30% using steriledeionized water. Serum vialswereamendedwith50,000dpm(100mgkg−1)of[1-14

C]n-hexadecane,[1-14C]naphthalene,or[9-14C]phenanthrene(specific

activities12,6.2,and8.2mCimmol−1,respectively;Sigma–Aldrich,

Mississauga, Ont.). The final mineralization set-up consisted of sixexudate/eluatetreatments(Altaiwildryeexduates,alfalfa exu-dates, and non-planted control eluates from PAH-spiked and non-spiked systems, as outlined in Section 2.1) of four repli-cateseach,assessedinseparatemineralizationmicrocosmswith thethree differenthydrocarbons(72microcosmstotal). Abiotic controls(n=3)foreachhydrocarbontreatmentwereestablished using autoclaved soil (2×1-h with a 1 week resting interval). Non-radioactiveduplicatemicrocosms(72microcosmstotal)were establishedforallmineralizationmicrocosmsforuseinsubsequent molecularanalyses.

2.5. Microbialcommunityassessment

TotalcommunityDNAwasextractedfromallnon-radioactive mineralization microcosms (two separate extractions of 0.50±0.01gsoilpermicrocosm)aspreviouslyoutlined(Phillips et al., 2006). Briefly, this methodused a combination of bead-beating,proteinaseK,andsodiumdodecylsulphatetolysecells. Proteinsandcellulardebriswereprecipitatedusing7.5M ammo-nium acetate, and DNA was subsequently precipitated using isopropanol, re-suspended in TE (pH 8.0), and purified using polyvinylpolypyrrolidone(PVPP)columns.DNAyieldwas quanti-fiedpre-andpost-PVPPpurificationonethidiumbromide-stained 0.7%agarose gelsbycomparison witha highDNA mass ladder (Invitrogen)andbyspectrophotometerevaluation.

Microbial community structure in each treatment replicate was examined by DGGE analysis of PCR-amplified 16S rRNA genefragments.PCRamplificationwasperformedusinguniversal eubacterial16SrRNAgeneprimers(Leeetal.,1993;Röllekeetal., 1996;supplementaryTableS2),and followingthePCRprotocol outlinedinPhillipsetal.(2006).PooledPCRreplicateswere concen-tratedandprecipitatedwith0.1volume3Msodiumacetateand2.5

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volumes100%ethanolat−20◦Covernight,re-suspendedin15␮l

ofTEbuffer(pH8.0),andquantifiedonethidiumbromide-stained 1.4%agarosegelsbycomparisonwitha100bpladder(Invitrogen). DGGEwasperformedonaBio-RadDCodesystem(Bio-Rad, Missis-sauga,Ont.)essentiallyasdescribedbyLawrenceetal.(2004).For eachgel,600ngofamplified16SrRNAgeneproductwasloaded perlaneontoan8%acrylamidegelwitha40–60%urea-formamide denaturing gradient.Electrophoresis wasperformedfor 16hat 80Vand60◦C.TheresultinggelswerestainedwithSYBRGreen

I(Sigma–Aldrich,Mississauga,Ont.)inTAEbufferandvisualized usingadigital geldocumentationsystem(GelDocMega; BioSys-tematica,Devon,UnitedKingdom).Alltreatmentreplicateswere assessedforintra-treatmentbandingpatternsimilarityon sepa-rateDGGE’spriortocombiningreplicatesforfinalinter-treatment comparativegels.

QuantitativePCR(Q-PCR)wasperformedonmicrobial commu-nityDNAextractedfromallmineralizationmicrocosms.The16S rRNAgene(Leeetal.,1993;Röllekeetal.,1996)andthreegenes involved in hydrocarbon degradation, catechol 2,3 dioxygenase (Luzetal.,2004),naphthalenedioxygenase(Baldwinetal.,2003), and alkane monoxygenase (Powell et al., 2006)were assessed. EachseparateassaywasperformedatleasttwicetoverifyqPCR reproducibility.PCRassays were performedonanABI7500 RT PCRsystem(AppliedBiosystems,FosterCity,Calif.)using Quanti-TectSYBRgreenPCRkits(Qiagen,Mississauga,Ont).Amplification reactionsweresetupaccordingtothemanufacturer’sprotocol. Primer concentrations, amplification conditions, and positive-control strains are listed in supplementary Table S2. Absolute quantificationwasperformedbycomparisonwithstandardcurves generatedfrompositive-controlstrains(supplementaryTableS2). Ten fold dilutions of DNA standards ranging from 102 to 106

