Microbial megacities fueled by methane oxidation in a mineral spring cave


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Microbial megacities fueled by methane oxidation in a mineral spring cave

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Microbial megacities fueled by methane oxidation in a mineral spring cave
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The ISME Journal
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Karwautz, Clemens
Kus, Günter
Stöckl, Michael
Neu, Thomas R.
Lueders, Tillmann
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Microbiota ( local )
Biofilms ( local )
Mineral Spring Cave ( local )
Methane Oxidation ( local )
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Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.
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The ISME Journal, Vol. 12 (2017-09-26).

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OPEN ORIGINALARTICLEMicrobialmegacitiesfueledbymethaneoxidationin amineralspringcaveClemensKarwautz1,GünterKus2,MichaelStöckl1,ThomasRNeu3andTillmannLueders11InstituteofGroundwaterEcology,HelmholtzZentrumMünchen – GermanResearchCentrefor EnvironmentalHealth,Neuherberg,Germany;2BavarianEnvironmentAgency(LfU),Department10: GeologicalSurvey,Hof/Saale,Germanyand3DepartmentofRiverEcology,HelmholtzCentrefor EnvironmentalResearch-UFZ,Magdeburg,GermanyMassivebiofilmshavebeendiscoveredinthecaveofaniodine-richformermedicinalspringin southernGermany.Thebiofilmscompletelycoverthewallsandceilingsofthecave,givingriseto speculationsabouttheirmetabolism.Herewereportonfirstinsightsintothestructureandfunction ofthebiofilmmicrobiota,combininggeochemical,imagingandmolecularanalytics.Stableisotope analysisindicatedthatthermogenicmethaneemergingintothecaveservedasanimportantdriverof biofilmformation.Theundisturbedcavernatmospherecontainedupto3000p.p.m.methaneandwas microoxic.Ahighabundanceanddiversityofaerobicmethanotrophsprimarilywithinthe Methylococcales ( Gammaproteobacteria )andmethylotrophic Methylophilaceae ( Betaproteobacteria ) werefoundinthebiofilms,alongwithasurprisingdiversityofassociatedheterotrophicbacteria.The highestmethaneoxidationpotentialsweremeasuredforsubmergedbiofilmsonthecavernwall. Highlyorganizedglobularstructuresofthebiofilmmatrixwererevealedbyfluorescentlectin staining.Weproposethattheextracellularmatrixservednotonlyasanelectronsinkfornutrientlimitedbiofilmmethylotrophsbutpotentiallyalsoasadiffusivebarrieragainstvolatilizediodine species.Possiblelinksbetweencarbonandiodinecyclinginthispeculiarhabitatarediscussed. TheISMEJournal (2018) 12, 87 – 100;doi:10.1038/ismej.2017.146;publishedonline26September2017IntroductionNaturalmicrobiotaoftenorganizeasbiofilms,where structuralfeaturesandmicrobialinteractionsgive risetoanenhancedabilityofbiofilmmicrobiotato beactiveandpersistunderchallengingenvironmentalconditions.Extensivebiofilmproductionhas beenpreviouslyreportedmostlyforenergy-rich surfacewatersystemsdominatedbyphototrophic primaryproduction(Battin etal. ,2016)orin engineeredwatersystems(Boltz etal. ,2017).In subsurfaceandgroundwatersystems,biofilmsare largelyconsideredoligotrophic(Grieblerand Lueders,2009;Ortiz etal. ,2014).Nonetheless,a numberofcavesandkarsticsystemshavebeen reportedtohostbiofilmsrichinmicrobialdiversity andwithelevated,mostlylithotrophicbiogeochemicalactivities(Holmes etal. ,2001;Engel etal. ,2010; Jones etal. ,2010;Rusznyák etal. ,2012;Barton etal. , 2014;Riquelme etal. ,2015). Inthisstudy,wereportonanexceptionally extensiveandmassivebiofilmformationthathas recentlybeendiscoveredinasemiartificialcaveofa historicmedicinalspringinSulzbrunn(Schott, 1858),situatedinprealpinesouthernGermany. Subaerialandsubmersedmicrobialbiofilmscompletelycoverthewallsandceilingofthisseminaturalcave(Figure1),givingrisetoextensive pendulous,mucousstructuresofupto15cmin