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ORIGINALPAPERAnalysisofintactladderanephospholipids,originatingfromviableanammoxbacteria,usingRP-LC-ESI-MSIngelaLanekoff&RogerKarlssonReceived:4April2010/Revised:3June2010/Accepted:8June2010/Publishedonline:17June2010#TheAuthor(s)2010.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractSincethediscoveryoftheanaerobicammoniumoxidizing(anammox)bacteria,manyattemptshavebeenmadeinordertoidentifytheseenvironmentallyimportantbacteriainnaturalenvironments.
Anammoxbacteriacontainauniqueclassoflipids,calledladderanelipidsandherewepresentanovelmethodtodetectviableanammoxbacteriainsedimentsandwastewatertreatmentplantsbasedontheuseofaladderanelipidbiomarker.
Intactladderanephosphatidylcholine(PC)lipidsareanalyzedusingreversed-phaseliquidchromatography–electrosprayionization–massspectrometry.
Followingextractionfromthecomplexsedimentmatrix,reversed-phaseLCisusedtoseparateladderanePClipidsbasedontheirtailgrouphydrophobicityaswellastheiretheroresterlinktotheglycerolbackboneinthesn-2position.
WeinvestigatethepresenceofintactladderanelipidsinnaturalsedimentsdisplayinganammoxactivityandillustratetheuseofaspecificintactmembraneformingPClipidasabiomarkerforviableanammoxbacterialcells.
Thepresentedmethodcanbeusedtoelucidatethewhereaboutsofviableanammoxbacteria,subsequentlyenablinganestimationofanammoxactivity.
Thiswillgreatlyincreasetheknowledgeofanammoxbacteriaandtheirimportanceintheglobalnitrogencycle.
KeywordsLadderanelipids.
LCMS.
Biomarker.
Anammoxbacteria.
NitrogencycleIntroductionItisestimatedthat25%to50%ofthetotalglobalmarinenitrogengas(N2)productionproceedsviatheanaerobicammoniumoxidation(anammox)reaction[1,2].
Locally,theanammoxcontributionmightbeevenhigher[2].
Bacteriaperforminganammoxwerediscoveredinthe1990sinawastewaterreactorandhavesincebeenfoundinamultitudeofenvironments[3–7].
Anammoxbacteriaconvertbiologi-callyavailablenitrogenspecieslikeammonia,nitrateandnitriteintoN2,whichisreturnedtotheatmosphere.
Inthissensetheyarecrucialforsustainingthenitrogenbalanceinourenvironment[8]andgreateffortshavebeenmadeinthesearchforevidenceoftheirpresenceandactivityinnaturalenvironments.
15N-tracerstudieshavebeenusedtodetermineratesofanammoxbymeasuringtheformationof29N2followingincubationof15N-labelednitrate[9,10].
Togetherwithpredictionsfromnutrientandoxygenprofiles,thismethodhasbeenveryusefulinelucidatingwheretheanammoxreactiontakesplace[11–16].
However,studiesusing15N-labelscannotalonebeusedtostatewhethertheanammoxbacteriaareinvolvedornotsincetheanammoxreactionalsocantakeplacethroughalternativeroutes[17].
Using16SrRNAgeneanalysisandmoleculartechniquessuchasfluorescenceinsituhybridization(FISH)andquantitativepolymerasechainreaction(qPCR),anammoxbacteriahavebeenfoundbothinsedimentsandwaterpillars[7,11,13,18,19].
Furthermore,FISHhasbeenasuccessfulmethodologynotonlyfordeterminingthepresencebutalsothediversityofanammoxbacteriaspecies.
However,FISHisamethodthatreliesonfluorescencemeasurementsthusmakingquantitativemeas-urementsproblematicsincemineralandorganicparticlestendtoquenchthefluorescencesignalwhensedimentI.
Lanekoff:R.
Karlsson(*)DepartmentofChemistry,UniversityofGothenburg,Kemivagen10,41296Gothenburg,Swedene-mail:rogerk@chem.
gu.
seAnalBioanalChem(2010)397:3543–3551DOI10.
1007/s00216-010-3913-3samplesareanalyzed.
ThequantitativedeterminationofanammoxbacterialabundancethroughqPCRisalsoassociatedwithissues.
