Methanotrophs microbiology fundamentals and biotechnological applications eun yeol lee

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Microbiology Monographs

Series Editor: Alexander Steinbüchel

Eun Yeol Lee Editor

Methanotrophs

Microbiology Fundamentals and Biotechnological Applications

MicrobiologyMonographs

Volume32

Serieseditor

AlexanderSteinbüchel Münster,Germany

Moreinformationaboutthisseriesat http://www.springer.com/series/7171

EunYeolLee Editor

Methanotrophs

MicrobiologyFundamentals andBiotechnologicalApplications

Editor EunYeolLee DepartmentofChemicalEngineering

KyungHeeUniversity

Yongin-si,Gyeonggi-do RepublicofKorea

ISSN1862-5576ISSN1862-5584(electronic)

MicrobiologyMonographs

ISBN978-3-030-23260-3ISBN978-3-030-23261-0(eBook) https://doi.org/10.1007/978-3-030-23261-0

© SpringerNatureSwitzerlandAG2019

Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpart ofthematerialisconcerned,specificallytherightsoftranslation,reprinting,reuseofillustrations, recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionor informationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar methodologynowknownorhereafterdeveloped.

Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthis publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexempt fromtherelevantprotectivelawsandregulationsandthereforefreeforgeneraluse.

Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbook arebelievedtobetrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsorthe editorsgiveawarranty,expressorimplied,withrespecttothematerialcontainedhereinorforanyerrors oromissionsthatmayhavebeenmade.Thepublisherremainsneutralwithregardtojurisdictionalclaims inpublishedmapsandinstitutionalaffiliations.

ThisSpringerimprintispublishedbytheregisteredcompanySpringerNatureSwitzerlandAG. Theregisteredcompanyaddressis:Gewerbestrasse11,6330Cham,Switzerland

Preface

Methanotrophsareaerobicproteobacteriathatcanutilizemethaneassolecarbonand energysource.Inecology,methanotrophshaveanessentialroleintheglobalcarbon cyclebyremovingmethanegeneratedgeothermallyandbymethanogens. Methanotrophshavebeenemployedasthebiocatalystformitigatingmethaneas greenhousegasandremediatinghalogenatedhydrocarbonsinsoilandunderground water.Recently,methaneisconsideredthenext-generationcarbonfeedstockfor industrialbiotechnologybecauseofitshighabundanceandlowprice.Methanotrophs canbeusedasthebiocatalystfortheproductionofchemicals,fuels,andbiomaterials frommethaneunderenvironmentallybenignproductionconditions.

Despitethegrowingimportanceofbasicandappliedresearcheson methanotrophs,therewasnocomprehensivetextbookforseniorundergraduateand postgraduatelevels. Methanotroph:MicrobiologicalFundamentalsandBiotechnologicalApplications waswritteninanattempttogivethereadersasystematicand comprehensiveoverviewforbothbasicandappliedaspectsofmethanotrophs.Thus, thisbookcanbeusedasareferenceformicrobiologistsandbiochemistsdealingwith ecology,environmentalfunctioning,andunderstandingofphysiologyandmetabolismofmethanotrophsaswellasthefundamentalsandapplicationsofmethane monooxygenases.Thisbookisalsovaluabletobiotechnologistsandbiochemical engineerswhoresearchontheomics-basedunderstandingofmethanemetabolism, metabolicengineeringforstrainimprovement,methanobactinbiosynthesis,and environmentalapplicationsofmethanotrophs.

Iwouldliketothankthepeoplewhowereinstrumentalinthewritingofthisbook. Firstofall,Iwouldliketothankthecontributorsfortakingtheirvaluabletimein writingthechapters.IappreciateverymuchProfessorAlexanderSteinbüchelofthe InstituteofMicrobiologyatMünsterUniversity,the MicrobiologyMonographs SeriesEditor,forgivingmetheopportunitytopublishthisbook.Iwouldliketo acknowledgeC1gasrefineryR&DprogramoftheNationalResearchFoundationof Koreaforsupportingtheresearchfundfordevelopmentofmethanotrophicplatform v

strainsforbioconversionofmethaneandmethanol.Lastly,Iamgratefultomy belovedfamilyandlabmembersfortheirconsiderationandsupportsothatIcould concentrateonwritingbookscomfortably.

Yongin-si,Gyeonggi-do,RepublicofKoreaEunYeolLee April2019

Contents

MethanotrophEcology,EnvironmentalDistributionandFunctioning ...1

PaulL.E.Bodelier,GermanPérez,AnneliesJ.Veraart, andSaschaM.B.Krause

EnrichmentandIsolationofAerobicandAnaerobicMethanotrophs ...39

Sung-KeunRhee,SamuelImisiAwala,andNgoc-LoiNguyen

TheBiochemistryofMethaneMonooxygenases ...................71

SunneyI.ChanandSeungJaeLee

Multi-omicsUnderstandingofMethanotrophs ....................121 YueZhengandLudmilaChistoserdova

Diversity,Physiology,andBiotechnologicalPotential ofHalo(alkali)philicMethane-ConsumingBacteria ................139 SnehalNariyaandMarinaG.Kalyuzhnaya

MetabolicEngineeringofMethanotrophsfortheProduction ofChemicalsandFuels ......................................163

OkKyungLee,DiepT.N.Nguyen,andEunYeolLee

Methanobactin:ANovelCopper-BindingCompoundProduced byMethanotrophs .........................................205

JeremyD.SemrauandAlanA.DiSpirito

EnvironmentalApplicationsofMethanotrophs ...................231 AdrianHo,MiyeKwon,MarcusA.Horn,andSukhwanYoon

Index ...................................................257

ListofContributors

SamuelImisiAwala DepartmentofMicrobiology,ChungbukNationalUniversity, Cheongju,RepublicofKorea

PaulL.E.Bodelier DepartmentofMicrobialEcology,NetherlandsInstituteof Ecology(NIOO-KNAW),Wageningen,TheNetherlands

SunneyI.Chan NoyesLaboratory,CaliforniaInstituteofTechnology,Pasadena, CA,USA

InstituteofChemistry,AcademiaSinica,Nankang,Taipei,Taiwan

LudmilaChistoserdova DepartmentofChemicalEngineering,Universityof Washington,Seattle,WA,USA

AlanA.DiSpirito RoyJ.CarverDepartmentofBiochemistry,Biophysicsand MolecularBiology,IowaStateUniversity,Ames,IA,USA

AdrianHo InstituteforMicrobiology,LeibnizUniversitätHannover,Hannover, Germany

MarcusA.Horn InstituteforMicrobiology,LeibnizUniversitätHannover, Hannover,Germany

MarinaG.Kalyuzhnaya DepartmentofBiology,SanDiegoStateUniversity,San Diego,CA,USA

ViralInformationInstitute,SanDiegoStateUniversity,SanDiego,CA,USA

SaschaM.B.Krause JohannHeinrichvonThünenInstitute,FederalResearch InstituteforRuralAreas,ForestryandFisheries,Braunschweig,Germany

MiyeKwon DepartmentofCivilandEnvironmentalEngineering,KAIST, Daejeon,SouthKorea

EunYeolLee DepartmentofChemicalEngineering,KyungHeeUniversity, Yongin-si,SouthKorea

OkKyungLee DepartmentofChemicalEngineering,KyungHeeUniversity, Yongin-si,SouthKorea

SeungJaeLee DepartmentofChemistryandInstituteforMolecularBiologyand Genetics,ChonbukNationalUniversity,Jeonju,RepublicofKorea

SnehalNariya DepartmentofBiology,SanDiegoStateUniversity,SanDiego, CA,USA

DiepT.N.Nguyen DepartmentofChemicalEngineering,KyungHeeUniversity, Yongin-si,SouthKorea

Ngoc-LoiNguyen DepartmentofMicrobiology,ChungbukNationalUniversity, Cheongju,RepublicofKorea

GermanPérez DepartmentofMicrobialEcology,NetherlandsInstituteofEcology (NIOO-KNAW),Wageningen,TheNetherlands

Sung-KeunRhee DepartmentofMicrobiology,ChungbukNationalUniversity, Cheongju,RepublicofKorea

JeremyD.Semrau DepartmentofCivilandEnvironmentalEngineering, UniversityofMichigan,AnnArbor,MI,USA

AnneliesJ.Veraart DepartmentofAquaticEcologyandEnvironmentalBiology, InstituteforWaterandWetlandResearch,RadboudUniversity,Nijmegen,The Netherlands

SukhwanYoon DepartmentofCivilandEnvironmentalEngineering,KAIST, Daejeon,SouthKorea

YueZheng DepartmentofChemicalEngineering,UniversityofWashington, Seattle,WA,USA

CASKeyLaboratoryofUrbanPollutantConversion,InstituteofUrban Environment,ChineseAcademyofSciences,Xiamen,China UniversityofChineseAcademyofSciences,Beijing,China

MethanotrophEcology,Environmental DistributionandFunctioning

PaulL.E.Bodelier,GermanPérez,AnneliesJ.Veraart, andSaschaM.B.Krause

4.1ControllingAbioticFactors..