gene copynumbers werepreparedfrom DNA extractsofthese strains(supplementaryTableS2).Genecopynumberswere calcu-latedfromtheconcentrationsofpositive-controlstrains,quantified spectrophotometrically,assumingamolecularmassof660Daper dsDNAbp,6.0Mbppergenomeandonecopypergenome(Park andCrowley,2006).Standardcurveswerelinearover4–5orders ofmagnitudewithR2values>0.990andE>0.83.Correctproduct sizeofstandardsandsampleswereperiodicallyverifiedonagarose gels.Q-PCRplateswereset-upsuchthateachgeneforallreplicate microcosmswasassessedinasinglereaction(Smithetal.,2006), andeachplateincludedafullrangeoftherelevantDNAstandards. 2.6. Statisticalanalyses

StatisticaltestswereperformedusingSPSSsoftware(SPSS13.0, Chicago,Illinois).Datawereexaminedforoveralltreatmenteffects usingANOVA,followedbyaTukeytest(variancesequal)oraGames Howelltest(variancesunequal)todeterminewhethersignificant differencesoccurredbetweentreatments. Homogeneity of vari-ancewasassessedusingtheLevenestatistic.Relationshipsbetween parameterswereassessedbystepwisemultipleregression anal-ysisandSpearman’srankcorrelation.Relationshipsinmicrobial communitystructurebetweentreatmentswereassessedby clus-teranalysis ofnon-weightedDGGE bandingpatterns,usingthe JaccardsimilaritycoefficientandtheUPGMAclusteringmethod (BioNumericssoftware,AppliedMaths).

3. Results 3.1. Plantgrowth

Alfalfaandwildryegrewwellinbothnon-amendedandPAH amendedsand.Attheendofthestudyallplantsappearedhealthy, withnovisiblechlorosisorothersignsofstress.Allwildryeand

controlsystemswerefreeofcontaminationattheendofthestudy. Mostalfalfasystemshowever,harbouredbacteriaatapproximately 104CFUml−1.Pantoeaagglomerans(SIM0.391)wasfoundinall

PAHsystemswhileMicrobacteriumesteraromaticum(SIM≥0.623) wasfoundintwonon-PAHsystems.Bothofthesebacteriahave previouslybeenrecoveredfrombeneaththeseedcoatof surface-sterilizedalfalfaseeds(MolineandKulik,1997).

3.2. PAHquantification

Traceamountsofphenanthreneweredetectedintheeluates ofseveralPAH-amendedmicrocosms.Theeluate/exudatesofone alfalfareplicate,onecontrolreplicate,andtwowildryereplicates contained2.9,1.9,8.0,and1.1␮gl−1phenanthrene,respectively.

3.3. Plantexudatequalificationandquantification

PAH-stressedwildryereleasedtwiceasmuchtotalorganic car-bon(TOC)asotherplants,atapproximately90mgTOCg−1root.The

quantityofidentifiedexudatesvariedgreatlybothbetweenand withintreatments(Fig.1).Individualorganicacidsrangedfrom less than 1␮gg−1 root tomorethan 300␮gg−1 root. Although

exudation patterns were highly variable, several trends were observed.Higherconcentrations oforganicacidswerefoundin PAH-stimulatedwildryetreatmentsthanin non-stimulated sys-tems,whiletheoppositewasfoundinthealfalfatreatments.Amino acid and phenolic compounds were detected at relatively low concentrationswithhighvariabilityinalltreatments(Fig.1).As withorganicacids,higherlevelsofbothgroupsweredetectedin wildrye-PAHversuswildryeexudates,withtheoppositeoccurring inthealfalfatreatments.