lengthalsoknownassnottites(HoseandPisarowicz, 1999).Todate,microbialsnottiteshavemostlybeen describedtoharborlow-diversitycommunitiesof lithotrophsinacidophilic,thermophilicorsulfidic habitats(Bond etal. ,2000;Holmes etal. ,2001; Northup etal. ,2003;Jones etal. ,2010;Ziegler etal. , 2013).Suchextremeconditionsdonotseemto prevailinSulzbrunn.Thusourobjectivewasto understandtheprimarybiogeochemicaldriversof thispeculiarmicrobialhabitat. TheSulzbrunncaveislocatedintheAllgäuAlps (Bavaria,Germany)atanaltitudeof875mabovesea level.Thecaveliesinawell-jointedsandstoneofthe Weissach-SchichtenofthesubalpineLowerFreshwaterMolasse.Withinaradiusof18kmfrom Sulzbrunn,naturalgashasbeenrepeatedlyobserved toemergefromdeepdrillholesthatreachTertiary formationsofthesubalpineMolasse.Theporous sandstoneofBausteinschichtenfromtheLower Correspondence:TLueders,InstituteofGroundwaterEcology, HelmholtzZentrumMünchen – GermanResearchCentrefor EnvironmentalHealth,IngolstädterLandstrasse1,Neuherberg 85764,Germany. E-mail:tillmann.lueders@helmholtz-muenchen.de Received7April2017;revised23July2017;accepted1August 2017;publishedonline26September2017 TheISMEJournal(2018)12, 87 – 100www.nature.com/ismej

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MarineMolasseisidentifiedasareservoirformigratinghydrocarbons.Thesubjacentorganic-richclaysareapotentialsourceofbrineswithhighiodineconcentrations(HesseandSchmidt-Thomé,1975).Waterentersthecaveintheformofupwellingmineralspringwater,aswellaspercolatingseepagewater.Undernormalhydraulicregimes,thissemi-artificialcaveisapproximatelyhalf-filledwithwater,withthewaterlevelcontrolledbyasimpleoverflowsystem.Historic(Schott,1858),aswellasrecentwateranalyses(LfU—BavarianEnvironmentAgency,personalcommunication2014),reporthighiodineloadsofupto23mgl1emergingwiththemineralspringwater,whichmixeswithrecentmeteoricgroundwaterinthespringcavern.Thesehighiodinelevels,whichexceedregularfreshwaterconcentrationsbyathousand-fold(Whitehead,1984),aswellaselevatedsalinityinthemineralspringwater,areanindicatorofupwellingformationwater,whichhasbeenincontactwithoil-andgas-ladensedimentdeposits(Luetal.,2015).ThesourcesofhydrocarbonsinthesubalpineMolassebasinareautochthonic,originatingfrommesozoicsediments(Hiltmannetal.,1999)overthrustedbyMolasseformationsduringthealpineorogeny.Fossilizedalgalbiomassistypicallyhighlyenrichediniodine,whichhasbeenfoundatconcentrationsofupto150mgl1inadrilledartesianwellinthearea(LfU,personalcommunication2014).InSulzbrunn,thesedimentsoftheLowerMarineMolassearesituated41000mbelowthesurface.Itcanbeassumedthattheupwellingofiodine-richwatersoccurstogetherwithnaturalgasesseepingfromdeeperhydrocarbonformationsalongdeeplypene-tratingfaultsystems.Althoughthemicrobiotaofmarinegasseepshavebeenintensivelyinvestigated(Ruffetal.,2015;Pauletal.,2017),comparablylittleinformationisavail-ableaboutsuchsystemsintheterrestrialsubsurface.Aerobicmethanotrophsandbiofilmshavebeenpreviouslyfoundingroundwateranddrinkingwatersystems,wheretheycanbeinvolvedintheoxidationofmethaneormethylatedcompounds(Newbyetal.,2004;Stoeckeretal.,2006).TheMovileCaveinRomania,receivingdeepthermalwatersrichinhydrogensulfide(Sarbuetal.,1996),alsohostsmicrobialmatsofactivemethanotrophs(Hutchens 8m1.2m16mHillslopeMineral spring waterMixed cave waterBedrockCemented gallOutßowPercolating seepage water Snottites50 cmSubaerialSubmersedWater level5 cm10 cm 2 cm Figure1(a)Conceptualcross-sectionoftheSulzbrunnspringcavernanditswaterflows(nottoscale).Insetimages:(b)Collectingnaturalgasseepingfromthespringpoolinaninvertedglassbottleusingafunnel.(c)Naturalbedrockandtheemptiedspringpoolduringbiofilmandmineralspringwatersampling.(d)Thefilledspringpoolwithsurroundingnaturalbedrockandman-madegallerycoveredwithbiofilms.(e)Thethreedistinctbiofilmcompartmentssampledinthisstudy:submersedandsubaerialwallbiofilms,snottitesattheceiling.(f)Close-upofthick,slimysnottites.(g)Close-upofsubaerialbiofilmsatupperwallandceiling.(h)Close-upofsubmersedwallbiofilms. MethanotrophicmicrobialmegacitiesCKarwautzetal88 TheISMEJournal