Abstract Thedynamicsofmethaneconcentrationsintheatmosphereinrecent decadeshasdemonstratedmanyanomalieswhicharepoorlyunderstood.Theonly biologicalwayofdegradingthispotentgreenhousegasisbymicrobialoxidation. Aerobicmethanotrophicbacteria(MB)playanimportantroleinmanyecosystems worldwidedegradingmethanebeforeitcanescapetotheatmosphere.Thisgroupof bacteriahasintensivelybeenstudiedasamodelmicrobialfunctionalguildbecause thereisastronglinkbetweentheconsumptionofmethaneandthecompositionof MBcommunities,facilitatingthestudyofmicrobial “behavior” intheenvironment. ThesestudieshaverevealedastrongbiogeographyofMBwhichisdisplayedintheir phylogenynotonlyonthebasisofsinglefunctionalmarkergenesbutalsoon

P.L.E.Bodelier(*)·G.Pérez

DepartmentofMicrobialEcology,NetherlandsInstituteofEcology(NIOO-KNAW), Wageningen,TheNetherlands e-mail: P.Bodelier@nioo.knaw.nl

A.J.Veraart

DepartmentofAquaticEcologyandEnvironmentalBiology,InstituteforWaterandWetland Research,RadboudUniversity,Nijmegen,TheNetherlands

S.M.B.Krause

JohannHeinrichvonThünenInstitute,FederalResearchInstituteforRuralAreas,Forestryand Fisheries,Braunschweig,Germany

© SpringerNatureSwitzerlandAG2019

E.Y.Lee(ed.), Methanotrophs,MicrobiologyMonographs32, https://doi.org/10.1007/978-3-030-23261-0_1

genomesequencebasis.Novelenvironmentalcontrollingfactorshavebeenrevealed (e.g.rareearthmetals)aswellasnovelorganismswithasyetunknowntraitsfor MB.Theresistanceandresilienceofmethaneconsumptionandmethaneconsuming communitieshavebeenshowntodependonspeci ficcommunitymembers.The currentknowledgeonenvironmentaldistributionandofMBhasledtoproposea life-historyscheme,classifyingMBcommunitiesontheircollectivetraitsratherthan singlyontheircapacitytheoxidisemethanealone.

1Introduction

Workingtowardsasustainablefutureforourplanetrequiresadrasticchangeinthe wayssocietyaddressesitsprimaryneeds:energy,food,resourcesandclimate.Finite stocksoffossilfuelsandcorrespondingrelatedclimateissuesleavesonlylow-carbon economieswithhighcontributionofrenewablesasalternatives,asrecentlyacknowledgedandratifiedinthe “ParisAgreement“ duringtheUnitedNationsFramework ConventiononClimateChange(http://unfccc.int/resource/docs/2015/cop21/eng/ l09r01.pdf).ToreachtheParistargetsdrasticreductionsinemissionsofgreenhouse gases(GHG)arenecessary(80%below1990levels),includingobtaining20%ofour energyfromrenewablesources.Inthiscontext,methane(CH4)playsakeyrole, beinga24timesmorepotentGHGthancarbondioxide,andcontributing40%to globalwarmingona20-yeartimescale(IPCC 2014).Hugeamountsofmethaneare releasedintotheatmosphereassociatedtogasandoilproductionandbybiological productioninnaturalandagriculturalwetlands,landfills,wastewatertreatmentand agriculturalproductionsystems,lakes,oceansandtermites(Deanetal. 2018). However,alargeportionofthismethanehasalreadybeenmitigatedbeforeescaping totheatmospherebytheactivityofmethane-oxidizingbacteria(MB)whichutilise methaneascarbonandenergysource(BodelierandSteenbergh 2014b).Thisreaction,turningmethaneintobiomassandmetabolitesisofhighvalueforproductionof bio-products(Fig. 1),isuniquetothesebacteria,andrequiresasetofenzymeswitha rangeofspecialfeatures(seeChaps. “Diversity,Physiology,andBiotechnological PotentialofHalo(alkali)philicMethane-ConsumingBacteria” and “MetabolicEngineeringofMethanotrophsfortheProductionofChemicalsandFuels”).Centralare themethanemonooxygenase(MMO)variants(particulate(pMMO)andsoluble methanemonooxygenase(sMMO))whichhaveaverybroadsubstraterange,making themhighlysuitableforapplicationsinsyntheticorganicchemistryandfordegrading environmentalcontaminantssuchastrichloroethylene(TCE)(Piejaetal. 2017). Theirimportanceforclimateandindustryhasspurredintensiveresearchintothe environmentaldistribution,diversity,ecology,physiologyandgenomicsofMB.The long-standingnotionthatthesemicrobesonlybelongtotheProteobacteria,andare exclusivelyobligatorymethanotrophicandaerobic,hasbeenprovenwronginthelast twodecades.NovelVerrucomicrobial(Dunfieldetal. 2007;vanTeeselingetal. 2014),anaerobic(Ettwigetal. 2010;Haroonetal. 2013;in ‘tZandtetal. 2018),

Fig.1 Methanecontainingbiogasoriginatingfrome.g.wasteorbeing flaredofduringenergy productioncanbeturnedintosustainablestorageofcarbonandenergy.Byusingnaturalconsortia ofmethaneconsumingmicrobesorengineeredstrains,methanecanbeturnedintoindustrially relevantchemicalswhichcanbestoredandtransportedeasilyandusedfortheproductionof valuablebio-basedchemicals

facultative(CrombieandMurrell 2014),and/ordenitrifyingMB(Dametal. 2013; Kitsetal. 2015)havebeendiscovered.Also,numerousenvironmentalstudies facilitatedbythedevelopmentofcultivationindependentassessmentmethodshave revealedavastasyetuntappeddiversityofMBwithdistinctbiogeography,indicatingalinkbetweenMBtraitsandhabitat(Knief 2015)ofinterestforfuturebiotechnologicalapplications.

Crucially,MBhavealsorecentlybeenshowntoperformbetterinnaturalaswellas syntheticmultispeciescommunities(Hoetal. 2014;Schnyderetal. 2018)depending oninteractionsandexchangesofnutrientswithothercommunitymembers(Zheng etal. 2014;Iguchietal. 2011)orbyexchangeofvolatileorganiccompounds(Veraart etal. 2018).However,thecultivationandindustrialhandlingofMBremaindifficult, resultinginonlyahandfulofstrainscurrentlyexploitedforindustrialpurposes.Next tothis,globalclimatemodelsstillgivepoorpredictionsoffutureatmospheric methaneconcentrations(Deanetal. 2018;Nisbetetal. 2014),whichdisplaya rangeofdistinctdynamicsandanomaliesinthepastdecades.Lackofadeeper understandingoftheecologyandfunctioningofMBinnaturalandman-made habitatsunderliesmanyoftheissueswehaveinusingMBinbio-industryandto explainatmosphericmethanedynamics.Thepresentchapterwillhighlightfeaturesof theecologyandenvironmentaldistributionofMBwithafocusontheaerobicMB.