3.4. Impactofexudatesonhydrocarbonmineralizationpotential Distincttrendswereobservedinthemineralizationassays.Plant rootexudateshadarepressiveeffectonthemineralizationofall hydrocarbonscomparedtocontroleluates(Table1;supplementary Fig.S1).Lag times(definedasthetime to5% measurable min-eralization)ofexudateamendedtreatmentswereusuallyhigher, maximummineralizationrateswerelower,andcumulative min-eralizationlessthanthatofcontrol-eluateamendedtreatments.In generalhowever,exudatesderivedfromPAH-stimulatedwildrye plantsexhibitedfewerrepressiveeffectsthanotherexudatesonthe mineralizationofallassessedhydrocarbons.Mineralization param-etersinthesewildrye-PAHamendedmicrocosmswereusuallynot significantlydifferentfromthoseofcontrolmicrocosms(Table1). Aprimingeffectwasobservedincontrol-PAHcomparedtocontrol microcosms,thoughthesedifferencesinmineralization parame-terswereusuallynotstatisticallysignificant(Table1).

A strong relationship was found between lag time, rate, and cumulative mineralization for each individualhydrocarbon (p≤0.001forallcorrelations,datanotshown).Stepwisemultiple regressionsrevealedthatspecificrootexudatecomponents, includ-ingtheorganicacidsmalonate,acetateandfumarate,andtheamino acidalanine,weresignificantlyassociatedwiththese mineraliza-tionparameters(Table2).Inparticular,malonatewasassociated withdecreasedmineralization,bothwithincreasedlagtimesforall hydrocarbonsanddecreasedrateandextentofhexadecane miner-alization.

3.5. Impactofexudatesandhydrocarbonsonmicrobial communities

Quantitative PCR (Q-PCR)was performedonmicrobial com-munity DNA extracted from all treatment replicates. The 16S rRNAgeneandthreegenesinvolvedinhydrocarbondegradation,

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Fig.1.Organicacid,aminoacid,andphenoliccontentofalfalfaandAltaiwildrye(wildrye)rootexudates.Thetag-PAHindicatesthattheexudateswerecollectedunder phenanthrene/pyrenestimulatedconditions.Dataarepresentedasmeans(n=4)witherrorbarsrepresenting±1standarddeviation.Differencesaresignificantatp≤0.05 whereindicated.

catechol2,3 dioxygenase,naphthalene dioxygenase,and alkane monoxygenase,werequantifiedbycomparisontostandards. Stan-dard curves for the four genes had average linear correlation coefficientsof 0.999,0.987,0.995,and 0.993,respectively.Each primer set yielded DNA bands of the appropriate size for the assessedgenesequence,withnoobservableprimer–dimer forma-tion(datanotshown).Theabsenceofnon-specificamplification andprimer–dimerformationwasconfirmedbymeltcurveanalyses (datanotshown).

Theadditionofcontroleluatesorplantrootexudatesdidnot resultinsignificantchangesingeneralmicrobialcommunity struc-ture(Fig.2).DGGEbandingpatternsandtherelativeabundanceof

individualbandswerehighlysimilarinalltreatments.The abun-danceof16SrRNAgenewasalsoconsistentbetweenalltreatments (Table3),indicatingthatnoneoftheeluates/exudateshada signif-icantimpactonoverallbacterialpopulationabundance.

Although bacterial population densities did not statistically differbetweentreatments, thereweresignificantdifferences in copy numbers of both catabolic genes involved in PAH degra-dation (Table 3). The abundance of these catabolic genes was highlypositivelyinter-correlated(r>0.939,p<0.001).Both cate-chol2,3dioxygenase(C2,3O)andnaphthalenedioxygenase(nahAc) genecopy numberswereincreasedinthose microcosmswhich exhibitedincreasedPAHmineralization,andtheirabundancewas

Table1

Hydrocarbonmineralizationparametersofsoilmicrocosmsamendedwithplantbulkexudatesorcontrol(non-planted)eluatesandoneofthreehydrocarbons.

