Characterization of hydrogenotrophic methanogenic Archaea ... · To begin enrichments, serum...
Transcript of Characterization of hydrogenotrophic methanogenic Archaea ... · To begin enrichments, serum...
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CharacterizationofhydrogenotrophicmethanogenicArchaeaenrichedfromTrunkRiver
BrittniL.Bertolet
MicrobialDiversity2018-MarineBiologicalLaboratoriesWoodsHole,MA
INTRODUCTION
Inlandlakes,rivers,andreservoirsareincreasinglyrecognizedfortheircontributiontoatmosphericmethane(CH4)concentrations.Globally,inlandwatersareestimatedtoproduced25%ofthecontinentallandsinkinCH4everyyear(Bastvikenetal.2011).However,inachangingworld,theseestimatesaredifficulttoprojectandthisislargelyduetoalackofprocess-basedmodelsthatconsiderthebiologicalcomplexityofthesebiogeochemicalprocesses.Ininlandfreshwaterandbrackishecosystems,CH4isprimarilyproducedbiologicallybymethanogenicArchaea(hereafterreferredtoas“methanogens”),whichregulatetherateofsedimentdiagenesisandcarbonfate(Borreletal.2011).Despitethis,fewecosystemmodelsofCH4productionexplicitlyconsiderthemicrobialcommunityandlittleisknownabouthowdifferencesbetweenmethanogensmaycontributetovariationinecosystemCH4production.
Biologicalmethanogenesisoccursviathreemajormetabolicpathways,characterizedbytheirelectrondonorsandterminalacceptors:acetoclastic(usingacetate),hydrogenotrophic,(usingH2/CO2),andmethylotrophic(usingmethyl-compoundsassubstrates)(Liu&Whitman2008).Infreshwaterandbrackishsediments,methaneisassumedtoresultprimarilyfromtheacetoclasticpathway(Conrad1999),withmanysuggestingthatthegenusMethanosaetatodominatelakesedimentcommunities(ZeppFalzetal.1999;Chanetal.2002).However,communitybiomarkersurveys(16SrRNAandmcrAgenes)increasinglydocumenthigher-than-expectedrelativeabundancesofknownorputativehydrogenotrophicmethanogensinlakesandsoilsenvironments(Nussleinetal.2001;Conradetal.2010).Althoughsequence-basedanalysesdonotdirectlyconsidertheactivecomponentofamicrobialcommunity,thepervasivenessofthesegroupsacrossregionsandecosystemssuggeststheymaybefunctionallyimportantconstituentsofsedimentmethanogencommunities.Unfortunately,fewmethanogenshaveeverbeenisolatedfromsedimentsandmuchisstilltobeunderstoodaboutthedistributionofphysiologicaltraits,suchassubstrateuse,growthrate,andCH4yield.
Inthepresentstudy,IsoughttoisolateandcharacterizehydrogenotrophicmethanogensfromTrunkRiversedimentsusingbothphysiologicalassaysandgenomics.Althoughisolationwasunsuccessful,Ideterminedhowthemethanogenicconsortiumrespondedtotheavailabilityofalternativeelectrondonorsusingexperimentalincubationswithformateadditions.Additionally,usingshotgunmetagenomics,fivemetagenomeassembledgenomes(MAGs)wereconstructedandusedtodeterminebothphylogenyandmetabolicpotentialofthemethanogenicconsortium.
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METHODS
Samplecollectionandenrichment:Sedimentwascollectedfromthesediment-waterinterfaceofTrunkRiver(TR),a
brackishriverinWoodsHole,MA(Lat:41.534850,Long:-70.641458).Sedimentwastransportedbacktothelaboratoryandplacedinananaerobicchamberuntilinoculation.