ObservationshaveshownthatbothRNAandDNAcanberelativelystableaftercelllysis[20,21]resultinginenhancedsignalsduetofossilRNAandDNA[22,23].
Hence,thistechniquemightnotreflecttheactualamountofviableanammoxbacteria.
Ourapproachistodeterminethepresenceofanammoxbacteriabyusingaspecificintactmembraneformingphosphatidylcholinelipidfromthefamilyofso-calledladderanelipidsasabiomarkerforthesebacteriainsediment[2,3,6,7,13,24].
Ofallbacterialcellsexaminedtodate,ladderanelipidshaveonlybeenfoundinanammoxbacteria[25].
Ladderanelipidscontainlinearlyconcatenatedcyclo-butanestructuresattheendofthehydrocarbontails,formingaladder-likestructure(Fig.
1).
Thesehighlystrainedstructuresarefoundasphosphatidylcholine(PC),phospha-tidylethanolamine(PE),andphosphatidylglycerol(PG)lipidsaswellasfattyacids(FA)andlysophospholipids[26–28].
Theintactladderanelipidsexistpredominantlywithanetherlinkagetothesn-2positionofaglycerolbackbone,viaanalkylchain,whilethesn-1positionislinkedviaeitheranetheroresterbond[26,29].
Capillarygaschromatography(GC)massspectrometry(MS)hasbeenusedtoanalyzeladderanephospholipidsasladderaneFAMEs(fattyacidmethylesters)afterderivatization[30].
However,thechromatogramsobtainedsufferedfromverybroadpeaks,containingseveralsmallpeakswithalmostidenticalmassspectra.
Thissuggestedthatthestrainedcyclobutanestruc-tureoftheladderaneFAwasthermallyunstableanddegradedduringGCanalysis[30,31].
AnimprovedmethodfortheanalysisofladderaneFAsbasedonnormal-phasehigh-performanceliquidchromatography(NP-HPLC)–atmosphericpressurechemicalionizationtandemmassspectrometry(APCI-MS/MS)waslaterdeveloped[32],aswellasamethodfortheanalysisofaladderanemonoetherPClysolipid[33].
However,asthesetechniquesrelyontheanalysisofFAsandlysophospholipidstheyhavenotprovidedadirectlinktotheamountoflivinganammoxbacteria.
In2009,Jaeschkeetal.
[33]showedthattheC20-[3]-ladderanemonoetherPC,alysophospholipidretain-inganintactPCheadgroupbuthavingonlyonealkylchain,wasabetterbiomarkerforviableanammoxbacteriathantheladderaneFAs.
However,lysophospholipidsaswellasFAsareonlypresentinverylowamountsinlivingcellssincethemajorityofthemembraneisconstitutedofintactlipidscontainingtwonon-polarhydrocarbontailgroupslinkedtoapolarheadgroup.
Duringdegradation,theintactmembraneformingphospholipidsreadilylosetheirheadgroupbutalsooneofthealkylchains,especiallyifitislinkedtotheglycerolbackboneviaanesterbond,resultinginfattyacidsandlysophospholipidsasdegradationproducts.
Inseveralarticles,ithasbeenshownthatintactmembraneformingphospholipidsisareliabletoolfordetectingviablebacteriainsediments[34–40],duetotherapiddecompositionofphospholipidsaftercelldeath.
EarlierworkperformedbyBoumannetal.
[26]hasdemonstratedtheseparationanddetectionofintactladderanelipidsextractedfrombacterialcellculturesandwastewatertreatmentplants,usingnormal-phaseLCandMSdetection.
ThemethodseparatesintactphospholipidsintoPC,PEandPGspecies,butitdoesnotallowforseparationofthelipidsbasedontheirtailgroupsorbondtotheglycerolbackbone.
Earlierstudiesindicatethattherearemanydifferenttypesofladderanelipidspecies,duetoawiderangeofhydrocarbonchainsbondedtotheglycerolbackboneeitherviaanetheroresterbondinthesn-1position.
Whenaspecificintactlipidistobeusedasabiomarkerforviableanammoxbacteria,itisofutmostimportancethatthemethodofuseseparatesthelipidsintoindividualladderanelipidspecies.