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etal. ,2004;Chen etal. ,2009).Recently,theroleof microbialmethaneoxidationwithincaveandkarst ecosystemshasbeenaddressedglobally(FernandezCortes etal. ,2015;McDonough etal. ,2016;Lennon etal. ,2017).Methane-drivencommunitiescan compriseamultitudeofinteractionsbetweenmethanotrophs,methylotrophsandheterotrophs(Beck etal. ,2013;Kalyuzhnaya etal. ,2013;Oshkin etal. , 2015;Paul etal. ,2017).Energeticconstraints imposedbythevariouspathwaysofcarbonassimilationunderlowconcentrationsofoxygenand methanehavebeenshowntotriggersubstantial exopolysaccharideproductioninmethanotrophs (Linton etal. ,1986;Strong etal. ,2015),whichcould potentiallyexplainsuchmassivebiofilmproduction. Theappearanceanduniformdistributionofthe snottites(Figure1)pointedtowardtheuseofagaseous substrateforgrowth.Thuswepositthatdeepgaseous energyinputsemergingwiththeupwellingwater, possiblylightalkanesormethane,couldbeamajor driverofbiofilmformationintheSulzbrunncavern. Wehypothesizethatthesnottites,aswellassubaerial andsubmersedbiofilms,onthewallshouldbe dominatedbyalowdiversityofautotrophscapitalizingontheavailableenergyinputs.Thecompartmentalizationofthecaveandpossibledistinctionsin substratesupplyshouldbereflectedindistinctbiofilm subtypes,substrateturnoverratesandisotopicsignatures.Finally,weaskwhetherpossiblelinksbetween methaneandiodinecyclingcanbeinferredforthis peculiarmicrobialhabitat.MaterialsandmethodsSiteandsamplingThesemiartificialSulzbrunncave(47.67°N;10.39°E) isaccessiblebydescending8mviaametalladder (Figure1).Enteringthecavewithoutbreathing equipmentisonlypossibleduringcoldseasons,as limitedairexchangethroughthechutedentrance causesmicrooxicconditions,especiallyduringwarmeroutsidetemperatures.Biofilmsfromboththe man-madecementedgalleryandthedistalbedrock wallsweresampledin2consecutiveyears(November2012andDecember2013)formolecularanalyses.InOctober2015,additionalbiofilmsamples weretakenformicroscopy.Inordertosample biofilmsandmineralspringwaterfromrockfissures attheendofthecave,waterfillingthegallerywas pumpedout(Figure1b).Biofilmsampleswere collectedfromthreedifferentcompartments:submersedbiofilms(~30cmfromthebottomofthe wall),subaerialbiofilms(~20cmbelowceiling),and snottitestakenfromtheceiling(Figures1eandf).At eachsamplingtimepoint,thethreecompartments weresampledinreplicatesalongahorizontal transectspanning16mofthecavegallery.Biofilm samplesweredirectlytransferredbyscrapinginto sterilepolypropylenetubes(Falcon,Becton Dickinson,FranklinLakes,NJ,USA),andallsamples werefrozen( 20°C)within6haftersamplingfor molecularanalyses. Watersamplesofpercolatingseepagewater, mineralspringwaterandthemixedcavewaterwere collectedinsterile,1-literglassbottlesformicrobiologicalandphysicochemicalanalyses.Mineralspring waterwasrepeatedlytaken(November2012,December2013)fromastainless-steelsamplingflume installedatafissureatthebackofthespringcave, whereasseepagewaterwascollected(December2013) withasterileglassfunnelfromtheceiling.Mixedcave water(Figure1d)wascollectedduringseveraloccasions(October2012,November2013,December2014, October2015)usingaRuttnersampler(KCDenmark A/S,Silkeborg,Denmark).Watersampleswerefiltered usingsterilefiltertops(0.2 m;Corning,Corning,NY, USA).Microbialcellswithinthewatersampleswere countedusingSybrGreenforDNAstainingonaflow cytometer(BeckmannCoulterFC500,Beckmann Coulter,München,Germany)aspreviouslydescribed (Bayer etal. ,2016).PhysicochemicalanalysesTemperature,pH,dissolvedoxygenandspecific conductivityofwatersamplesweremeasuredwith calibratedfieldsensors(Hach,Düsseldorf, Germany).Watersampleswereanalyzedfordissolvedorganiccarbon(DOC)usingaTOC-V(Shimadzu,Neufahrn,Germany).Priortoinjection,DOC samples(0.45 mfiltered)wereautomaticallyacidified(pH o 