Priortoinoculation,selectivemediatoenrichforhydrogenotrophicmethanogenswaspreparedanaerobically.Toprepare1literofmedia,5mLof1Mammoniumchloridesolution,0.1mLof100mMpotassiumphosphate(pH7.2)solution,5mLof1MMESbuffer(pH5.5),1mLoftraceelements,0.1mLof1%Resazurin,and10mLof100Xfreshwaterbase(consistingof100gNaCl,40gMgCl2.6H2O,10gCaCl2.H2O,50gKClperliter)wereaddedperliterofdeionizedwater.Themediawasthenboiledfor10minutesunderastreamofN2/CO2(80%:20%)gastoboiloutoxygen.ThemediawasalsocooledunderN2/CO2.Oncecooled,1mLofmultivitaminsolution,25mLof1Msodiumbicarbonate,and1mLof1Msodiumsulfidewereadded.Mediawasthenbroughtinsidetheanaerobicchamber,andoncethemediabecameclear,25mLofmediawasdispensedinto100mLserumbottles.Serumbottleswerethencrimpedshutwithair-tightrubberseptaandautoclaved.Aftercoolingfromtheautoclave,0.5mLofantibacterialsolution(1gRifampcinSVper100mLwater)wasaddedtoeachbottle.
Tobeginenrichments,serumbottlescontainingsterileanaerobicmethanogenmediawerebroughtintotheanaerobicchamberanduncrimped.Approximately1goffreshsedimentwastheninoculatedintoeachbottleandserumbottleswerecrimpedshutwithair-tightrubbersepta.Atthegassingstation,headspacewasreplacedwithaH2:CO2gasmixture(20%:80%),usingthreeroundsofrepeatedvacuumandgasreplacement.Afterheadspacereplacement,allenrichmentswerestoredinthedarkat30degreesCelsius,wheretheyremainedforthedurationofthestudy.
Liquidenrichmentswerepassagedevery7-9daysinthesameliquidmediausingananaerobicsyringe.Atotalofthreepassageswereconductedbeforeisolationstrategieswereemployed.Ateachstepofpassage,productionofCH4wasdeterminedthroughgaschromatographywithaflameionizationdetector(FID)andenrichmentswerevisualizedforauto-florescenceat420nmusingaflorescentmicroscopeandtheAlexa488filter.EnrichmentswithoutCH4productionwerediscarded.Isolationapproaches:
Toisolatehydrogenotrophicmethanogens,anumberofdifferentstrategieswereemployed.First,enrichmentswereseriallydilutedwiththeintenttodilute-to-extinctioninliquidmedia.Thisoccurredinthesamemediadescribedpriorinthesame100mLserumbottleswithaH2/CO2headspace.Toobtaincolonies,agarshakes,flat-bottombottles,andplateswereprepared(Fig.1).Agarosewasaddedtothemethanogenmediadescribedabovetoafinalconcentrationof1%agarforeachmethod.
Shakeswereconstructedin25mLBalchtubes,inwhich9mLofsterileanoxic1%agarmediawereaddedtoeachandmixedwith1mLofinoculumbeforesolidification.MediaandinoculationwaspreparedunderunderastreamofN2/CO2atthegassingstation.Fortheflat-bottombottles,mediawaspreparedasdescribedaboveandinoculumwasspreadonthesurfaceoftheagaraftersolidificationandoccurredintheanaerobicchamber.Forplates,two
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differentstrategieswereemployed:topagarandnotopagar.Forallthreemethods,inoculumwasseriallydilutedtodeterminebestinoculationconcentration.Additionally,forallthreemethods,headspacewasreplacedwiththesameH2:CO2gasmixtureandreplenishedeverydaytocontinuouslysupplythenecessarysubstrates.Allcultureswerestoredinthedarkat30degreesCelsiusforthelengthofthestudy.