2TaxonomyandPhylogeny

Currentlydescribedspeciesandtheirphylogenyhaveextensivelybeendescribed recently(DedyshandKnief 2018)andwewillnotgointodetailedtaxonomyhere. However,insightsfromcomparativegenomicsandmanydiscoveriesinthelasttwo

decadeshasresultedinamuchmoredifferentiatedviewonthismicrobialguild. Sincethe firstisolationofabacteriumcapableofgrowthonmethaneassoleenergy andcarbonsource(Söhngen 1906),obligateaerobicMBhavetraditionallybeen studiedandconceptuallybeentreatedasacoherentfunctionalmicrobialgroup.The firsttaxonomicframeworkforMBemergedaftertwodecadesofpioneeringworkin thelastcentury(Whittenburyetal. 1970;Bowmanetal. 1993)basicallydividing MBintwomaingroupsbasedonphysiological,morphological,ultrastructural,and chemotaxonomiccharacteristics.TypeIMBdistinguishedfromtypeIIbyhaving intracytoplasmicmembranes(ICM)structuredasvesiculardiscswhiletheseare arrangedalongtheperipheryofthecellaspairedmembraneswithtypeIIMB.Next tothis,typeIMB fixcarbonviatheribulosemonophosphatepathway(RuMP)while typeIIusetheserinepathway.Furthermore,bothgroupsclearlydifferedintheir fattyacidcompositionwithinthepolarlipidfractionofthecellmembranes(PLFA), withC16dominatedPLFApredominantlyintypeIandC18intypeII(Bowman etal. 1993;Bodelieretal. 2009)withevenPLFAwhichtodatehaveonlybeen detectedinMB(C16:1ω8candC18:1ω8c)enablingtheseparatedetectionofMB fromotherbacteriainenvironments(Bodelieretal. 2013).Besidestheability fix molecularnitrogen,whichatthattimeseparatedtypeIfromtypeII,theproduction ofrestingstages(cystsorspores)havealsobeenconsideredastaxonomicfeatureto distinguishmorebetweengeneraratherthanmaintypes.Theseparationintwomain groupsatthattimewasalsoreflectedinthephylogenybasedon16SrRNA,placing typeIwithinthe Gammaproteobacteria andtypeIIinthe Alphaproteobacteria (Bowmanetal. 1993).However,thesuccessfulcultivationofmanynewspecies (Knief 2015;DedyshandKnief 2018)generatedinformationthatrenderedthis dichotomousclassificationsystemnotrepresentativeforthephylogenomicand physiologicalviewwehavenow.Internalmembranesystemsdonotoccurinall MB(DunfieldandDedysh 2014;Vorobevetal. 2011);PLFAspeci ficfortypeIMB occuralsointypeII(Dedyshetal. 2007);nitrogen fixationalsooccursintypeIMB (Methylobactertundripaludum)andMBhavebeendiscoveredwhichhavepartsor evencompletegenesetsforbothtypesofcarbon fixationpathways(Nguyenetal. 2018),althoughithasnotbeendemonstratedthatbothpathwayscanbeoperational ineithertypeIortypeIIMB.

Despitetheinadequacyofthe “typeI/II” classificationsystemasanintegrated reflectionofphysiology,biochemistryandmorphology,thesystemisstillinuse sincecurrentphylogenybasedon16SandMMO(particulate(pMMO)andsoluble (sMMO)methanemonooxygenase)(Knief 2015;DedyshandKnief 2018)reflects thisclassification.CurrenttaxonomicradiationofaerobicMBcoverstaxonomically classifiedpurecultures,MBwithacandidatestatusduetothefactthatthesestrains areonlyavailableashighlyenrichedculturesanduncultivatedlineagesrepresented byenvironmental pmoA sequences(seeFig. 2).MostMBtypestrainsandclassified generafallwiththe Gammaproteobacteria inthefamilies Methylococcacea (type Ia/Ib)and Methylotermaceae (typeIc),whilethe Alphaproteobacteria harborthe familiesof Methylocystaceae (typeIIa)and Beijerinckiaceae (typeIIb)(Fig. 2). WiththediscoveryofMBinthephylumofthe Verrucomicrobia (Dunfieldetal. 2007;vanTeeselingetal. 2014;Poletal. 2007)belongingtothefamilyofthe

Fig.2 PhylogenyofdescribedaerobicMBbasedon16SrRNA( a )andtherelatednessofuncultivatedlineagesinrelationdescribedrepresentativesonthebasis ofthe pmoA gene( b ).FromDedyshandKnief( 2018 )withpermission

Methylacidophyliceae atypeIIIclassifierwasnecessarytobeinlinewiththe “phylogeny-based” typing.WiththeadventofthisMBphylumanotherbiochemical variationwasintroduced,bythefactthattheseMBareautotrophic, fixingcarbon usingtheCalvincycle(Khademetal. 2011).

Thediscoveryoftheabilitytogrowonmethaneofanalreadydescribed filamentousbacterium Crenothrixpolyspora (Stoeckeretal. 2006)belongingtothe Gammaproteobacteria wasanothersurprisewithintheMBrealm,addinganother family(Crenotrichideae)totheMBtreewithadeviating pmoA,similarto amoA of ammoniaoxidizers.Lateranovelgenuswithinthisfamilywithwasdescribed Clonotrix (Vigliottaetal. 2007),whichsimilarto Crenotrix hasnotbeenbrought intopureculturerenderingtaxonomicallyacandidatusstatus.

Althoughstrictlyspeakingbeingananaerobe,microbesbelongingtothenovel NC10phylumandrepresentedbythe Candidatus Methylomirabilisoxyfera,canbe consideredaerobicMBsincetheygenerateoxygenfromthereductionofnitriteand thesubsequentdisproportionationofnitricoxide(NO)(Ettwigetal. 2010)anduse thatoxygentooxidizemethaneaerobically.Recently,novelspeciesbelongingtothe Methylomirabilis genushavebeenidentified(Grafetal. 2018;Versantvoortetal. 2018)butasyethasnotbroughtintopureculture.

NexttotheMBinpureculturesorenrichmentsarangeof pmoA-basedlineages havebeenidentifiedwhichmostlyclusterwithtaxonomicallydescribedorganisms butalsorepresentarangeofsequencesdistinctfromexisting pmoA sequences (Knief 2015;DedyshandKnief 2018)(Fig. 2).Foralmostalloftheseclustersit remainstobeelucidatedwhatorganismsarebehindthesesequences.However, recentlyforthesocalledUSCα (foruplandsoilcluster α)usingsinglecellsequencingofactivelyatmosphericmethaneoxidizingcells,agenomesequencewas obtaineddemonstratingthecloseassociationofrepresentativesofthisclusterwith thegenus Methylocapsa (Pratscheretal. 2018).

Recently,microbialtaxonomyhasbeenrevisedbytheinclusionofwholegenome sequencesoftaxonomicallydescribedmicrobesaswellasmetagenomeassembled genomes(MAG)fromenvironmentalsamples(Parksetal. 2018).Thisextensive effortcompared120singlecopyubiquitousproteincodinggenesofalmost95,000 genomes.Thisanalysesledtoasubstantialrevisionoftaxonomyremovingmanyof theinconsistenciesandpolyphyleticcasesofincongruencyofphenotypicandsingle genebasedphylogeny.AlsoforMB,changesweresuggested,ofwhichthepropositionofdividingtheorderof Methylococcales intothreefamiliesbeing Methylomonadaceae (Ia), Methylococcaceae (Ib)and Methylothermaceae (Ic)(Parksetal. 2018).Anindetailwholegenomeanalyses,comparingANI(average nucleotideidentity),dDDH(digitalDNA-DNAhybridization)andAAI(average aminoacididentity)ofexistingMethylococcalesgenomesalsoledtoreclassification ofpolyphyleticMBgenera(Orataetal. 2018)leadingforexampletothenewgenus Methylotuvimicrobium.ForacompleterevisionoftheMBtaxonomyandphylogeny, theamountofsequencedtypestrainsisstillnotcompleteenough.Itisobviousthough fromtheGTDB(http://gtdb.ecogenomic.org/)database,wherenon-MMOcarrying bacteriaareclassifiedintoMBorders(e.g. Cyclocystaceae intotheorderof Methylococcales)aswellasrecentevolutionarystudiesofcoppercontaining

monooxygenases(OsborneandHaritos 2018;Khadkaetal. 2018)thatmethane oxidationisatraitandpropertythathasbeenpassedonbyseverallateralgene transferevents.However,stillremnantsandrelatedfunctions,necessaryfor performingmethanotrophyarestillpresentinancestors.Thelattermayleadto morereshuf flingasgenomesbecomeavailableandleadmoreandmoretothe realizationthatwearenotlookingsimplyatafunctionalguildbutatgroupof microbeswithmanymoreevolutionaryselectedtraitsthanmethaneoxidationalone.