Mineralizationmicrocosm Lag***(days)† Maximumrate***(%14CO

2day−1) Cumulative***%mineralized

Phenanthrene

Control 5.3(1.0)cd 1.8(0.1)b 36.0(5.4)ab

Control-PAH‡ 4.1(0.5)d 2.3(0.2)a 44.5(2.0)a

Alfalfa 9.9(0.5)a 1.2(0.2)b 22.9(2.7)c

Alfalfa-PAH 8.5(2.0)ab 1.2(0.1)b 25.5(4.1)c

Wildrye 10.3(0.6)a 1.3(0.2)b 24.2(2.3)c

Wildrye-PAH 6.9(1.7)bc 1.5(0.3)ab 31.2(4.9)bc

Naphthalene

Control 1.6(0.3)b 11.6(4.9)ab 62.5(5.4)ab

Control-PAH 1.5(0.0)b 17.2(1.8)a 70.5(11.5)a

Alfalfa 4.9(0.9)a 5.6(1.2)b 51.8(4.1)b

Alfalfa-PAH 2.8(1.6)ab 7.9(3.5)b 58.8(4.5)ab

Wildrye 5.4(0.6)a 6.1(0.4)b 51.8(1.7)b

Wildrye-PAH 2.0(0.4)b 7.5(3.3)b 61.6(4.1)ab

Hexadecane

Control 3.3(1.2)bc 3.1(0.4)ab 48.8(2.1)a

Control-PAH 2.9(0.3)c 3.7(0.3)a 48.8(3.4)a

Alfalfa 5.6(1.3)ab 2.0(0.5)bcd 39.2(6.5)ab

Alfalfa-PAH 4.9(1.4)bc 2.2(0.6)bcd 41.1(4.8)ab

Wildrye 7.4(1.0)a 1.2(0.2)d 30.8(4.5)b

Wildrye-PAH 4.4(1.1)bc 2.5(0.7)bc 44.3(5.6)a

Dataarepresentedasmeans(n=4)with±1standarddeviationinparentheses.

Meansinasinglesub-columnfollowedbyadifferentletteraresignificantlydifferentat***p0.001.

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Table2

Linearmultipleregressionforhydrocarbondegradationparameters(mineralizationandgenecopynumber)asafunctionoftheconcentrationofspecificrootexudate components.

Hydrocarbontreatment

Constant±standardized␤-coefficients R2

Phenanthrene

Lag**† 9.298+0.521(malonate)0.495(alanine) 0.500

Rate* 1.238+0.458(fumarate) 0.153

Cumulative NS

16SrRNA‡*** 7.591+0.402(glycine)0.551(azelaic)+0.683(aconitate)−0.459(succinate) 0.755

C2,30*** 7.269−0.734(malonate)+0.343(alanine)+0.329(acetate) 0.750

nahAc*** 5.758−0.713(malonate)+0.474(alanine)+0.268(acetate) 0.785

Naphthalene

Lag** 3.340+0.819(malonate)−0.553(hydroxymalonate) 0.573

Rate NS

Cumulative* 58.781+0.433(acetate) 0.187

16SrRNA** 7.518−0.768(malonate)−0.404(glycine)+0.390(maleate) 0.708

C2,30** 7.225−0.615(malonate)+0.370(acetate) 0.514

nahAc** 6.077−0.659(malonate) 0.434

Hexadecane

Lag*** 4.777+0.586(malonate)+0.438(fumarate)−0.364(alanine) 0.730

Rate** 2.037−0.606(malonate)+0.381(alanine) 0.497

Cumulative** 47.109−0.601(malonate)−0.429(fumarate) 0.606

16SrRNA NS

alkB NS

Regressionequationsignificantat:*p≤0.05;**p≤0.01;***p≤0.001.

Lag,daysto5%mineralization;rate,maximumrateofmineralization(%14CO

2day−1);cumulative,cumulative%mineralized. ‡Genecopynumbersg−1drysoil:16SrRNA,eubacterial16SrRNA;C2,3O,catechol2,3dioxygenase;nahAc,naphthalenedioxygenase.

stronglycorrelatedwiththemineralizationparameters(Table4). In naphthalene mineralization microcosms, there were signifi-cantcorrelations betweenthese catabolic genes and 16S rRNA genes (r>0.663, p<0.001) and between 16S rRNA gene abun-dance and mineralization parameters (Table 4). However, no comparablecorrelation wasobserved inmicrobial communities from phenanthrene mineralization microcosms. As seen with mineralization parameters, specific exudate components were significantly associated with gene copy numbers (Table 2). In particular, malonate was associated with reduced C2,3O and nahAccopynumbersinbothPAHtreatments,whilealanineand acetate were associated withincreased copy numbersof these genes.