Usingnormal-phaseLCseparation,however,itisimpossibletodistinguishbetweenindividualspecieshavingdifferenttailgroupsbutsharingthesameheadgroup.
ThemethodbyBoumannetal.
[26]hasnotbeenusedforanalyzingintactmembraneformingladderanelipidsfromanammoxbacteriainsedimentsamples.
Theanalysisoflipidsoriginatingfromsedimentsiscomplicatedbythepresenceofmanydifferentphospholipidspeciesaswellasamuchhigheramountofcontaminatingorganicmaterialascomparedtowastewatertreatmentsamples.
ThisputshighdemandsontheLCmethodtoseparateoutspecificindividualphospholipidstobeusedasbiomarkersinsedimentsamples.
Here,wehavedevelopedamethodusingaspecificintactmembraneformingladderanePClipid(m/z816,Fig.
1IV)asabiomarkerforviableanammoxbacterialcellsinnaturalsedimentsdisplayinganammoxactivity.
ThemethodenablesPClipidstobeseparatedbasedontheirtailgrouphydrophobicityaswellastheirattachmenttotheglycerolbackbone,whichcanbeeitherthroughanether–etherorester–etherbond.
ThisenablesanalysisofspecificladderanePClipidsinanammoxbacteriafromnaturaloceansediments.
HereweshowthatthepresenceofthespecificladderanePClipidatdifferentdepthsinasedimentcorecorrelateswellwithestablishednutrientandoxygenprofilesinsedimentsaswellasprofilesofanammoxbacterialabundanceestimatedusingmoleculartechniquesincludingFISHandqPCR[33].
ExperimentalsectionChemicalsPhospholipidreferencesamples1,2-didecanoyl-sn-glycero-3-phosphocholine(10PC),1,2-dipalmitoyl-sn-glycero-3-phospho-choline(16PC),1,2-diphytanoyl-sn-glycero-3-phosphocholine(4Me16PC),1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine3544I.
Lanekoff,R.
Karlsson(DE16PC),and1,2-distearoyl-D70-sn-glycero-3-phosphocho-line(D7018PC)werefromAvantiPolarLipids.
ForLCMSmeasurementsLCMSgrademethanol(MeOH)and2-propanol(isopropylalcohol,IPA)werefromRiedel-deHan,formicacidfromFlukaandammoniafromMerck.
Sodium(meta)periodate,chloroform,n-hexane,ammoniumbicarbonate,anddiethyletherwerefromSigma-Aldrich.
Dichloromethane(DCM)anddichlorodimethylsilanewerefromFlukaandNH2solid-phaseextraction(SPE)columns(500mg,3mL)fromStrata.
MaterialsFormethoddevelopment,anammoxbacteriagrowinginbiofilmsonplasticbiofilmcarriers(AnoxkaldnesAB,Lund,Sweden)wereobtainedfromHimmerfjrdsverketswastewatertreatmentplantinStockholm,Sweden.
LadderanePClipidsextractedfromanammoxbacteriabiofilmswereusedasreferencestodeterminetheLCretentiontimeduringsedimentanalysis.
Sedimentcoresamples(dividedintodepths0–3,3–6,6–9,and9–12cm)wereobtainedfromGullmarsfjordenoutsideLysekilonthewestcoastofSwedenandstoredat4°Cforafewdaysuntilanalysiswasperformed.
BiofilmextractionToextractladderanelipids,threedifferentprotocolswereinvestigated.
ThefirstprotocolisdescribedbyZinkandMangelsdorf[41]whereMeOH:DCM:10mMammoniumacetate(AmAc)isusedintheratioof2:1:0.
8.
ThesecondfollowstheFolchprocedureusingamixtureofchloroform:MeOH(2:1)[42]andthethird,fromHaraetal.
[43],usesamixturebasedonn-hexane:isopropanol(3:2).
Priortoextractionthebiofilmsampleswerecentrifugedandcarefullyweighedtoensurethatacomparisonbetweenthedifferentlytreatedbiofilmsamplescouldbemade.
Inallmethodsweusedultrasonication,asdescribedbelow,andextractedthelipidsthreetimeswithacentrifugationstepinbetween.
Supernatantswerecombinedandcollectedinseparateseparationfunnels.