2),spargedwithoxygentoremove inorganiccarbonandanalyzedbyhigh-temperature combustion(Mathis etal. ,2007).Majorcations (calcium,magnesium,potassium,ammonium, sodium)andanions(nitrite,nitrate,chloride,bromide,sulfate)weremeasuredonaDX-100(Dionex, Germering,Germany)ionchromatographas described(Stoewer etal. ,2015).Totaliodineconcentrationsinwatersampleswereanalyzedbyion chromatography – inductivelycoupledplasmamass spectrometry(MichalkeandWitte,2015).Analysis ofwaterstableisotopeswascarriedoutbycavity ring-downspectrometry(PicarroL2120-I,Picarro, SantaClara,CA,USA)for2Hand16Oandwithliquid scintillationcountingfor3Handwasusedto estimatewatermixingratios(Stoewer etal. ,2015). Gassampleswereeithertakendirectlybycollectinggasbubblesemergingfromthedistalspringpool intowater-filled,invertedbottlesorbypumping fromtheundisturbedcaveatmospheretotheoutside ofthecavernviatubinginstalledattheceiling. Tubingvolumewasflushed5×beforesamplecollection.MethaneandCO2werequantifiedwithin24h aftergassampling,whilegasforisotopeanalysiswas keptinthedarkat4°Cinappropriateglasscontainersuntilmeasurement.CH4andCO2werequantified byinjecting250 lofgasviaaHayeSepDcolumn (80 – 100mesh,6m×1/8')toagaschromatograph(GC)equippedwithheliumionizationand thermalconductivitydetectors(SRIInstruments, Methanotrophicmicrobialmegacities CKarwautz etal89 TheISMEJournal

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BadHonnef,Germany).Methanedissolvedinwater wasquantifiedusingtheheadspaceequilibration method(KampbellandVandegrift,1998).13Cand2H abundanceofgaseswasmeasuredbyseparationona RT – QPLOT(30m×0.32,Restek,BadHomburg, Germany)columnandmeasuredusingaTRACE GCUltra(ThermoFisherScientific,München, Germany),coupledtoaFinniganMAT253IRMS (ThermoFisherScientific)viaaFinniganGC CombustionIIIInterfaceaspreviouslydescribed (Bergmann etal. ,2011). Forisotopicandelementalanalysis,biofilmswere lyophilizedandgroundtopowder.13Cand15N abundancewasmeasuredonanelementalanalyzer (EA;EuroVectorSPA,Redavalle,Italy)witha combustionunit(Hekatech,Wegberg,Germany) connectedtoaMat253IRMSaspreviously described(Bernstein etal. ,2010).Traceelement analysisoflyophilizedbiofilmswascarriedoutfor totaliodine,phosphorous,ironandsulfurby inductivelycoupledplasma – opticalemissionspectrometry(Hou etal. ,2006).Furtherdetailsonthe physicochemicalandisotopicanalysesareavailable inSupplementaryInformation.ActivitymeasurementsMethaneoxidationratesweremeasuredfortriplicates offreshbiofilmsamplestakenfromthethreedifferent compartments.Forthis,~6gofbiofilmsampleswere dispersedin20mlofnitratemineralsaltmethanotrophmedium(Whittenbury etal. ,1970)filledinto 250mlglassbottlesandsealedwithbutylrubber stoppers.Inall,16000p.p.m.ofCH4(Linde, München,Germany)wasaddedtotheair-filledbottles andmethaneoxidationwasfollowedovertimeusing GCmeasurementsdescribedabove.Theincubations werecontinuouslyshaken(150r.p.m.)andkeptinthe darkat12°C.Gasanalyseswerecarriedoutafter0,24, 48and72hofincubation.Methaneuptakerateswere normalizedtobiofilmfreshweight(gFW).BiofilmvisualizationStructuralfeaturesofthebiofilmswereexaminedby confocallaserscanningmicroscopyofextracellular polymericsubstancesandglycoconjugateswith fluorescentlylabeledlectinsasdescribed(Zhang etal. ,2015).Biofilmswereusedfresh,fixedin paraformaldehydeorembeddedinO.C.T.compound (Tissue-Tek,SakuraGmbH,Staufen,Germany). Sampleswerecutintothinsectionsusingarazor bladeoraCM3050Scryotome(Leica,Wetzlar, Germany).Variousnucleicacid-specificstains,for example,SybrGreen,Syto9andSyto60(Molecular Probes,Leiden,TheNetherlands)wereusedfor stainingofbacterialcells.Glycoconjugateswere contrastedwithfucose-specificAALlectin(Vector Laboratories,Burlingame,CA,USA)labeledwith Alexa-488,Alexa-568orAlexa-633fluorochromes (MolecularProbes).Aconfocallaserscanningmicroscope(TCSSP5X,Leica)equippedwithasuper continuumlightsourceandcontrolledbytheLAS