Figure1.Agarshakes(A),flat-bottombottles(B),andpetriplates(C)usedtoisolatehydrogenotrophicmethanogencolonies.Formategrowthexperiment:
TodeterminehowtheavailabilityofformateaffectedCH4productionratesoftheenrichmentmethanogenicconsortium,experimentalincubationswereconstructedin25mLanaerobicBalchtubes(Fig.2).EachBalchtubecontainedafinalvolumeof12mL(10mLofselectivemethanogenmedia,1mLofinoculationfromasinglesourceenrichment,and1mLofadditionalmediabasedontreatment).Theconsortiumwassubjectedtothreedifferenttreatmentsinwhichtheavailabilityofelectrondonorswasmanipulated.TreatmentsincludedH2-only,formate-only,andH2-formate.Fortreatmentsreceivingformate,formicacidwasaddedtoafinalconcentrationof2mMformate.Additionally,theformate-onlytreatmentcontainedaN2/CO2headspaceinsteadofH2/CO2.Finally,1mLofsterileultra-purewaterwasaddedtotheH2-onlytreatmentstokeeptotalvolumeconstantacrosstreatments.
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Figure2.Experimentalincubationsfortheformategrowthexperiment.
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CH4productionwasmonitoredeverydayuntiltheconcentrationofCH4wasunchangingandstationaryphasewasobtained.H2/CO2wasneverreplenished.Incubationswerevisualizedusingmicroscopytodetermineanycursorychangesinrelativeabundancesofuniquecelltypes.
TocalculateCH4productionrates,peakareawasfirstconvertedtopartspermillion(ppm)ofCH4usingastandardcurveproducedonJuly7th,2018.TheconcentrationofCH4(ppm)wasthenconvertedtomicromolar(umol)usingtheidealgaslawandnormalizedbythevolumeofthemedia(mL).TodeterminetherateofCH4production,onlydatafromthegrowthstageoftheexperiment(8/13/2018-8/15/2018)wasusedinalinearregressionagainsttime.Thus,theCH4productionrateisreportedasumolCH4mL-1hr-1.TodeterminedifferencesinCH4productionratesbetweensamples,aone-wayANOVAwasperformedinR.
Metagenomicanalyses:
DNAwasextractedusingaPowerFecalextractionkitfromtheinitialenrichmentsafter21daysofgrowth.DNAwassequencedonanIlluminaHiSeq.RawreadsweretrimmedusingTrimmomatic,co-assembledusingMegaHit,mappedusingBowtie,andbinnedusingconcoct.Oncebinned,MAGswerecheckedforcompletionusingCheckM,whichconsidersthepresenceofsinglecopygenes.TodeterminethephylogenyofconstructedMAGS,16ribosomalproteinsusedinHugetal.2016wereextractedusingaphylogeneticworkflowdescribedinGrahametal.2018.ThisworkflowusesacuratedreferencedatabaseofthesameribosomalproteinsextractedfromavailablemethanogengenomesfromNCBI.Severalphylogenetictreeswereconstructedwiththeentiredatabase,aswellasonlysubsetsofthedatabase.Finally,MAGswereannotatedusingtheRapidAnnotationusingSubsystemTechnology(RAST)platformandmetabolicmodelswerebuiltusingModelSeedinKbase.
RESULTSANDDISCUSSION
Microscopycharacterizationandisolation:ThreeuniquemethanogeniccelltypeswereconfirmedintheTRenrichmentsthrough
microscopy(cocci,rod-like,andspiralshaped)(Fig.3).Additionally,2non-florescentcelltypeswerealsoobserved,withonehighlymotilenon-florescentcelltype.Allcelltypeswereobservedineverypassage,withnonoticeabledifferencebetweenpassagesafterafewdaysofgrowthhadoccurred.However,furtherquantification,eitherwithflowcytometryorspecificFISHprobes,wouldhavegreatlybenefittedthis
Dilution-to-extinctioninliquidmediawasunsuccessfulinproducingpurecultures.CultureseitherproducedCH4andcontinuedtohaveamixedconsortium,ornoCH4wasproducedandfewcellswereeverdetected.Shakesandflat-bottombottleswerealsounsuccessfulinproducingcolonies,howeverallculturesproducedCH4.Theagarplateswerethemostsuccessful.AlthoughIneverobtainedcolonies,onthelastdayofincubation,smallspeckswerenoticeableonthesurfaceoftheagarthatmayhaveproducedcoloniesiflongergrowthwaspossible.Thispotentialforcolonyformationislikelyduetotheincreasedsurfaceareaoftheplates,aswellasthelargeamountofheadspaceavailableintheplatecanisters(Fig.1C).Itisalsoworthnotingthatthesespeckswereobservedonplateswithouttopagarandinoculatedwithundilutedinoculum,sodilutionsarelikelyunnecessaryinfutureattempts.