Norelationshipwasfoundbetweenexudatecompositionand thetotalcopynumberofalkanehydroxylase(alkB)genes,which werecomparablebetweenalltreatmentsattheendofthestudyand averagedapproximately106genecopiespergramofsoil(Table3).

AlthoughnosignificantdifferencewasobservedinalkBabundance, ingeneraltotalalkBcopynumberswerepositivelycorrelatedwith 16SrRNAgenenumbers(r=0.779,p<0.001).

Fig.2.DendrogramanalysisoffinalDGGEbandingpatternsfromallexudateand eluateamendedmineralization(14C-labelednaphthalene,phenanthrene,and

n-hexadecane)microcosms.TreatmentsincludedalfalfaandAltaiwildrye(wildrye) exudatesandnon-plantedcontroleluates.Thetag-PAHindicatesthattheoriginal exudates/eluateswerecollectedunderphenanthrene/pyrene stimulated condi-tions.

Table3

Totalgenecopynumbersofsoilmineralizationmicrocosmsamendedwithplant bulkexudatesorcontrol(non-planted)eluates.

Mineralizationmicrocosms Genecopynumbersg−1drysoil(logscale)

16SrRNA C2,3O***† nahAc***

Phenanthrene

Control 7.57(0.11)a 7.78(0.14)a 6.59(0.12)a

Control-PAH‡ 7.52(0.19)a 7.84(0.17)a 6.63(0.22)a

Alfalfa 7.58(0.09)a 7.03(0.26)c 5.63(0.26)bc

Alfalfa-PAH 7.54(0.09)a 7.29(0.45)abc 5.84(0.50)bc

Wildrye 7.65(0.04)a 7.09(0.09)bc 5.59(0.16)c

Wildrye-PAH 7.69(0.07)a 7.64(0.32)ab 6.29(0.03)ab

Naphthalene

Control 7.37(0.20)a 7.75(0.32)a 6.57(0.39)ab

Control-PAH 7.42(0.10)a 7.88(0.11)a 6.74(0.16)a

Alfalfa 7.27(0.23)a 6.92(0.33)b 5.41(0.41)cd

Alfalfa-PAH 7.37(0.07)a 7.26(0.35)ab 5.91(0.42)bcd

Wildrye 7.22(0.10)a 6.80(0.04)b 5.30(0.06)d

Wildrye-PAH 7.38(0.15)a 7.27(0.46)ab 6.11(0.38)abc

Mineralizationmicrocosms Genecopynumbersg−1drysoil(logscale)

16SrRNA alkB

Hexadecane

Control 7.60(0.03)a 5.95(0.05)a

Control-PAH 7.65(0.20)a 6.06(0.46)a

Alfalfa 7.69(0.17)a 5.93(0.24)a

Alfalfa-PAH 7.47(0.13)a 5.63(0.33)a

Wildrye 7.63(0.13)a 5.87(0.20)a

Wildrye-PAH 7.57(0.18)a 6.07(0.20)a

16SrRNA,eubacterial16SrRNA;C2,3O,catechol2,3dioxygenase;nahAc, naphtha-lenedioxygenase;Dataarepresentedasmeans(n=4)with±1standarddeviation inparentheses.

Meansinasinglesub-columnfollowedbyadifferentletteraresignificantly

differentat***p≤0.001.

Thetag-PAHindicatesthattheoriginalplantexudates/controleluateswere

collectedunderphenanthrene/pyrenestimulatedconditions.

4. Discussion

Phytoremediation researchers often find that specific plants may promote degradation under oneset of conditions but not under another. To date, we know relatively little about how

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Table4

Spearman’srankcorrelationcoefficients(n=24)forhydrocarbonmineralization parametersandmicrobialgeneabundanceineluateandexudateamendedsoil microcosms.