3EnvironmentalDistribution

MBplayacrucialroleinmethanemitigationfromanumberofimportantmethane emittingsourceslikewetlands.Hence,inhabitatswhereyouhavehighmethaneand oxygeninclosevicinityMBareexpectedto flourish.However,therearemany habitatswhereMBwouldnotbeexpected.Inthissection,theenvironmental distributionofMBaswellastheirbiogeographywillbediscussed.Recent findings inafewkey-habitatswillbehighlighted.

3.1Biogeography

Principally,MBcanbefoundinallhabitatsweremethaneisavailable.Thiscanbe methaneproducinghabitatswithcloseproximityofanoxicandoxichabitats (e.g.wetlands,peat,lakesediments,marinesediments,landfills,etc.),butalsoin allotherhabitatsincontactwithanormalatmospherewhichcontainsapproximately 1.86ppmofmethane.ThedirectconnectionbetweenMBandclimatechangehas spurredthousandsofstudiesfocusingonenvironmentaldistributionofMB,their activityandthefactorscontrollingthelatter.Manyoftheseaspectshavebeen describedinarangeofexcellentreviewpapers(e.g.Knief 2015;Hansonand Hanson 1996;Conrad 1996, 2007;BodelierandLaanbroek 2004;Semrauetal. 2010;Kolb 2009;BodelierandSteenbergh 2014a, b;Hoetal. 2013a, b).Thewealth ofstudiesisalsoduetothefactthatthepresenceandactivityofMBcanbescreened verystraightforwardbydetectingthepresenceofthe pmoA/sMMO geneorby measuringthemethaneoxidationpotentialofincubatedsamples(e.g.Bodelier etal. 2013).Nexttothis,arangeofstudieshasshownthatcommunitycomposition ofMBislinkedtotheconsumptionofmethane(Schnyderetal. 2018;Bodelieretal. 2013;Nazariesetal. 2011;Levineetal. 2011)whichisenforcedbythepossibilityto trace 13C-labelledmethaneintaxonomicallyrelevantcompounds(e.g.PLFA,DNA, proteins)therebyassessingtheactivespeciesincomplexenvironments(Bodelier etal. 2013;Boschkeretal. 1998;Daebeleretal. 2014).Hence,thedirectlink betweenecosystemfunctionanddiversityoftheresponsiblespecieshasmadeMB amodelgroupwithinthe fieldofenvironmentalmicrobiology,beingmaybethebest describedenvironmentalmicrobialfunctionalgrouporguild.Using pmoA based

analysisofallcultivatedanduncultivatedMBdiversity,(Knief 2015)KniefexecutedacomprehensivebiogeographyanalysesofallMBdiversityknownatthat moment.Itshowednotonlythatmorethanhalfofallenvironmentalsequenceswere relatedtoknowncultivatedspeciesbutalsothatMBdisplaydistinctbiogeography andhabitatpreference(Knief 2015).Typically,aquatichabitats(freshandmarine) tendtoharborlineagesof pmoA sequenceswhichdonotoccurelsewhere,while terrestrialenvironmentslikesoilcontainMBspecieswithamuchbroaderenvironmentaldistribution,maybereflectingthelargerenvironmentalheterogeneityand influencingbioticandabioticfactorsaffectingMBfunctioningandsurvivalin terrestrialecosystems.Typicalexceptions,arethe pmoA sequencelineages(typeId e.g.USCα,USCγ)associatedwiththeuptakeofatmosphericmethaneinsoilswhich werefoundalmostexclusivelyinuplandsoilsaswellastheVerrucomicrobialMB whichonlyoccurinextreme(lowpH,hightemperature)terrestrialsoils.Thestudyby Kniefwasbasedon371studiesbasedonthe pmoA gene.WeutilizedtheEarth MicrobiomeProject(EMP)(Thompsonetal. 2017)databasewhichisthemost comprehensivedatacollectiononmicrobial16SrRNAsequencingreadsandassociatedmetadataonvarioushabitats,tocharacterizepatternsofmethanotrophsacross differentbiomesandhabitatsonanevenbiggerscale(Table 1).

WhencomparingdistributionofMBgenerainterrestrialandaquaticbiomes (Fig. 4;Table 2)itisobviousthatterrestrialhabitatsarepopulatedbothbytypeI andIIMB.Themoststrikingdifferencebetweenterrestrialandaquaticbiomesisthe farlessevenrelativeabundancesofdetectedgeneraintheaquatichabitatswhichis duetoastrongrepresentationofthegenera Methylobacter, Methylocaldum ,and Methylomonas (Fig. 4).

Incontrast,proportionsofabsolutereadcountsweremoreevenlydistributed amongmajormethanotrophicgenerainterrestrialbiomes(Fig. 4;Table 2),possibly reflectingthedifferencesinselectivefactorsinterrestrialandaquatichabitats.An exceptiontothebroaddistributionofthegenerabelongingtothefamily Methylococcaceae isthegenus Methylosoma,whichexclusivelyisfoundinaquatic systems.Incontrasttothegenerallydisturbedrepresentativesofthegenera Methylobacter, Methylocaldum,and Methylomonas,membersofthefamily Methylocystaceae,moreinparticularofthegenus Methylocystis aremorestrongly associatedwithterrestrialthanwithaquaticbiomes(Fig. 4)althoughmanyisolates havebeenobtainedfromaquaticenvironments(Heyeretal. 2002).Membersofthe genus Methylosinus canbefoundpredominantlyinaquatichabitats.Withinthe familyof Beijerinckiaceae,thegenus Methylocella isonlyprevalentinterrestrial biomes.Representativesofthisgenusdonotcontainthe pmoA geneandthereforeare generallyunderrepresentedinstudiesofmethanotrophs(Rahmanetal. 2010). Methylocella isafacultativemethanotrophs,whichcanuseotherhydrocarbons suchasacetate,ethaneandpropane(Farhanetal. 2018),whichmayberelatedto itsenvironmentaldistribution.IthastobenotedthattheanalysesoftheEarth Microbiomedatasetarebasedonrelativelyshort16SrRNAsequencingreads (90–151bp),butevenwhenlookingonlyatsequencesrelatedcultivatedrepresentativestheanalysesofKnief(2015)isconfirmed,demonstratingclearbiogeographyof MBgenerawhichistheresultsofevolutionandselectionbasedonmanymoretraits thanthecapacitytooxidizemethane(Hoetal. 2013a, b, 2017a).Averyrecentstudy

Table1 Environmentsin researchstudiesoftheEarth MicrobiomeProject(EMP) (Thompsonetal. 2017)that includedmethanotrophic genera