AFsoftware(ver.2.6.1.,Leica)wasusedforimaging. Imageswerecollectedat1 msectioningintervals usingthe25×NA0.95wiand63×NA1.2wi objectivelenses.Signal-to-noise-ratioswereoptimizedusingtheglow-over-underlookuptable. Multichannelimagedatasetswereprojectedby usingtheImarissoftware(ver.8.2.0,Bitplane, Zürich,Switzerland).DNAextractionandmolecularanalysesDNAwasextractedfrombiologicallyreplicated samples( n =2 – 4).Frozenbiofilmswereresuspendedin1×phosphate-bufferedsalinebuffer anddisruptedbysonication(CuryandKoo,2007). Repetitive(3×)sonication(35kHz,SonorexRK102; BandelinElectronicGmbH&Co.,Berlin,Germany), shakingandspinning(5500 g for10minat4°C)was usedtointerrupttheextracellularmatrix.DNAfrom cellpelletsandfiltersfromwatersamplingwas extractedfollowingthepreviouslypublishedprotocols(Pilloni etal. ,2012). QuantitativePCRofbacterialrRNAgenesinDNA extractedfrombiofilmandwatersampleswas performedaspublished(Pilloni etal. ,2011).In addition,apreliminaryscreeningofthediversityof methanotrophmarkergenesinbiofilmswascarried outbyterminalrestrictionfragmentlengthpolymorphismfingerprintingasdescribedin SupplementaryMethodsandSupplementaryTable S1.Barcodedampliconsofbacterial16SrRNA genes,coveringtheV1 – 3region,weregenerated andsequencedonaFLX+GenomeSequencer(454 LifeSciences,Roche,Indianapolis,IN,USA)as previouslydescribed(Pilloni etal. ,2012;Karwautz andLueders,2014)butanalyzedandclassifiedusing theSILVAngsdataanalysisplatform(Pruesse etal. , 2012;Quast etal. ,2013).Defaultsettingswereused forqualitycontrol,de-replication,operationaltaxonomicunit(OTU)clusteringandclassificationona 97%sequenceidentitylevel.TaxonomicassignmentswerebasedontheSILVAdatabaserelease 123(24July2014).Thesequencingdatasetwas furtherprocessedusingthephyloseqpackage (McMurdieandHolmes,2013)withintheRenvironmentversion3.1.2(RDevelopmentCoreTeam, 2013).Meanabundanceofthemostprevalenttaxa ( 4 3%relativeabundance)fromreplicatesamples wereplottedasKronaplots(Ondov etal. ,2011). Furtherdetailsonmolecularanalysesareavailable inSupplementaryInformation.Allsequencingdata havebeendepositedwiththeEBIsequenceread archiveundertheBioProjectIDPRJEB14605.ResultsGeochemicalcharacterizationofcavewater, atmosphereandbiofilmsThemineralspringwaterwassaline,microoxicand carriedonlylowamountsofDOC,nutrients, Methanotrophicmicrobialmegacities CKarwautz etal90 TheISMEJournal

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phosphateandalternativeelectronacceptors,such asnitrateorsulfate(Table1).Totaliodineconcentrationwas~20mgl 1inthespringwaterand dilutedto~3mgl 1inthemixedcavewater.The temperatureofthemixedcavewaterwasbetween8 and11°Cdependingonthetimepointsofsampling, whilethemineralspringseemedconstantat~7°C. ThepHofthemixedcavewaterwascircumneutral (pH~7.5).Analysisofthewaterstableisotopes( 18O and 2H)allowedestimationofratiosofmixing betweendeepspringwaterandrecentmeteoric waterinthecavern,basedonendmembermixing calculations.Ontwoofthewatersamplingdates,the contributionofmeteoricseepagewaterfromthe surfacewasestimatedtobeat58±17%or54±18% ofthemixedcavewater,respectively. Naturalgasdirectlybubblingfromrockfissures intothedistalspringpoolwascapturedand characterizedbyGC-isotoperatiomassspectrometry.Isotopicsignaturesidentifieditasthermogenic methane,indicatedbyits 13Cand 2Hvaluesof 43.6±0.2 ‰ (Figure2)and-164.9±2.2 ‰ ,respectively.Highmethaneconcentrationsofupto50% weremeasuredintheemerginggas.Average methaneconcentrationsinthemixedcavewater andinthecaveatmospherewere6000and3000p.p. m.,respectively.ThecarbonisotopesignatureofCH4intheundisturbedcaveatmospherewas 36.8±0.1 ‰ 13C.Meancarbondioxideconcentrationsof8000p.p.m.inthecaveairwerealsoclearly elevatedcomparedwithambientbackgrounds.The 13CofCO2intheseepinggasandinthecave atmospherewas 33.2±0.1 ‰ and 25.2±0.1 ‰ , respectively(Figure2).Dissolvedoxygenconcentrationsinthespringwaterandmixedcavewaterwere 3.2%and4.3%inaverage,respectively(Table1). Samplingoftheundisturbedcaveatmospherevia