Allbiofilmsamplesweresubjectedtoliquid–liquidextraction(LLE),asdescribedintheoriginalpapers[41–43],andSPE[44].
Thephospholipidfractionswerefullyevaporatedinasilanizedtesttubeunderastreamofnitrogengas,re-dissolvedin200μLmobilephase,andstoredforashortperiodoftimeundernitrogenatmosphereat80°CpriortoLCMSanalysis.
TheFolch[42]andHara[43]procedureswerelessefficientthantheZinkprocedure[41]andwerethusnotusedforthesedimentextractions.
SedimentincubationandextractionAsedimentcorewasobtainedfromGullmarsfjorden(110mbelowseasurface)anddividedintofour3-cm-thicksamplesbetween0and12cmofdepth.
Anaqueoussolutioncontaining5%sodium(meta)periodate(150ml)wasaddedto150gofwetsediment.
Thesedimentslurrywassonicatedfor2min,usinganultrasonicprocessor(SonicsVibracell,Model501)at70%,witha7-spulseanda5-srest.
Theslurrywasincubatedat4°Cforaperiodof1-weektobreakclustersofbacteriaandtolysecells[45,46].
Afterincubation,thesamplewastransferredtotwo250-mLcentrifugeflasks(TeflonFEP,Nalgene)andcentrifugedat3,220gfor15minusinganEppendorfCentrifuge5810R.
Thesupernatantswerecombinedandcollectedinaseparationfunnel.
MeOH(75ml)wasaddedtoeachcentrifugeflaskandthesamplewassonicatedandcentrifugedusingthesameconditionsasabove.
TheMeOHsupernatantswerealsocombinedandcollectedinaseparateseparationfunnel.
Tofurtherextractlipids,theZinkprocedure[41]wasused.
Thesamplewasagaintreatedwithultrasonicationasdescribedabovebutwith75mLofMeOH:DCM:AmAc(2:1:0.
8)threetimesperflaskwithcentrifugationstepsinbetween[41].
Thesupernatantswerecombinedandcollectedinseparateseparationfunnels.
Fig.
1SchematicillustrationsofladderanePClipidsinvestigatedinthisstudy.
(I)Dietherlipidatm/z800,(II)dietherlipidatm/z802,(III)ether/esterlipidatm/z814,(IV)ether/esterlipidatm/z816.
Notethesequentiallylinkedcyclobutane/cyclohexanestructuresattheendofthehydrocarbontailsIntactladderanephospholipidsfromviableanammoxbacteria3545SedimentsamplecleanupprocedurePriortoLLE,DCM,andAmAcwereaddedtoalltheseparationfunnelsuntiltheratiosofthesolventswereMeOH:DCM:AmAc1:1:0.
9.
Afterphaseseparation,theDCMphaseswerecollectedinaroundbottomflask.
ThewaterphasewassubjectedtotwomoreextractionswhereDCM:MeOHwasaddedinaratioof2:1[41].
Again,theDCMphaseswerecollectedandcombinedwiththepreviousextracts.
TheDCMintheroundbottomflaskwasreducedusingarotavaporatorfromBüchi,andfullyevaporatedinasilanizedtesttubeunderaflowofnitrogengas.
Thesamplewasre-dissolvedin12mLof2:1n-hexane:chloroformandstoredundernitrogenatmo-sphereat80°CuntilsubjectedtofurthercleanupbySPE.
FourSPEcolumns(StrataNH2(55μm,70A)500mg,3mL)persamplewereconnectedtoavacuumbox(Resprepvacuummanifoldwith12ports)andactivatedwith12mLMeOH.
Thecolumnswerecondi-tionedwith12mLofn-hexanepriortoloadingthesample.
Awashwith12mLchloroform:IPA(2:1)elutedcholesterylesters,triglycerides,cholesterols,diglycerides,andmonoglycerides.
FAswereelutedwith2%aceticacidindiethylether.
Finally,thephospholipidswereelutedwith12mLofMeOH,combinedandfullyevaporatedinasilanizedtesttubeunderastreamofnitrogengas[44].
Thecleansamplewasre-dissolvedin200μLmobilephase,andstoredundernitrogenatmosphereforashortperiodoftimeat80°CpriortoLCMSanalysis.