Hydrocarbonmicrocosm Genecopynumbers†

16SrRNA C2,30 nahAc Phenanthrene Lag‡ 0.002 −0.810*** −0.866*** Rate 0.073 0.619*** 0.711*** Cumulative −0.018 0.759*** 0.809*** Naphthalene Lag −0.525** −0.916*** −0.952*** Rate 0.349 0.671*** 0.653*** Cumulative 0.457* 0.806*** 0.839***

Hydrocarbonmicrocosm Genecopynumbers†

16SrRNA alkB Hexadecane Lag −0.118 −0.317 Rate 0.135 0.356 Cumulative 0.123 0.335 Significantat:*p≤0.05,**p≤0.01,***p≤0.001.

Genecopynumbersg−1drysoil:16SrRNA,eubacterial16SrRNA;C2,3O,catechol

2,3dioxygenase;nahAc,naphthalenedioxygenase.

Mineralizationparameters:Lag,daysto5%mineralization;rate,maximumrate

ofmineralization(%14CO

2day−1);cumulative,cumulative%mineralized.

differentplantspeciesimpactthestructureand functionofsoil microbialcommunities,andhowtheseimpactsaffectthe degra-dationpotentialofthesystemasawhole.Weaddressedthisissue byexamininghowdifferencesinexudationpatternsimpactthe degradationpotentialofmicrobial communitiesindigenoustoa weatheredhydrocarboncontaminatedsoil.Rootexudatescollected fromAltaiwildrye and alfalfainitially repressedmineralization ofall assessed hydrocarbons,resultingin increased lagperiods anddecreased overall mineralizationratescompared toeluates derivedfrom controltreatments (Table 1).In general,exudates collectedfromwildryegrownunderhydrocarbonstresswerethe leastrepressive,whilethosecollectedfromnon-stressedwildrye hadthegreatestrepressiveeffect.Otherstudieshavereporteda comparableinhibitoryeffectforrootexudates.L.perenneexudates havebeenfoundtoinduceadistance-dependentrepressiveeffect onPAHdegradation(Corgiéetal.,2004,2006).Rentzetal.(2004)

foundthat both realexudates fromawide range ofplants and single-compoundpotentialrootexudatesubstratesrepressedthe phenanthrenedegradingactivityofaP.putidastrain.Althoughin ourstudycumulativemineralizationofbothhexadecaneand naph-thaleneinmosttreatmentsreachedparityafter28days(Table1), theinitialandvariablerepressive effectsoffer insightsintothe underlying mechanisms of plant-microbe interactions in these systems.

Previousstudieshavelinkedincreasedhydrocarbon degrada-tion in exudate amended systems to non-specific increases in bacterialpopulations(Da Silvaetal.,2006;Kamathetal.,2004; Rentzetal.,2004;Tuomietal.,2004).Inthisstudyhowever,DGGE analysisrevealednodetectabledifferenceinthetypeorrelative abundanceofthedominantbacterialspeciesfoundinthe differ-enttreatments(Fig.2),and16SrRNAgenequantificationshowed nostatisticallysignificantshiftsinmicrobialpopulationabundance (Table3).Whileshiftsinnumericallyminoryetactivemicrobial species(Corgiéet al.,2006)may haveoccurred, theywere not detected.Previousresearchonthesoilusedinthisstudy(Phillips etal., 2006,2008)hasshown that over timeplants do exert a selectiveinfluenceonbothrhizospherecommunitystructureand populationdensity.However,theindigenousmicrobial commu-nityincludesmanyspeciesadaptedtohydrocarboncontaminated environments(Phillipsetal.,2006andsupplementaryFig.S2)anda

greaternutrientinputmayberequiredbeforechangestothe over-allcommunitystructureoccur(Griffithsetal.,1999).Theadded levelsofexudatesweresufficienthowever,toelicitchangestothe metaboliccapacityofthesoilmicrobialcommunities.Ourresults showthatthesechangesindegradationpotentialwereassociated withspecificdifferencesinexudationpatterns.