Numberofstudies

Freshwater2757

Soil1581

Rhizosphere389

Biofilm387

Freshwatersediment328

Marinesediment321

Sand(biofilter)99

Organicmaterial(both)71

Mucus(humanandanimal)46

Feces(humanandanimal)35

Dustandair34

Animalnest28

Sebum(humanandanimal)22

Undergroundwater17

Seaandhypersalinewater15

Lakestromatolitemat13

Bodily fluid(animal)4

Brackishwater3

Saliva(humanandanimal)3

Animalexcreta3

Environmentalcategoriesweresimplifiedbutlargelyfollowedthe terminologyusedintheEMPmetadata.PleaserefertoFig. 3 for detailedmethodology

investigatingthebiogeographyofMBin697soilsamplesdistributedoverScotland providesstrongevidencethatevenonaregionalscaleclimo-edaphicfactors(Temperature,moisture,soilphysico-chemistry)influencesdistributionoftypeIIMBand USCα relatedsequencetypes(Nazariesetal. 2018).Land-use,moisture/rainfall, nutrientsandmetalswerethemostimportantfactorsinfluencingdistribution.The authorsofthisstudyusedtheclimo-edaphicrelationshipstomapMBdistribution overScotland,deliveringpotentialimportantinformationtoinformmodelspredicting greenhousegasemissionanduptakeinsoilunderfutureclimateorland-usechange scenarios(Nazariesetal. 2018).Similargeostatisticalapproacheswereusedonaplot scaletomapactivity,abundanceanddiversityofMBcommunitiesinawetlandsoil (Bodelieretal. 2013;Krauseetal. 2013;Wangetal. 2012)representingahydrologicalgradient.Onaplotscale(10m 10m),coveringthewhole floodingand subsequentmoisturegradient,methaneoxidationactivityanddistributionofthe activetypesofMBwereverywellexplainedbymoistureandthesubsequent availabilityofmethane(Bodelieretal. 2013;Wangetal. 2012).However,ona smallerscale(1m 1mand20cm 20cm)spatialdistributionwasaffectedby other,morelocalparametersnotdirectlyinfluencedbythelargerscalehydrological gradients(Krauseetal. 2013).Hence,MBdisplayclearbiogeographysubjectedto largescaledifferencesinabioticfactors,butonasmallscalelocalfactorsmayplaya moreprominentroleinnichedifferentiationandspeciationdependingontherespectiveenvironmentalproperties.

Fig.3 EnvironmentsinresearchstudiesoftheEarthMicrobiomeProject(EMP)(Thompsonetal. 2017)thatincludedmethanotrophicgenera.Environmentalcharacterizationwassimplifiedand basedonavailablemetadataoftheEMPproject.Fordatageneration,we firstusedtheoverview table “ChooseYourOwnEMPBIOMTable” fromtheEMPFTPsitetoselectaclosedreference OTUbasedquality filteredbiomedataset(filename:emp_cr_silva_16S_123.qc_filtered.biome). Subsequently,acustomizedR(RDevelopmentCoreTeam 2008)andQIIMEscriptswasusedto extractallmethanotrophicgenera,readcounts,andmetadata.Scriptsandrawdatacanbeprovided uponrequestandother filesareprovidedat ftp://ftp.microbio.me/emp/release1/ .Sampleprocessing, sequencing,andcoreamplicondataanalysiswereperformedbytheEarthMicrobiomeProject (www.earthmicrobiome.org ),andallampliconsequencedataandmetadatahavebeenmadepublic throughtheEMPdataportal(qiita.microbio.me/emp)

3.2SelectedHabitats

Consideringthewidedistributiondescribedabove,itwouldbeachallengeto describeMBinallhabitatsweretheyaredetected.Hence,inthischapterwewill focusonafewclimaterelevanthabitatswithanemphasisonnovelrecent developments.

3.2.1MethaneSinkHabitats

MBaretheonlybiologicalwayofmitigatingmethaneproducedinanoxichabitats butalsototakeupandoxidizemethanefromtheatmosphere.Uptakeofmethanein oxicuplandsoils(forest,grassland,deserts)represents6%ofthesinkpartintotal

Fig.4 Distributionofmethanotrophicgenerainterrestrial(a)andaquaticbiomes(b)accordingto metadataoftheEMPproject(Thompsonetal. 2017). TheseOTUswereonlyclassifiedtofamily levelintheEMPproject(Thompsonetal. 2017).Differentmethanotrophicgeneraweregroupedby theirphylogeneticgroupsofthefamilies Methylocystaceae, Beijerinckiaceae , Methylococcaceae , Methylothermaceae, Methylacidiphilaceae ,andCandidatedivisionNC10.Circleswerearranged usingtheRfunctioncircleProgressiveLayoutintheRpackagepackcirclesversion0.3.3 (RDevelopmentCoreTeam 2008).Thesizeofeachcircleisproportionaltotheabsoluteread counts.Circlesweredetachedforbettervisibility.Dataisbasedon4.067,99316SrRNAgene sequencingreads(Table 2)from6156studiespublishedintheEarthMicrobiomeProject(Thompson etal. 2017).TheoriginaldataisshowninTable 2.PleaserefertolegendofFig. 3 fordetailed methodology

globalmethanebalance(Deanetal. 2018)andisaprocessproposedlycarriedoutby asyetuncultivatedMBbelongingtotypeIdandIIbsequenceclusters(Dedyshand Knief 2018),betterknownasUSCγ andUSCα andrelatedclusters.Theprocess carriedoutbythesesocalled “highaffinity” MB(degradingmethanetoconcentrationsbelow1.8ppm)isaffectedbyarangeofenvironmentalparameters(CH4,O2, moisture,ammonium,agriculturalpractices,land-usechange,plants,organicacids

Table2 Absolutenumberofsequencingreadsofmethanotrophicgenerainaquaticandterrestrial biomes

TaxonomyAquatic_biomeTerrestrial_biome

Methylocystaceaea 182651272

Methylocystis 682830242

Methylosinus 106567165801

Methylocapsa 15241638

Methylocella 756061966

Methyloferula 14251457

Methylococcaceaea 173025272

Methylobacter 209128699226

Methyloglobulus 3470220175

Methylomarinum 161136

Methylomicrobium 2658918095

Methylomonas 382196158153

Methylosarcina 15815

Methylosoma 32430564

Methylosphaera 26785

Methylovulum 460198630

Methylocaldum 60483728607

Methylococcus 1460212032

Methylogaea 6022148

Methylothermus 60

Candidatus.Methylomirabilis 5485514

Methyloceanibacter 195858

aTheseOTU’swereclassifiedtothefamilylevel.Differentmethanotrophicgeneraweregroupedby theirphylogeneticgroupsofthefamilies Methylocystaceae, Beijerinckiaceae , Methylococcaceae , Methylothermaceae, Methylacidiphilaceae ,andCandidatedivisionNC10.PleaserefertoFig. 4 for detailedmethodology

andmonoterpenesetc.)ashasbeendescribedinarangeofstudies(Kolb 2009; Nazariesetal. 2011;Dunfield 2007;KolbandHorn 2012;Maureretal. 2008; Menyailoetal. 2010;Wieczoreketal. 2011).Recoveryafterdisturbancecantake decadesandmaydependonthediversityofMBpresent(Levineetal. 2011).Dueto thefactthatatmosphericmethanealoneisnotsufficienttogrow,allattemptstogrow andisolateatmosphericmethaneMBhassofarfailedandhence,novelinformation onthedistributionandfunctioningofthesemicrobeshasbeenverysparse.Novel habitatspotentiallycontributingtoatmosphericmethanedegradation,havebeen reportedrecently.Glacierfore fieldsoils(Naueretal. 2012),higharticcryosols (Lauetal. 2015),thawingpermafrost(Singletonetal. 2018),limestone(Waring etal. 2017),Lava(Pratscheretal. 2018)andkarstcaves(Zhaoetal. 2018).Genomes relatedtoUSCγ (Edwardsetal. 2017)andUSCα (Pratscheretal. 2018;Singleton etal. 2018)havebeenrecoveredfromarticsoilandfromforestsoils.Thesegenomes haveconfirmedtherelatednessofUSCα withgenus Methylocapsa andforUSCγ with Methylocaldum .ThenovelUSCα genomesrecoveredfromamireinNorthern

Swedenrevealedthepotentialforutilizationofacetate,confirmingwhatwasalready detectedbystableisotopeprobingearlier(Pratscheretal. 2011)aswellasthe presenceofgenesnecessaryforCOconsumption(Singletonetal. 2018).Pratscher etal.(2018)wasabletovisualizethe firstUSCα cellfromforestsoilsusinga combinationof fluorescencecellsortingusingacombinationofasuicidesubstrate and fluorescentinsituhybridization.Inthiswaysufficientcells,activelyoxidizing atmosphericmethanecouldbecapturedtoperformagenomeanalyseswhich revealedthatthesemicrobesonlyhaveXoxF(lanthanidedependent)methanol dehydrogenaseonly,genesinvolvedinexopolymerproductionandexcretionas wellasthecapacitytoformtrehalose.Thelattermaypointtowardsadaptationtodry orcoldconditionsandgrowthinbiofilms.Thelatter fittedwiththehighabundance ofthesemicrobesinsubterraneancavewallbiofilmsincavesglobally(Pratscher etal. 2018),asurveywhichwaspossiblewith16SrRNAprimersdevelopedusing thegenomeinfo.Allinall,thesenewdevelopmentspointintothedirectionthat atmosphericMBthriveinoligotrophichabitats,wheretheymakepartofpioneering microflora.