LiquidchromatographyandmassspectrometrySeparationwasachievedusingaGeminiC18column(50*2mm,3μmparticlediameter,hybridsilicagelcolumnforreversed-phaseHPLC)withaGeminiC18guardcolumn(4*2mm),fromPhenomenex.
Thetemper-atureofthecolumnwaskeptconstantat30°CusingacolumnovenLCO102fromEcom.
ThemobilephasesinthegradientconsistedofA;4%water,0.
1%formicacid,and0.
1%ammonia(25%)inMeOHandB;isopropanol(IPA)containing0.
1%formicacidand0.
1%ammonia(25%).
Theinitialcompositionwithaflowof0.
4mL/minwas100%Awhichwasloweredto70%Abetween10and20minoftheseparation.
At21min,theflowwasloweredto0.
2mL/minand100%Bwasusedtowashthecolumnfor10minbeforereturningtotheinitialconditions.
Totalanalysistime,includingwashingstep,foreachsamplewas40min.
Theanalyteswereelutedwithin20min.
Analysisofbiofilmsamplesduringmethoddevelopmentwasperformedusinga600SControllerand626PumpfromWatersanda234autosamplerfromGilsoncoupledtoaMicromassQuattroLC,triplequadrupole,withelectrosprayionization(ESI).
MS/MSanalysisenabledparentalionscanningofthePCheadgroup,atm/z184,todetectPClipids.
Argonwasusedascollisiongas,thepotentialoverthecapillarywas4.
0kVandtheconevoltagewas90V.
Sourceblocktemperaturewasheldat150°Canddesolvationtemperatureat300°C.
SedimentsampleanalysiswasperformedusingaPerkin-ElmerSeries200autosamplerandLCpumpcoupledtoaMicromassQToF,quadrupoletime-of-flightwithESIscanningbetweenm/z100and900.
AsaretentiontimereferenceforladderanePClipidsabiofilmsampleextractwasconsecutivelyanalyzedusingtheQToFset-up.
ResultsanddiscussionLadderanePClipidextractionandsamplecleanupAnammoxbacteriahavebeenshowntoformbiofilmswhenculturedinwastewaterreactorsandmostlikelygrowinclustersinoceansedimentsaswell.
Biofilmandclustersaremostlyformedviaaprotein-polysaccharidematrix[47,48]andbiofilmformationmakesitmoredifficulttolysecellsinordertoextractlipidsfromthecellmembranes.
Forthisstudy,severalchemicalandphysicaltreatments,includingboiling,hydrogenperoxideorhypochloratetreatments,mechanicalhomogenizationandsonicationwereattemptedinordertoseparateandlysecellsinbiofilms(datanotshown).
Thesuccessofthesetreatmentsweremonitoredusingfluorescencemicroscopybystainingthecellswith4′,6-diamidino-2-phenylindole.
Furthermore,theextractedladderanePClipidswereanalyzedusingLCMSpostextraction.
Thebestresults,showedbylackofladderanelipidsinsamplesextractedasecondtime,wereachievedwhenanammoxbacteriainbiofilmswereincubatedwitha5%solutionofperiodatepriortoprobesonication,asdescribedintheexperimentalsection[45,46].
NosignificantdifferenceintheladderanePClipidcompositionorquantitydependingontheperiodateincubationtime(2daysversus7days)wereobserved,showingthatnodecompositionoftheladderanePClipidoccurredduringincubation(datanotshown).
InoceansedimentsfromGullmarsfjorden,approximately3%ofallbacteriahavebeenfoundtobeanammoxbacteria[4].
Thelowcellabundanceandthecomplexityofthesedimentsamplematrixputshighdemandsontheextractionmethod.
TooptimizetheyieldofladderanePClipidsintheextractionstep,wecomparedthreeextractionprotocols,usingcarefullyweighedtriplesamplesofanammoxbacteriainbiofilms.
Thesolventmixturesinthecomparedmethodswere;chloroform:MeOH[42],n-hexane:isopropanol[43]andMeOH:DCM:AmAc[41].
Afterextractionandsamplecleanup,allsampleswereanalyzedusingthedescribedLCMSmethod.
InFig.
2,theareaoftheintegratedpeaksof3546I.