gastubesindicatedmicrooxicconditions(O2: 15.1±4%, n =5)understeadystate. ThepHofbiofilmsampleswassimilartothatof thewater(pH~7.5).Elementalanalysisoffreezedriedbiofilmsrecoveredalightcarbonisotope signature( 44.4 ‰ ),directlycorrespondingtothe seepingmethaneitself,especiallyforthesubmersed biofilms(Figure2).Snottitesandsubaerialbiofilms showedasignificantlyheavier 13C( 30.7±1.1 ‰ ). ThiswasinbetweenthesignaturesofCH4andCO2inthecaveatmosphere,ratherthandirectlyreflectingseepingCH4.Asimilarpatternofisotope signatures,albeitlesspronounced,wasrecovered Table1Waterphysicochemistryandmicrobialcellcountsfordifferentwaterbodiesinthecave MixedcavewaterMineralspringwaterMeteoricseepagewater MedianMin. – max.MedianMin. – max.MedianMin. – max. Physicochemistry Temperature7.97.2to11.77.28.6aEC( Scm 1)21002020to22006200b5890to6900b526apH7.57.2to7.67.6b7.2to8.3b8.3aO2(mgl 1)4.32.3to5.53.22.3to3.48.5aNutrients,electronacceptors DOC(mgl 1)1.20.8to1.40.7b0.5to0.9b0.6aNO3 (mgl 1)1.90.8to3.4bdbdto0.25.64to5.6 PO4 3 (mgl 1)bdbdbdbdto0.020.010.01to0.02 SO4 2 (mgl 1)2.11.1to3bdbdto1.74.23.4to4.2 FeTotal(mgl 1)NA0.87c0.46to1cNA Watermineralization Na+(mgl 1)93.951.9to328.6649.8581.3to1131.155to18.8 K+(mgl 1)1.61.2to34.64.2to8.611to1.4 Mg2+(mgl 1)22.820.7to140.724.423.1to55.32322.2to23.3 Ca2+(mgl 1)97.262.1to205.151.550.2to111.982.276.9to82.5 Cl(mgl 1)15477.5to606.61301.71237.9to2248.31.11.1to4.6 Br(mgl 1)1.30.7to4.619.216.1to29.40.010.01to0.03 I(mgl 1)c3.20.9to520.520to30.7NA Waterisotopes 18Od 10.5 10.8to 10.2 8 9.6to 7.7 11 11.8to 10 2Hd 73.9 74.7to 73.2 67.2 69.1to 66.5 75.4 82.3to 68 TU7.14.88.3aCellcounts 1.6×1063.7×105to1.9×1063.1×1032.7×103to1.9×1066.7×1036.6×103to2.4×105Abbreviations:bd,belowdetectionlimit;DOC,dissolvedorganiccarbon;EC,electricconductivity;NA,notanalyzed;TU,tritiumunitsdefinedas ratioof13Hatomto1018Hatoms.aSinglemeasurementinDecember2012.bThisstudy( n =4)androutinemonitoringdatafromLfU( n =4).cLfUmonitoringdata( n =4)fromOctober2011toApril2012.dThisstudy( n =3)andLfUmonitoringdata( n =4). Methanotrophicmicrobialmegacities CKarwautz etal91 TheISMEJournal

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alsofornitrogenstableisotopes(Figure2).Nitrate wasthemostprevalentnitrogenspeciesinthewater (Table1),whereasammoniumandnitritewere belowdetectionlimits.Totalnitrogencontentwithin thebiofilmsdecreasedtowardtheceiling(Table2). However,distinctnitrogenspeciesinwaterand biofilmswerenotindependentlyanalyzed. Ahightotalcarboncontentof~372mggDW 1on averagewasmeasuredforthesnottites(Table2). Nitrogenandphosphorusconcentrationswerelow, especiallyforsnottitesontheceiling,resultinginan elementalcompositionof37%C,0.6%Nand0.01% PandhighC:N(61),C:P(4727)andN:P(82)ratios. Thesenutrients,plusironandsulfur,werecomparablymoreabundantinsubmersedbiofilms.In contrast,totaliodineconcentrationsofbiofilmswere highestattheceilingbutvariedsubstantially throughoutsamplinglocationsandwere 482.1±197.6 ggDW 1onaverage(Table2).Methaneoxidationpotentialsinlaboratorybiofilm incubationsPotentialmethaneoxidationrateswerequantified forbiofilmsofthethreecompartmentstosubstantiateourhypothesisofmethaneasanimportantdriver ofbiofilmformation.Freshbiofilmsamplesdispersedinnitratemineralsaltmediumwereamended with16000p.p.m.CH4inheadspace.Forallbiofilm samples,substantialmethaneoxidationrateswere recordedunderlaboratoryconditions.Theywere ~3 molgbiofilmFW 1day 1forthesubaerialbiofilms andsnottitesandalmostanorderofmagnitude higher(~26 molgbiofilmFW 1day 1)forthesubmersed biofilms(Table2).VisualizationofbiofilmstructuresFirststructuralinsightsintothebiofilmmatrices weregeneratedbyconfocallasermicroscopyin combinationwithlectinstainingofglycoconjugates (Figure3).Imagedatarevealedunusuallylarge capsule-likestructures,whichseemedtoembed smallnumbersofcellsandwhichwerepartly connected(Figure3a).Oftentheseglycoconjugates