However,atmosphericmethaneoxidationisnotrestrictedtotheseexotic,enigmaticmicrobesbutpotentiallycanbecarriedoutbysomewell-knownlowaffinity (i.e.thriveathighmethaneconcentrations)MBwhichhavebeendemonstratedto oxidizeatmosphericmethaneinpurecultureincubations(KniefandDunfield 2005) althoughtheseMBcannotgrownorsurviveonatmosphericmethanealone. Recently,itwasdemonstratedthatagriculturalsoilscanbeturnedintoastrong sinkforatmosphericmethaneuponadditionoforganicamendments(e.g.compost, sewagesludge;aquaticplantmaterial)(Hoetal. 2015, 2017b).Although,the mechanismsresponsibleforthisphenomenonarenotyetclear,theuseofstable isotopeprobingincombinationwithPLFA(phospholipidfattyacid)demonstrated thatconventionalMB(i.e. Methylocystis; Methylosinus; Methyloferula )were responsiblefortheuptakeofmethaneintheseagriculturalsoils(Hoetal. 2019). Sincecellspecificactivitieswerelowerthanwhatisneededformaintenanceofcells, leadingtotheconclusionthattheseMBneedalternativesourcesofenergy.ConventionalMBinrice fieldsoilwereabletoturnthesoilintoapersistentandstrong sinkforatmosphericmethaneafterbeingspikedwithhighconcentrationsofmethane(Caietal. 2016).

Consumptionofmoremethane(i.e.repeatedincubationwith10,000ppmv)ledto asoilcommunitythatwasabletotakeupmethaneformorethana100days, demonstratingthatevenawetlandsoilcanturnintoasinkformethanewithout involvementofhighaffinityMB.UtilizationofstoragecompoundslikePHB(poly hydroxylbutyricacid)thatcanaccumulateduringperiodsofhighmethaneavailabilitywerehypothesizedtobethesourceofenergytocontinue pmoA activityunder atmosphericmethanesupply.Thismaypartiallyalsoexplaintheeffectofadditionof organicamendmentsinagriculturalsoil,whichmaypromoteinternalmethane productioninsoilaggregatesfuelingconventionalMBwithenergybutpotentially alsowithnutrientsandotheressentialmineralslikemetals(Fig. 5).Hence,with respecttosoilmethanesinksrecentnoveldevelopmentsoffernewviewsonfunctioningofhighaffinityMBbutalsopointtowardsthepotentialofasyetnon-methane

Fig.5 Proposedmechanismsofresidue-stimulateduptakeofatmosphericCH4 byagricultural soils.Additionoforganiccarboncanleadtoanaerobicconditionsmediatedbytheaggregate structureinthesoilwhichleadstointernalCH4 productionstimulatinggrowthofobligatoryand facultativeMBormethylotrophscarryingsMMOgenes.Thisstimulationmaybeenhancedby nutrients(N,P,metals,traceelements)presentintheresidues.Thesestimulatedcellswillhavea highercapacitytoco-oxidizeCH4 fromtheatmosphere

sinkhabitatstobeexploredfortheirroleinclimatechangemitigationscenariosby assessingthefunctioningofconventionalMBandtheirroleinatmosphericmethane uptake.Thelatterwillbenecessarysinceclimatechangephenomena(lowerrainfall, hightemperature)mayincreaseatmosphericmethaneconsumption(Festetal. 2017).

3.2.2Wetlands

Duetotheirtight-aquaticterrestrialcouplingandlargeorganicmatteraccumulation, wetlandsarehotspotsofbiogeochemicalprocessing.Thehighmethanogenicactivity intheiranoxic,carbon-richsoilshasmadethemlargestsourceofglobalatmospheric methane,emitting142-284TgCH4 peryear(Kirschkeetal. 2013).Thisprovidesan idealhabitatforanaerobicandaerobicmethanotrophs,whichbothareabundantin wetlands,andcanreduceCH4 emissionsby10–90%(Segers 1998).Thefunctioning ofaerobicmethanotrophsinwetlandshasbeenreviewedextensively(Conrad 2007; BodelierandSteenbergh 2014a, b;Dedysh 2009;Bridghametal. 2013;Bodelier 2011)mainlyalsoduetotherelevanceofthesemicrobesinmitigationofmethane emissionfromricepaddies(e.g.Bodelieretal. 2000;Krugeretal. 2002;Chowdhury andDick 2013).MBabundanceandcommunitycompositioninwetlandslargely dependonlocalenvironmentalconditions,inpartdeterminedbytheecosystemtype. Wecandistinguishseveraldifferenttypesofwetlands,includingpeatlands(bogsand fens,arcticpermafrost),saltmarshesandestuaries,tropical(natural)wetlands

Internal CH4 hypothesis

Smart residue mixes

includingriver floodplains,agriculturalwetlandssuchasricepaddies,andgeothermalwetlands.Methanotrophcommunitieshavebeenbestdescribedfortemperate and(ant)arcticecosystems,althoughsomeresearchhasbeendoneinthetropics, whichalsoharbourlargepeatlandareas.Inpeatlands,aerobicproteobacterial methanotrophsdominate,withmembersofthealphaproteobacteriadominatingin oligotrophic,acidic(pH < 5)bogs,whereasingeneralgammaproteobacteria dominateinminerotrophic,nutrient-richandonlymildlyacidic(pH > 5)fens (reviewedbyVerbekeetal. 2018).Wetlandsoccuroveracontinuumofhydrologicalandgeologicalconditions,andthereforeoftenliesomewhereinbetweenthese twoconditions,thusharbouringamixtureofalphaandgammaproteobacteria. Withinthealphaproteobacteriaoccurringinbogs, Methylocystis strainshavebeen foundtobemostactive,while Methylocella and Methylocapsa speciesarealso abundant(Verbekeetal. 2018).Withinthegammaproteobacteria, Methylobacter, Methylomonas and Methylomicrobium speciesarethemostabundant(Verbeke etal. 2018).Geothermalwetlandsarecharacterizedbyhightemperaturesand tendtobeveryacidic(pH < 4)duetosulfuroxidation.Methanotrophsisolated fromthesewetlandshavebeenfoundtobelongtothe Verrucomicrobia,andare currentlydescribedasthecandidatecluster “Methylacidiphilum” andthecandidate genus “Methylacidimicrobium” (Sharpetal. 2014).

Typically,aerobicMBarepresentatorabovetheoxic-anoxicinterfaceof wetlandsoils(Reimetal. 2012;Vaksmaaetal. 2017b).However,inrecentyears MBhavealsobeendetectedinassociationwith Sphagnum mosses(Larmolaetal. 2010;Kipetal. 2010),eitheroccurringinair-filledporeswithin Sphagnum tissues, orassociatedtotheirroots.ItwassuggestedthatMBliveinsymbiosiswithpeat mosses(Raghoebarsingetal. 2005)providingthemosswithCO2 whilereceiving photosyntheticoxygenfromthemoss.Otherstudiesreportedonmethanedependent nitrogen fixationbymossessuggestinganimportantroleforaerobicMBinpeat growth(Larmolaetal. 2014),whichwascontradictedbyotherstudiesfailingto observeaneffectofmethaneadditiononN2 fixationbymosses(Koxetal. 2018).As yetconclusiveevidenceformechanismofsymbiosisbetweenMBandpeatmosses inmissing(HoandBodelier 2015)asaretheresponsiblemicrobes(Koxetal. 2018). Asimilar,potentiallyfarreachingsymbiosisisclaimedforMBandriceroots,where diazotrophicMBintherhizosphere fixnitrogenusingmethaneasenergysourceand transferringthisnitrogentothericeplants(Ikedaetal. 2014).Agenesimilartothe oneinvolvedinplant-fungalsymbiosiswasproposedtobeinvolvedintheactivation ofrice-MBnitrogentransfer(Baoetal. 2014).However,besidesthelocalizationof typeIIMBproteinsinriceroottissue(Baoetal. 2014)nodirectproofforthis symbiosisisavailable,whichcanbeofgreatinfluenceforthesustainablecultivation ofriceemittinglessmethanewhilelessnitrogenfertilizerisrequired.Withrespectto thelatter,therecentlydiscoveredabilityofsomeMBtodenitrify(Dametal. 2013; Kitsetal. 2015)shedsnewlightoninteractionsbetweenmethaneandnitrogen cyclinginwetlandsystemsandbeyond.Therecentlydescribedgenomesequenceof thedenitrifyingmethanotroph Methylomonasdenitrificans FGJ1revealsgenes involvedinoxygenscavengingunderhypoxicconditions,hypothesizedtomaintain oxygenprovisionforthe pmoA underlowoxygenconditions(Orataetal. 2018).