Lanekoff,R.
KarlssonthreechosenladderanePClipids,foundatm/z802,814,and816,arecompared.
TheresultshowsthatthehighestyieldofladderanePClipidsisobtainedwhenthemixtureofMeOH:DCM:AmAc[41]isused.
LCmethodforladderanelipidsThedevelopedreversed-phaseLCmethodseparatesPClipidsbasedonthetailgrouphydrophobicity,makingtheladderanelipidselutelast.
Figure3bshowsthechromato-gramgeneratedduringseparationofthefivenon-ladderanePClipidstandardsshowninFig.
3a.
ThisconfirmsthattheLCmethodseparatesthePClipidsbasedonthelengthandhydrophobicityofthehydrocarbontailgroup.
Furthermore,themethodseparatesthePClipidsbasedonthelinktotheglycerolbond,wherethemorepolarether–ester-linkedlipidselutebeforetheether–etherlinkedlipidswiththesametailgroups.
Thisfeatureoftheseparationisespeciallyimportantwhenaspecificladderanelipidisbeingusedasabiomarker.
TheconcentrationsofalllipidsinFig.
3barethesame,buttheresponsevariesconsiderably.
Themostobviousdifferenceisthattheresponseoftheether–etherlipidisseveralmagnitudeslower.
ThisdecreasedsignalintensitycouldbeduetothelowerionizationorfragmentationefficiencyofthedietherPCduringmassspectrometricanalysis[49].
Thehigherdetectionlimitofdietherspeciesencouragedtheuseofanondietherspecieasbiomarker.
InFig.
3c,abiofilmsamplecontaininganammoxbacteriaisanalyzedusingparentalionscanningofthePCheadgroup.
Fig.
3ChromatogramsshowingtheLCseparationofladderanePClipids.
aStructuresoffivenon-ladderanePClipidsusedduringmethoddevelopment.
(1)10PC,(2)16PC,(3)DE16PC,(4)D7018PC,(5)4Me16PC.
bChromatogramofthenon-ladderanePCstandardsatthesameconcentrationanalyzedbyRP-LCQqQusingparentalionscanningofm/z184.
cChromatogramofabiofilmsamplecontaininganammoxbacteriaanalyzedbyRP-LCQqQusingparentalionscanningofm/z184.
Ladderanelipidselutelastinthechromatogram.
Themorepolarether–ester-linkedladderanePClipidsIIIandIVelutebeforetheether–etherlinkedIandIIFig.
2ComparisonofthreeextractionmethodsforladderanePClipidsfoundatm/z802(II),814(III),and816(IV),n=3.
TheRomannumeralsII,III,andIVdenotethestructuresillustratedinFig.
1.
*Denotesasignificantdifference(studentsttest<0.
05)bIntactladderanephospholipidsfromviableanammoxbacteria3547ThealmostidenticalladderanePClipidsatm/z814(III)andm/z816(IV),bothbeingester–etherladderanePClipids,coeluteinthepeakdenotedbyIII,IVinFig.
3c.
Asexpected,theladderanePClipidsatm/z800(I)andm/z802(II),alsoalmostidenticalbutbothdietherladderanePClipids,coelutelaterinthepeakdenotedbyI,II.
ThecoelutionofspeciesIIIandIVaswellasIandIIisacceptablesincetheseanalytesoriginatefromtheanammoxbacteriainconstantrelativeconcentrations,thuskeepinganeventualdiscriminationeffectduringanalysisconstant.
MassspectrometryofladderanelipidsForelectrospraymassspectrometry,PClipidsionizebestinthepositiveionmodeandreadilylosetheirheadgroupatm/z184whensubjectedtocollisioninduceddissociation.
AQqQ,usingparentalionscanningofthem/z184fragmention,wasusedforanalyteconfirmationduringmethoddevelopment.
Figure4ashowsthechromatogramofananammoxbiofilmsample.
ThetwopeakselutinglastcontaintheintactladderanePClipidsI–IVasshowninthecorrespondingmassspectra(Fig.
4b–c).
ThedetectionlimitsoftheladderanePClipidswasfoundtobehigherwhenusingparentalionscanningontheQqQcomparedtoscanningusingtheQToF.