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Figure3.WetmountfromTRmethanogenenrichments,visualizedunderafilter(Alexa488).Methanogensarevisualizedbasedonauto-florescenceat420nm,whichisduetothepresenceofredoxcofactorF420.Uniquecelltypesaredenotedwitharrows.EffectsofformateavailabilityonCH4productionratesofenrichments: FormateavailabilityhadasignificanteffectonconsortiumCH4productionrates(Fig.3B).Alltreatmentsweresignificantlydifferentfromeachother,andincubationswithbothformateandH2hadthehighestrateofCH4production.Interestingly,incubationsreceivingonlyformateasthesoleelectrondonorhadnegligibleCH4production.WhileatraceamountofCH4wasdetectedonthefirstdayofsampling,thisismostlikelyduetoresidualH2thatwasintroducedintheinoculationasCH4concentrationsdidnotincreaseotherwisethroughoutthelengthoftheincubation.However,thisisunconfirmed.
ItisworthnotingthatthesignificantincreaseinCH4productionwhenbothelectrondonorsarepresentisgreaterthanthesumoftheH2-onlyandformate-onlytreatments,suggestingsomeinteractionthatispromotingCH4productionwhenbothelectronacceptorsareavailable.Previousresearchershaveseenevidenceofformate-dependentreductionofCO2withH2inothermembersofMethanobacteriales(Yangetal.2016),whichmaybecontributingtohigherCH4productionrates.However,becausecommunityassemblywasnotdeterminedattheendoftheexperiment,itisdifficulttodeterminethemechanismbehindthiseffect.AnalysesofmetabolicmodelsfromtheconstructedMAGsmayprovidefurtherinsight.
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Figure3.AverageCH4concentrationintheheadspaceofexperimentalincubationsforeachdayandtreatment(A).CH4productionrateofeachtreatmentduringthegrowthphase(8/13/18-8/15/18)(B).Errorbarsrepresentstandarderror.Metagenomicanalysisofenrichments:
Shotgunmetagenomicsequencingoftheconsortiumenrichmentsretrievedfourmethanogenbins(M1-M4)andonebacterialbin(B1)(Fig.4).MAGsM1-M3werenearcomplete(90.1-98.9%)withlessthan1%contamination.MAGM4wasmoderatelycomplete(65.8%)withlessthan1%ofcontamination,andMAGB1wassemi-complete(82.7%)with1.3%contamination.TheseMAGsconstitutedonly25.4%ofthetotalbase-pairssequenced,andfurtherrefinementofotherbinscouldprovideadditionalcompleteorsemi-completeMAGs.
Figure4.Visualizationofcompletemetagenomeassembledgenomes(MAGs)constructedfromshotgunmetagenomicsequencingofTRsedimentenrichments.VisualizationwasgeneratedusingAnvi’o.
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Phylogeneticanalysisof16ribosomalproteinsextractedfromtheconstructedMAGsplacedallmethanogenMAGswithinknownhydrogenotrophicmethanogengenerawithhighconfidence(Fig.5).Fourdifferenttreesweregenerated,eachwithadifferentnumberofreferencegenomes,andalltreesdisplayedthesameplacementofMAGs.Thus,thephylogenyisvisualizedinFigure5withonlyasubsetofthereferencedatabase.
ThephylogeneticanalysisofMAGB1wasnotasconclusive.RibosomalproteinsplacedB1withintheTenericutesphylum,withintheMollicutesclass.Althoughno16Sor23SrRNAgeneswereassembled,one5SrRNAgenecouldbeextractedfromtheMAG,andthiswas92%identicaltothegeneinaTenericutesMAGassembledfromenrichmentsfromamethaneseep(Skennertonetal.2016).Membersfromthisgrouparehypothesizedtobeanaerobicfermenters,thatmaysupplymethanogenswithsubstratessuchasacetateandethanol.Itisinconclusive,however,whetherthisisatruesyntrophicrelationship,andfurtherresearchisstillneededtounderstandtheroleofthefree-livingTenericuteswithinmethanogeniccommunities.MembersofMollicuteshavealsobeenshowntopossessresistancetoRifampicinantibiotics,whichmayexplainthepresenceofthisgroupinourenrichments.