formedlargermultilayerstrandswithvoids(nonlectin-stainedzones)inbetween(Figure3b).Glycoconjugatestructuresappearedevenlydistributed withsingleembeddedbacterialcellsinsomeimages (Figures3aandb),whereasonlypartofthecells formedAAL-specificglycoconjugatesinothers (Figure3c).Multilayerglobularfeatureswithclusters ofcapsule-likestructures,aswellasglobuleswith higherlevelsoforganization,werealsoobserved (Figure3d).MolecularanalysisofwaterandbiofilmmicrobiotaMicrobialabundancesasdeterminedbyflowcytometrywere~3.1×103cellsml 1inthemineral springwaterandamuchhigher~1.6×106inthe mixedcavewater(Table1).Mixedcavewatercell countswerelargelyconsistentwith16SrRNAgene quantification(SupplementaryFigureS1),butquantitativePCRcountsforthespringwaterwerehigher thancellcounts.Genequantificationrevealedahigh abundanceofupto~3.6×109bacterial16SrRNA genesgbiofilmFW 1forthesubmersedbiofilms. PCRscreeningforfunctionalmarkergenesindicativeofmethanotrophsormethylotrophswasconductedasafirstqualitativetestforthepresenceof suchmicrobesinthesystem.Alltestedwaterand biofilmsampleswerePCRpositive(datanotshown) forgenesencodingthemethanoldehydrogenase ( mxaF )andparticulatemethanemonooxygenase ( pmoA )butnotforsolublemethanemonooxygenase. Cave CO2Cave CH4Seepage CO2Seepage CH4Subaerial Snottites Submersed-12-8 -4 0 -10-6-22N-stable isotope abundance of bioÞlms [10315N]C-stable isotope abundance of bioÞlms [10313C]-25 -30 -35 -40 -45 Figure2 Carbonandnitrogenstableisotoperatiosmeasuredfor distinctbiofilmcompartments.Stableisotopesignaturesof methaneandcarbondioxideeitheringasbubblesseepingfrom thespringpoolorinthecaveatmosphereareindicatedingray shading.Thelengthoftheboxplotsdepictsthequartilesandthe crosshairtherange(min.,max.)ofisotopemeasurementsfor snottites. Table2Elementalcompositionofdriedbiofilmsandpotentialmethaneoxidationratesoffreshbiofilmsat12°Cinthelaboratory BiofilmC (mgg 1) N (mgg 1) P (mgg 1) Fe (mgg 1) S (mgg 1) I ( gg 1) CH4ox.a( Mg 1day 1) Snottitesb372.3±806.1±10.1±0.11.7±1.11.9±0.60.5±0.23 Subaerialc283.88.70.218.14.40.23.1 Submersedc18311.90.5805.60.425.7an =3.bn =6.cn =2. Methanotrophicmicrobialmegacities CKarwautz etal92 TheISMEJournal

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TerminalrestrictionfragmentlengthpolymorphismfingerprintingofthepmoAgenepoolrevealedaconsiderablediversityofpmoAT-RFsinallbiofilms.Intotal,13ofthe30detectedpmoAfragmentsoverlappedbetweensamples,while14appeareduniqueforthemixedcavewater(notshown).BacterialcommunitycompositioninwaterandbiofilmsReplicateampliconsequencinglibrariesweregener-atedforthethreebiofilmcompartmentandwatersamples(Figure4).Dataprocessingresultedin482OTUsaffiliatedwiththebacteria(SupplementaryTableS2).Outofthese,363OTUswerefoundinbiofilmsamplesand297OTUsinwater,withanoverlapof178OTUs.BesidestheProteobacteria(especiallyAlphaproteobacteria,BetaproteobacteriaandGammaproteobacteria),Planctomycetes,Bacter-oidetesandVerrucmicrobiaalsocontributedsub-stantiallytothecommunities.Atotalof8phylawerepresentat41%abundanceinatleast1ofthesamples.Betaproteobacteriawereespeciallyabun-dant(59%)directlyinthespringwater,detectableatnotableabundanceinmixedcavewaterandsubmersedbiofilms(44%and25%,respectively),butalmostabsentfromsubaerialbiofilmsandsnottites.Incontrast,membersoftheAlphaproteo-bacteria,Planctomycetes,BacteroidetesandVerru-comicrobiaweregenerallymoreabundantinsubaerialbiofilmscomparedwithsubmersedsam-ples(Figure4).In-depthtaxonomicanalysisrevealedmanyknownmethylotrophicandmethanotrophicpopula-tions(Knief,2015)withinthebiofilms.PotentialmethylotrophicAlphaproteobacteriawereapparentasBeijerinckiaceae,Hyphomicrobiaceae,Rhodobac-teraceae,ErythrobacteraceaeandSphingomonada-ceae,detectedespeciallyinsubaerialbiofilms(Figure4).SeveralfacultativemethanotrophswithintheAlphaproteobacteria,suchasMethylocella,MethylorosulaandMethylobacteriumspp.werealsoidentifiedbutonlyatlowabundance(allo1%).WithintheBetaproteobacteria,membersoftheMethylophilaceae(manyofthemobligatemethylo-trophs)wereabundantinsubmersedbiofilms(14%)butwerealsofoundinmineralspringwater(4%)andmixedcavewater(8%)samples.DominanttaxawithinthisgroupwereaffiliatedtoMethylotenera 20 µm20 µm20 µm40 µm Figure3Visualizationofbiofilmstructuresinsnottitesbylasermicroscopyincombinationwithlectinstaining.