ThiscanpartlybeexplainedbythefactthattheladderanePClipidshavecomparablylonghydrocarbontailgroupswhichmakethemhardtofragment[46].
Therefore,weusedtheQToF,scanningbetweenm/z100and900,todetectladderanePClipidsinsedimentsampleswhichcontainsubstantiallyloweramountsthanbiofilmsamples.
InFig.
4d,asedimentsampleisanalyzedbyRP-LCQToFandtheselectedmasschromatogram,i.
e.
achromatogramwherethem/z816,correspondingtotheladderanePClipidIV,isextractedoutofthetotalionchromatogram,isshown.
ThepeaklabeledasIVistheladderanePClipidatm/z816andthecorrespondingmassspectrumofthatpeakisfoundinFig.
4e.
ThisclearlyshowsthepresenceofanammoxbacteriaintheoceansedimentsamplethatwastakenfromGullmarsfjordenonthewestcoastofSweden.
AscanbeseeninthemassspectrafrombothbiofilmandsedimentsamplestheRP-LCmethodclearlyseparatetheladderanePClipidsfromotherlipidspeciesinthecomplexsedimentsample.
Fig.
4ChromatogramsandmassspectraofintactladderanePClipids.
aChromatogramofabiofilmsamplecontaininganammoxbacteriaanalyzedbyRP-LCQqQusingparentalionscanningofm/z184.
ThetwopeakselutinglateinthechromatogramcontainstheladderanelipidsI–IVasshowninbandc.
bMassspectrumextractedfromthepeakat12.
5mindenotedIII,IVina.
TheintactladderanePClipidsIIIandIVatm/z814and816areidentified.
cMassspectrumextractedfromthepeakat16mindenotedI,IIinA.
TheintactladderanePClipidsIandIIatm/z800and802areidentified.
dSelectedmasschromatogramfortheintactladderanePClipidIVatm/z816inasedimentsampleanalyzedbyRP-LCQToFscanningbetweenm/z100and900.
eMassspectrumextractedfromthepeakat16mindenotedIVind.
TheintactladderanePClipidsIIIandIVatm/z814and816areidentifiedb3548I.
Lanekoff,R.
KarlssonDeterminationofaspecificladderanebiomarkerTheladderanePClipidatm/z816waschosenasabiomarkerforanammoxbacteriabasedontwoimportantcharacteristics.
First;theladderanePClipidatm/z816displaysthehighestabundanceinthemassspectrumwhenusingthedescribedmethod.
Second,theladderanePClipidatm/z816isanether–ester-linkedladderanelipid,whichwehaveobservedtobelessstableinsedimentwhencomparedtotheether–etherlinkedPClipids.
Thelowstabilityinsedimentenvironmentalsosuggeststhatthislipidwouldbettertracklivinganammoxbacteria.
Thiswasinvestigatedbyanalyzingtheamountofester–ether(e.
g.
,m/z816)andether–ether(e.
g.
,m/z800)linkedladderanePClipidsinfreshsedimentandagainafterstoringthatsamesedimentformorethan1monthinarefrigeratorandat20°C.
Theether–esterladderanePClipidswerefoundinreducedlevelsintheoldersedimentscomparedtothefresh,whereastheether–etherladderanePClipidswerefoundinthesameamountsregardlessofsedimenttype.
Thisobservationsuggeststhatthedegradationinsedimentfortheselipidsdoesnotoccuratsimilarratesandthatanester–etherlinkedladderanePClipidisasuitablebiomark-erforlivinganammoxbacteria.
Furthermore,thestabilityoftheether–etherladderanePClipidsoffersanexplanationtowhyotherstudies,analyzingladderanelipiddegradationproducts,havefoundladderaneFAsandladderanelysoli-pidsspreadthroughoutthesedimentdepthprofiles[33].
TodemonstratetheaccuracyoftheladderanePClipidatm/z816asbiomarkerforviableanammoxbacteriawehaveperformedtestsonbothsamplesfromthewastewatertreatmentplantHimmerfjrdsverketinStockholm,SwedenandasedimentcorefromGullmarsfjordenonthewestcoastofSweden.
BiofilmsamplesfromthreeofthereactorsatHimmerfjrdsverketpresumablycontaininganammoxbacteriawereanalyzed.