Hence,consumptionofmethanemaybeprovidingwetlandplantswithnitrogenby diazotrophicMBbutontheotherhandmayalsoleadtoNlossbydenitrifying MB.MBcommunitycompositionwillplayacrucialroleinthisdelicatebalance. Whenitcomestowetlands,inmanycases Methylobacter speciesarethedominant activeMBpresent(Bodelieretal. 2013;Smithetal. 2018).Recentlyithasbeen shownthat Methylobacter genomesretrievedfromfreshwaterwetlandsharbour genesfordissimilatorydenitrificationaswellasgenesforperformingunderhypoxic conditions,suggestingthatthephenotypediscoveredinthedescribedstrain Methylomonasdenitrificans isfunctionalinnatureandismorewidespreadamong typeIMB(Smithetal. 2018).Thepreferenceoflowoxygenconcentrationsfor growthandactivityhasbeendescribedasanichedifferentiatingfactorforMBin otherwetland(Reimetal. 2012)andlakesediments(Hernandezetal. 2015)with always MethylobacterspeciesbeingdominantandactiveMBatlowoxygen(Oshkin etal. 2015). Methylobacter wasalsothemostactiveandabundantMBspeciesin articpeat(Tveitetal. 2013, 2014)aswellasinthehighmethanefensedimentlayers alonganpermafrostthawgradient(Singletonetal. 2018).Thelatterstudyrepresents themostextensivemetagenomeandmetatranscriptomeanalysesofMBcommunitiestodate.Permafostwetlandsreleasehighamountsofmethaneuponthawing whichmakesmitigationbyMBofhighenvironmentalrelevance.Theinvestigated thawinggradientinamiresysteminNorthernSwedenrevealedaclearniche differentiationoftypeIIaandb(USCα)MBinthepermafrostpalsaandinthe acidicbog,whiletypeIMBdominatedthehighmethanedeeperfensedimentlayers (Singletonetal. 2018).TheretrievedMBgenomesrevealedmanyfeaturesof relevanceofMBsurvivalandfunctioninginthesehabitatsandalsoyieldevidence for Hypomicrobia tobelongtothetypeIIMBfamilies.Theauthorsconcludedthat methaneconcentrationwasastrongselectivefactorshapingtheobservedniche differentiation.Fairlyrecent,anoveldescribedMB, CandidatusMethylospira mobilis (typeIb)fromanacidicpeatbog,showeddistinctchemotacticbehaviour towardshighmethaneandlowoxygen,demonstratingthepotentialforMBto activelypositionthemselveswithintheenvironmentalgradientsinherenttowetland habitats(Danilovaetal. 2016).

Thelatterwillbequiteimportantgiventhemostrecentviewonmethane oxidationinwetlandsystemswhereaerobicMBdonothaveexclusiveaccessto methaneconsideringthepresenceofanaerobicmethaneoxidisers(AOM)whichcan mediatedanaerobicmethaneconsumptionbyvariouselectronacceptors,including iron,sulphate,andmanganese(Deanetal. 2018;Vaksmaaetal. 2017b;Welteetal. 2016).Anaerobicmethaneoxidationcanbecoupledtonitriteornitratereduction. Thishasbeenobservedinthecandidatespecies Methylomirabilisoxyfera(nitriteAOM), firstenrichedfromnitrite-richsediments(Ettwigetal. 2010)andtheANME2Darchaeallineage “Candidatus Methanoperedenaceae”,whichhasbeenrecently enrichedfromrice-paddysoils(Haroonetal. 2013;Vaksmaaetal. 2017a).Inrice paddies(Vaksmaaetal. 2017b)aswellasincoastalwetlands(Heetal. 2019), anaerobicmethaneoxidationratesaswellasnumberscanbesubstantialandindicate thatastrongnichedifferentiationofAOMandMBinwetlandsystemtogether formingeffective filtersforthemethaneproducedbyoxidizingfromsourceto

atmosphere.However,theeffectivenesswillstronglydependontheenvironmental gradientsbutalsoonthetraitsandcompetitiveabilitiesoftherespectiveAOMand MBpresent.

Sometimes,importantcontrollingfactorsareoverlookedintheseinteractions.In arangeofwetlandditchesastrongcorrelationwithmethaneaerobicmethane oxidationandphosphorusavailabilitywasfoundinthesurfacecmofthesediments whichweredominatedbytypeIMB(Veraartetal. 2015).Scanningtheliteratureon these findingsrevealedstrongpositiveeffectsofPonmethaneoxidationinarctic permafrostwetlands,alsodominatedbytypeIMB(Grayetal. 2014).BrowsingMB genomesontraitsrelatingtoPuptakeandutilizationrevealedaclearseparation betweentypeIandIIMB,pointingtowardsastrongerrelianceonPintypeIMB, makingPanimportantnichedifferentiatingfactorinwetlandsystems(Veraartetal. 2015).

3.2.3AquaticSystems

Aquaticsystemsarewaterbodiesclassifiedasfreshwater,transitional,coastalor marine,coastalandmarine.Themarineecosystemisthebiggestcoveringalmost 70%oftheEarthsurfacebutCH4 releasedfromthemtotheatmosphereisasmall sourcewithameanvalueof12TgCH4 year 1 incomparisontoothernatural environments(Saunoisetal. 2016).Onthecontrary,freshwaterecosystems(~4% Earthsurface)cancontributeupto100TgCH4 year 1 (Bastvikenetal. 2011). EstimationsorfuturetrendsinCH4 emissionslackrobustnessduetotherestricted numberofmeasurements,differentmethodologyusedandalsothelittleknowledge onthedynamicsofCH4 emissionsinsomesystems(Reayetal. 2018).Thereleaseof CH4 fromaquaticsystemstotheatmosphereinvolvesprocessessuchasdiffusion, ebullition(Walteretal. 2007),seepage,resuspension(Bussmann 2005)orbioturbation(OliveiraJunioretal. 2019)ofthesediments.Typically,methaneproduction occursinsedimentsoranoxicbottomwaterlayers.However,fairlyrecentitwas discoveredthatoxicwatersaresupersaturatedwithCH4 (Grossartetal. 2011;Karl etal. 2008).Thisphenomenonofthesocalled “methane-paradox” hasspurred aquaticmethanecyclingresearchtopinpointthepossibleabiotic(DelSontroetal. 2017;McGinnisetal. 2017)orbiotic(Grossartetal. 2011;Tangetal. 2016;Yan etal. 2019)factorsunderlyingthisphenomenon(Yanetal. 2019).Thelargeroleof freshwatersystemsinglobalmethaneemission,combinedwiththeunexpectedhigh methaneconcentrationsinoxicwatercolumnshasspurredattentionforMBin aquaticsystems.