Figure5.PhylogenyofmethanogenMAGsconstructedfromTRsedimentenrichments.Desulfurococcusamylolyticuswasusedasanout-groupandbootstrapvaluesarereported.
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Methanococcus maripaludis
Methanocella arvoryzae
TR M4
Candidatus Methanomethylophilus alvus
Methanothermus fervidus
Methermicoccus shengliensis
Methanobacterium formicicum
Methanobrevibacter smithii
Methanofollis ethanolicus
Methanosphaerula palustris
Methanosphaera sp. WGK6
Methanothermobacter marburgensis
Methanococcus aeolicus
Methanobacterium congolense
TR M2
Methanoplanus limicola
Methanococcoides burtonii
Methanocorpusculum bavaricum
Methanosaeta concilii
Methanobrevibacter wolinii
Methanobacterium paludis
Methanopyrus kandleri
Methanothermobacter thermautotrophicus
Methanobacterium lacus
Methanospirillum hungatei
TR M1
Methanococcus voltae
Methanoculleus bourgensis
Methanofollis liminatans
Methanothermococcus thermolithotrophicus
TR M3
Methanolinea tarda
Methanocorpusculum labreanum
Methanotorris formicicus
Methanoregula boonei
Candidatus Methanoperedens nitroreducens
Methanoregula formicica
Methanomassiliicoccus luminyensis
Desulfurococcus amylolyticus
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CONCLUSIONSANDFURTHERDIRECTIONS
HydrogenotrophicmethanogenicconsortiumfromTRsedimentswereenrichedandcharacterized.Atleast5distinctmicroorganismswerepresentandCH4productionratesweresensitivetotheavailabilityofelectrondonors.AdditionsofformatesignificantlyincreasedCH4
productionascomparedwithincubationsreceivingonlyH2.Thishasimplicationsforunderstandingmethanogenesisinnature,asrarelyarecommunitiesisolatedwithonlyoneavailableelectrondonor,andmaypointtoecologicallyimportantmetabolisms.Further,analysisofconstructedMAGsalsosupportedtheuseofformateasanimportantintermediateinthereductionofCO2tomethaneinthishydrogenotrophiccommunity.InbothMAGsM2andM3,formatedehydrogenaseswerepresent,whicharenecessaryforformatedonationofelectronstoheterodisulfideinthelaststepofmethanogenesis.However,furtherresearchisneededtoconfirmthemechanismforhowformateisbeingutilizedbythisconsortium.
Inthisstudy,thecoupleduseoffunctionalassaysandgenomicsanalyseswasapowerfultoolforunderstandingthemetaboliccapacityofmicrobialcommunities,butfurtherworkwouldgreatlybenefittheseconclusions.Particularly,understandingdynamicchangesinthemicrobialconsortiumafterexperimentationwouldhavehelpedsupporthypothesesforhowformateadditionsaffectedCH4production.Thiscouldhavebeenaccomplishedthrougheither16Ssequencing,flowcytometry,orFISHvisualizationoranyotherwaytoquantifymicrobialcommunities,andsubsequentexperimentsshouldconsideremployingsuchmonitoring.
ACKNOWLEDGMENTS
ThankyoutoMadelineLopezMunozandGeorgeO’Toole,whomadealltheanaerobicworkpossible.ElainaGrahamforcontributingsomuchcodeandsupportwiththebioinformatics.RachelWhitaker,GabriellaKovacikova,ScottDawson,NickiLimoli,andJamieHallforendlessencouragement.AlloftheMBLMicrobialDiversitycoursestafffortheincrediblehelpandsupportandknowledge.
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