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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.

AB

C

H2-only

H2+Formate

Formate-only

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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8/13/18 8/15/18 8/17/18Date

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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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