Thebiomarkerwaspresentinthesamplefromthereactorthatdisplayedanammoxactivity(datanotshown),shownbythegenerationofnitrogengasunderanoxicconditions,indicatingthepresenceofintactlivinganammoxbacteriainthereactor.
DetectingladderanePClipidsinthecomplexmatrixofsedimentisconsiderablymoredifficult.
Thecorrespondingmasschromatogramsofm/z816atthedepths0–3,3–6,6–9,and9–12cmareshowninFig.
5a.
Nutrientandoxygenprofiles,previouslyinvestigatedby,e.
g.
,Jaeschkeetal.
[33],indicatesthatanammoxbacteriaresideintheupperlayerofthesediment,closetotheborderbetweentheanoxicandoxiczone.
Accordingly,theladderanePClipidbiomarkeratm/z816wasfoundatthisdepth,asshowninthedepthprofileinFig.
5b.
Themorestableether–etherladderanePClipidatm/z800was,however,foundatalmosteverydepthinthecore,likelyduetotheslowerdegradationrateforether–etherPClipidsrelativetotheether–esterlipids,asdiscussedabove.
TheamountofladderanePClipidsfoundatthesedepthscanberelatedtoeachothersincetheladderanePClipidshavealinearresponse(integratedpeakareaversusconcentration)intheconcentrationrangeofinterest(datanotshown).
Inthis,examplethedepthsinthecorearerough,andthedepthatwhichtheanammoxbacteriaarefocusedisprobablyalotFig.
5Resultfromthesedimentdepthprofilestudy.
aFouroverlayedmasschromatogramsofm/z816.
Thesedimentsampledepthsareindicatedatthetraces.
ThebottomtraceisareferencesamplefromabiofilmcontainingladderanePClipids.
bDepthprofileofladderanePClipidIVatm/z816(ether–ester),indicatedbythetriangles,andladderanePClipidIatm/z800(ether–ether),indicatedbythecircles.
Depthslicesare3cmthick.
ForladderanePClipidIV,atm/z816,thepeakswerenormalizedtothemaximumareafoundatdepth0–3cmdenoted100%.
ForladderanePClipidI,atm/z800,thepeakswerenormalizedtothemaximumareafoundatdepth6–9cmdenoted100%Intactladderanephospholipidsfromviableanammoxbacteria3549narrower.
ThefindingofanammoxbacteriaatthissiteinGullmarsfjordenisconsistentwithpreviousobservations[4,50].
ConclusionsAmethodhasbeendevelopedtoanalyzeintactladderanePClipidsusingRP-LC-ESI-MS.
Inthisstudy,themethodwasusedtoidentifyaspecificintactladderanePClipidbiomarkerforviableanammoxbacteria.
Thechosenbiomarkerisanether–esterladderanePClipidwhichisfoundatm/z816.
Further,threedifferentmethodsforextractingladderanePClipidshavebeenevaluatedandamultistepsamplecleanupprocedureforladderanelipidsinsedimentsamplesisdescribed.
Whenusingthedescribedmethodonsedimentsamples,wefoundthattheselectedbiomarkerispresentonlyintheupperpartofthesedimentcore.
Thisfindingissupportedbyearlierobservationsthatanammoxbacteriaresidesintheupperpartofthesediment,justbelowtheoxiczone.
Thedescribedmethodprovidesanewtoolfortheanammox-interestedresearchcommunitytostudyviableanammoxbacteriainnaturalenvironments.
Thiswillleadtoanincreasedunderstandingofthepreferredbiogeochemicalenvironmentoftheanammoxbacteriaandhowtheircommunitystructureisaffectedbyongoingenvironmentalchanges.
Furthermore,thelocaliza-tionofviableanammoxbacteriaandaconcomitantobservationofanammoxactivitywillcontributetoagreaterunderstandingoftheglobalnitrogencycle.
AcknowledgmentsTheauthorswouldliketogreatlyacknowledgeProfessorStefanHulthandhisgroupattheDepartmentofChemistry,UniversityofGothenburgforfruitfuldiscussionsaswellashelpwithsamplecollection.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialLicensewhichper-mitsanynoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthor(s)andsourcearecredited.
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