Exudatepatternsareknowntobeinfluencedbyamyriadof factors,includingplantspecies(Ciéslínskietal.,1998;Lesuffleur etal.,2007),soilcomposition(Ciéslínskietal.,1998), environmen-talparameters(Henryetal.,2007),andthepresenceofxenobiotics (Bais etal.,2004).Similarly,wefounddifferencesintheoverall exudationpatternsbothbetweenplantspeciesandbetweenPAH stressedandnon-stressedplantsofthesamespecies(Fig.1).We alsofoundhowever, that thecomposition ofexudates released by replicatesof a given treatment washighly variable(Fig.1). Otherstudieshavenotedthatindividualplantsofthesamespecies (Lesuffleuretal.,2007)andthesamevariety(Ciéslínskietal.,1998; Micallefetal.,2009)mayhavesubstantiallydifferentexudation patternswhengrownunderthesameconditions.Thisintra-species exudatevariationhasbeenshowntohaveasignificantimpacton thecompositionofrhizospheremicrobialcommunities(Micallef etal.,2009)andonbacterialgeneexpression(Marketal.,2005). Withrespecttohydrocarbondegradation,suchexudatevariation mayhaveasignificantandspecificimpactonthedegradative fit-nessofthemicrobialcommunity.Inthecurrentstudy,increased concentrationsoftheorganicacidmalonateinexudateswere asso-ciatedwithdecreaseddegradationpotential(Table2).Incontrast, increasedconcentrationsoftheorganicacidacetateandtheamino acidalaninewereingeneralassociatedwithincreaseddegradation potential.

Exudatesmayspecificallyinfluencethedegradativefitnessof anexisting microbial communityviaa number ofmechanisms, includingshiftsincatabolicgeneexpression,generalmetabolic sta-tus,and catabolicgenetransfer (Da Silvaetal.,2006;vanElsas etal.,2003).Bothcataboliterepressionandinductionare partic-ularly importantphenomena forenvironmentalbacteria, which mustquicklyadapttorapidlychangingcarbonsourceswithoutloss ofcompetitivefitness.Althoughgeneexpressionwasnot specif-ically assessedin thecurrent study,ifcatabolic repression was thedominant factor we would expect tosee minimal changes in catabolic gene copy number, yet still see decreased hydro-carbonmineralization.Thisoccurredinhexadecanemicrocosms, wherealkanehydroxylasegenecopynumberswereequivalentin allmicrocosms(Table3)andnotcorrelatedwithmineralization parameters(Table4).Previousresearchhasshownthatrichcarbon sourcesinhibitalkanedegradationbycataboliterepressionofthe alkBoperoninaPseudomonasspecies(Yusteetal.,1998).Similarly, numerousorganicacids,includingfumarate,havebeenshownto repressPalkBpromoterexpressioninBurkholderiacepacia(Marín etal.,2001).Inourstudy,thedecreasedalkanemineralization asso-ciatedwiththeorganicacidsfumarateandmalonate(Table2)may beindicativeofcataboliterepression.

Similarrepressionmayhaveoccurredintheexudate-amended PAHmicrocosms,asexudatesfromawidevarietyofplantshave been shown to repress nahG, a gene involved in naphthalene dioxygenasetranscription(Kamathetal.,2004).However,wealso observed significantchanges in thecopy number ofboth cate-chol2,3dioxygenaseandnaphthalenedioxygenasegenes(Table3), andtheabundanceofthesegeneswashighlycorrelatedwithPAH mineralizationparameters(Table4).Bacterialgenecopynumber withinasoilmaybealteredbyseveralmechanisms,includingshifts ingeneralmicrobialpopulations,DNAreplicationpreceding bac-terialdivision,andplasmidtransfer.Innaphthalenemicrocosms, increasesin generalmicrobialpopulations mayhaveaccounted forsomeofthechangeindegradationpotential,as16SrRNAand catabolic gene copy numbers were highly correlated (r>0.600,