MarineCH4 oxidationcanbeperformedbyanaerobicandaerobicCH4 oxidizers (Ruffetal. 2019).Anaerobicoxidationisdrivenbysyntrophicinteractionbetween archaeaandSO4 2-reducing δ-Proteobacteria(Krukenbergetal. 2018).TheseconsortiaofANME-archaea(ANME1,2and3)andsulfate-reducingbacteria(SRB) dominatemethaneoxidationindeep-seasediments(Ruffetal. 2013;McGlynnetal. 2018;Durisch-Kaiseretal. 2005).Thisinteractiondependsoncontrolledgene expressiontoenhanceelectrontransferamongtheconsortiummembers(Krukenberg

etal. 2018;McGlynnetal. 2018).AerobicMBinmarineenvironmentsarecomprised by fivesocalleddeep-seaclustersallassociatedwithtypeIlineages(reviewedin Knief 2015).Asyet,thereisnoclearevidenceofthefactorsthatdriveniche differentiationofMBbelongingtotheseclusters(Ruffetal. 2013).Despitethelow amountofO2 indeepregionsofmarinesystems,aerobicmethanotrophsfromthe orderMethylococcalesarepresent(Ruffetal. 2013;Tavorminaetal. 2008).Ithas beenrecentlydescribedthatANME-archaeaandTypeIMBforma “methanotrophic microbiome” whereTypeIMBarethe firsttocolonizethedeep-seasediments(Ruff etal. 2019).RegardingTypeI,somelineagesrelatedtoterrestrialenvironmentsare presentinadeep-seafanbecauseoftheinputsofsoilderivedorganicmatter(Bessette etal. 2017).The pmoA phylotypesfoundinthewater-columndifferfromtheones inhabitantthesedimentsintermsofdiversityandabundance.Forinstance,the planktonicMBmembersaredistributedinthreecladesbelongingtotheorderof Methylococcaleswhereasthebenthiconesarelessdiverse(Tavorminaetal. 2008). Similartowetlands,MBcanbefoundinOxygenMinimumZones(OMZ),regionsin oceansandseaswhereO2 dropsdowntonanoorpicomolarlevels.Theseregions showanactiveCH4 oxidationwhereTypeIMBandNC10(Chronopoulouetal. 2017)seemedtoplayarelevantrole.

Freshwaterecosystemsharborenvironmentssuchaslakes,rivers,pondsand reservoirsplayingarelevantroleintheCcycleaswellasinthecyclingofother nutrients.Theyalsoprovidemanyservicesaswater,electricalenergyandfood supplyorcanserveasrecreationalareas(Vollmeretal. 2016).Still,theyarevery vulnerabletoclimatechangeespeciallyregardingtheincreaseoftemperaturewhich altersthethermalstructureandwaterchemistryofthesesystems(Yvon-Durocher etal. 2011).Amongthefreshwatersystems,lakesarethemoststudiedinrelationto CH4 dynamics.Lakescoverasmallarea(0.9%)oftheEarth’ssurfacebuthavea largecontribution(6–16%)tothetotalglobalCH4 budget(Tangetal. 2016; Bastvikenetal. 2004).Incomparisontootherenvironments,freshwatersystems hold “endemic” MBwhichwemaycalltypicalfreshwaterMB.Theselineages comprisesixsequenceclusterswithintypeIaandIbMB(Knief 2015;Dedyshand Knief 2018).MostoftheseclustersareubiquitousinFWsystems,exceptfortheso calledcluster4awhichisonlydetectedsofarinsedimentswhilecluster2ismore frequentinthewatercolumn(Knief 2015).FirstreportsonfreshwaterMBcommunitiesfocusedonthesediment(NaguibandOverbeck 1970;ReeburghandHeggie 1977;Cappenberg 1974).Thesestudies,demonstratedclearlythatsedimentsharboredmuchhighernumbersofMBthanthepelagicpartofthelake(Zhouetal. 2018).Inmostpelagiczonesoflakes,typeIMBbelongingtothegenus Methylobacter arethedominatingMBgroup(Bleesetal. 2014;Biderre-Petitetal. 2011;Kojimaetal. 2009),Recentlyanovelgenuswithinthefamilyofthe Methylococcaceae, CandidatusMethylomidiphilusalinensis,wasdemonstratedto bethemostabundantMBinahumiclakeinFinland(Rissanenetal. 2018).Nextto this,MBrelatedthe filamentousbacterium Crenothrixpolyspora werereportedtobe themainCH4 sinkintwostratifiedlakes(Oswaldetal. 2017),demonstratingthe lackofknowledgeonMBinfreshwaterhabitats.Incontrast,otherreportsfoundthat TypeIIrepresentativesmayplayarelevantroleinthelacustrineCH4 cycle.For

instance,inanalkalinelaketherewasanalmostequalabundanceoftypeIIMB, represented2%ofthewholemicrobialcommunity,whiletypeIaccountedbetween 3%and5%(Carinietal. 2005).Intheoxicwatersofanutrientdepriveddeeplake (Zigahetal. 2015)typeIIwasmoreabundantandactiveinnutrientthantypeI (Parksetal. 2018).Similarresultswerefoundinsub-arcticlakes(Heetal. 2012). Surprisingly,highabundanceoftypeIMBhavebeendetectedinanoxicpartsof thewatercolumnoflakes(Schubertetal. 2011)suggestingthattheyareeither inactiveorperforminganaerobicmethaneoxidation.However,thisisnotthecaseas theywereactivelytranscribing pmoA andnoarchaealAOMweredetectable(Blees etal. 2014;Rissanenetal. 2018;Miluckaetal. 2015).Thiswouldsuggestthatthey areinteractingwithothermembersofthemicrobialplanktoniccommunityorusing otherelectronacceptorsratherthanO2.IncubationswithlightincreasedtheCH4 oxidationinanoxicwatersamplesdominatedbyTypeIfromboreal(Rissanenetal. 2018)andtemperatelakes(Oswaldetal. 2015).Thisimpliesaninteractionbetween MBandphototrophs(oxygenicphotosynthesis)asdemonstratedrecentlyina numberoflakes(Miluckaetal. 2015;Oswaldetal. 2016).Hence,thelowamount oflightpenetratingintothedeeperparsoflakesissufficientforphototrophsto produceoxygenusedbymethanotrophstooxidizemethaneaerobically.Recently,it wasalsoreportedthatmembersoftheNC10phylum,i.e. Candidatus Methylomirabilisoxyfera canplayanimportantroleinmethaneoxidation.This bacteriumlinkstheCandNcyclesbycouplingCH4 oxidationwithNO2 /NO3 reduction(nitrate/nitrite-dependentanaerobicmethaneoxidation,n-damo).Ina freshwaterreservoir,clonelibrarieswerecomprisedoftypeI,IIMBwithadominanceofmembersoftheNC10phylum.Thelatterdominatedindeepwatersofthis system(Kojimaetal. 2014).Morerecently,abloomofNC10 “Ca. Methylomirabilis limnetica” reaching27%ofthewholebacterialcommunitydominatedthedeepest waterlayersofastrati fiedlake(Grafetal. 2018).Asimilarstronglinkbetween nitrogencyclingandmethaneoxidationwasfoundin15Indiandam-reservoirs. Using 15N-incubationsitwasshownthatCH4 amendmentboostedNlossfromthis systemswithmorethan10timesduetodenitri ficationbyaerobicMB(Naqvietal. 2018).

AnaerobicCH4 oxidation(AMO)alsotakesplaceinfreshwatersystemsand playsasignificantroleinfreshwaterCH4 dynamics.Thisprocesscanhappen simultaneouslywiththeaerobicoxidation(Arpetal. 2018)orseasonally(Roland etal. 2017).CH4 oxidizersdifferentfromtheProteobacterialwere firstdescribedin anoxicwaterlayersofLakePluβsee(Elleretal. 2005).Thisstudyshowedthe existenceofanaerobicmethane-oxidizingarchaea(ANME1and2)andtheroleof theanaerobicCH4 oxidationinafreshwatersystem.Differenttotheirmarine counterparts,thesearchaealmethanotrophsseemednottodependonsulfatereducingpartners,astherewasnoevidenceofphysicalattachmenttothem.Ina meromictic,temperateandeutrophiclake,CH4 anaerobicoxidationwascoupled withSO4 2 reductiononceNO3 isscarceorbothacceptorsmaybeusedconcomitantly(Rolandetal. 2017).Inatropicallake,anewclusterofANME(differentfrom themarinemembers)wasdescribedandassociatedtoSO4 2 (Zigahetal. 2015).