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p<0.001).Onefactorwhichmayhaveimpactedmicrobial abun-danceinthesemicrocosmsistheorganicacidmalonate,whichwas negativelyassociatedwith16SrRNAgenecopynumbers(Table2). Malonateisacellularrespirationinhibitorthatactsby competi-tivelyinhibitingtheactivesiteofsuccinatedehydrogenaseinthe citricacidcycle.Thusmalonatemayhaveimpactedthemetabolic statusofthesoilmicrobialcommunitiessuchthatreplicationwas inhibited.Ifdepressedmicrobialpopulationswerethedominant factorimpactingcatabolicgenecopynumberhowever,wewould haveexpectedtoseesignificantpopulationdifferencesbetween treatments.Aspreviouslydiscussed,thisdidnotoccur,norwas thereanycorrelationbetweenpopulationdensitiesandcatabolic genesinphenanthrenemicrocosms.

ManybacterialgenesinvolvedinPAHdegradationarecarried onconjugativeplasmids(Nojirietal.,2004).Thecatabolicgenes assessedinthecurrentstudyarecommonlyfoundonthesame plasmid,with C2,3Ooften present in multiplecopies (Liet al., 2006;Sentchiloetal.,2000).Whilegenelocationwasnotdirectly assessed,theveryhighcorrelationbetweennahAcandC2,3Ocopy numbers(r>0.939,p<0.001),independentof16SrRNAgenecopy number,suggeststhattheywerelocatedonthesameplasmid.Ifso, thenplasmidtransfermayhavebeenasignificantfactor influenc-ingthechangesindegradationpotential.Horizontalgenetransfer (HGT)isknowntooccurwithhighfrequencyintherhizosphere (Jussila et al.,2007; vanElsaset al.,1988,2003), stimulatedin partbyincreasedexudation(Mølbaketal.,2007).In ourstudy, nahAcandC2,3Ocopynumberswerepositivelyassociatedwiththe organicacidacetateandtheaminoacidalanine(Table2),two com-poundswhichhavebeenshowntodirectlyincreaseHGTevents (NielsenandvanElsas,2001).Wealsoobservedanegative asso-ciationbetweenthesegenes andtheorganicacidmalonate.As previouslydiscussed,malonatemayrepressthemetabolicstatus ofmicrobialcommunities.Severalstudiessuggestthatconjugative transferofplasmidsisassociatedwithincreasedcellularmetabolic activity(Lilleyet al., 1994;Smets et al.,1993; vanElsas et al., 1988),thereforeincreasedconcentrationsofmalonatewithinthe exudatescouldplayaroleindecreasingcatabolicgenetransfer. Asplasmidcopynumberwasnotdirectlyassessedinourstudy, nodefinitivelinkmaybemadewiththedecreaseddegradative capacity of some exudate-amended microcosms. However, this mechanismcertainlywarrantsfurtherinvestigationin phytoreme-diationstudies.

Ourfindingsprovideinsightintowhyspecificplantspeciesmay promotedegradationunderonesetofcircumstances,yetinhibit it underanother.Comparable differencesin exudationpatterns may have contributed to the heritable differences in crude oil degradationfacilitated bygenotypic clonesof alfalfainanother study (Schwab et al., 2006; vanElsas et al.,2003).Our results suggest that the patterns of exudation are the driving factors behindthe degradationtraits. Recentresearchwith Arabidopsis thalianaaccessions(Micallefetal.,2009)suggeststhatsome exu-dation traits are alsostably heritable. If so,then plant-specific selection and breeding for specificpatterns of exudation could greatlyfurthertheeffectivenessofphytoremediationtreatments, andremovesomeofthecurrentuncertaintyassociatedwithplant selection.

Acknowledgements

This research was supportedby a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and by an NSERC postgraduate scholarship to L.A.P. The authors would like to thank Talisman Energy Inc. for site access.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.apsoil.2011.10.009.

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Figure

Fig. 1. Organic acid, amino acid, and phenolic content of alfalfa and Altai wildrye (wildrye) root exudates
Fig. 2. Dendrogram analysis of final DGGE banding patterns from all exudate and eluate amended mineralization ( 14 C- labeled naphthalene, phenanthrene, and  n-hexadecane) microcosms

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