(a)Globularglycoconjugatesignals(green)surroundingindividualbacterialcells(red)(67sections).(b)Strandsofglycoconjugatesignals(red)withbacterialcells(green)andinterspersedvoids(58sections).(c)Biofilmsectionwhereonlypartofthebacterialcells(green)areshowntobeassociatedwithglycoconjugatesignals(red)(62sections).(d)Multilayerglycoconjugates(triple-stainedAAL)indicatingthreeordersoforganizationinsnottites:glycoconjugatecapsulessurroundingindividualbacterialcells,embeddedclustersofcapsules,andhigher-orderspheresofglycoconjugateclusters(59sections).Theselected3Dimageseriesareshownas2Dmaximumintensityprojections.SampleswerestainedwithAALlectin(a–d),aswellasnucleicacid-specificstains(a–c). MethanotrophicmicrobialmegacitiesCKarwautzetal93 TheISMEJournal

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spp.Mostnotably,adominanceof Hydrogenophilaceae (27%)and Gallionellaceae (14%)wasobserved inthespringwater,whiletheywerealmostabsent fromallothersamples.The Rhodocyclaceae also contributedtothebacterialcommunityofthespring waterandmixedcavewaterandsubmersedbiofilms. The Methylococcales ( Gammaproteobacteria ) representedthemostabundantknownmethanotrophicgroup(Knief,2015)acrossallsamples (Figure4).Intotal,15divergentlineageswerefound, withthe Crenotrichaceae and Methylococcaceae beingmostabundant.Dominanttaxawithinthe mixedcavewaterwereaffiliatedto Methylovulum sp.(28%). Crenothrix and Methylobacter spp.were abundantinsubmersedbiofilms(13%and10%, respectively)butapparentlyreplacedby Methylomicrobium spp.inthesubaerialbiofilms(4%).Membersofthe Pseudomonadaceae werealsoabundant Pseudomona s Methylomicrobium MethylovulumMethylobacterCrenothrix1.4% [±1] 2.2% [± 0.2] 1.9% [± 0.2] 1.3% [± 0.5]Deltaproteobacteria Planctomyces Pir4SM1A02 Porphyrobacter<1% 2.5% [± 1.2] < 1% [± 0.6] < 1%< 1% 2.6% [± 0.5] 1.2% [± 0.6] 1.2% [± 0.04] 1.3% [± 0.2] < 1%< 1%SUBAERIAL( n=2 )SNOTTITES( n=4 )SUBMERSED( n=3 )BIOFILM SAMPLESMIXED CAVE WATER( n=2 )MINERAL SPRING( n=3 )n.d.n.d.n.d.< 1% % 5 2% 0 5 0%100%BacteroidetesCytophagales Cytophagaceae Flavobacteriales Flavobacteriaceae Sphingobacteriales Saprospiraceae ArenibacterPlanctomycetaceae Phycispheraceae Phycispherales PlanctomycetalesPlanctomycetes NC10MethylomirabilisVerrucomicrobiaChthoniobacterales Chthoniobacteriaceae FukuN18AlphaproteobacteriaDB1-14 Rhizobiales Rhodobacterales Caulobacterales Rhodospirillales Rickettsiales Sphingomonadales Hyphomonadaceae Beerinckiaceae Rhodobacteriaceae RhodospirillaceaeHyphomicrobiaceae ErythrobacteriaceaeMethylococcaceae Legionellaceae Crenothrichaceae Legionellales Pseudomonadales Methylococcales Xanthomonadales PseudomonadaceaeGammaproteobacteria No afÞliationComamonadaceae Hydrogenophilaceae Methlyophilaceae Gallionellaceae RhodocyclaceaeBetaproteobacteriaHydrogenophilales Methylophilales Nitrosomonadales Burkholderiales RhodocyclalesSider oxydans Ferriphase lousSulfuricellaMethyloteneraGallion e llaSulfuritaleaFerribacter ium WATER SAMPLES Figure4 Bacterialcommunitycompositioninbiofilmandwatersamplesasshownby16SrRNAgeneampliconsequencing.All abundantphylaorproteobacterialclasses(relativeabundance 4 3%)areshownascompositeKronaplots(Ondov etal. ,2011)resolved downtothefamilylevel.Selectedtaxamentionedinthetextareshownatthegenuslevel.ThediameterofKronacirclesisscaledtothe meanabundanceofphylaorclasses.Variationsoftaxonabundancesbetweenreplicatesamples,eithergivenass.d.ortherangefromthe mean(if n =2),isshownasdiagonalbars. Methanotrophicmicrobialmegacities CKarwautz etal94 TheISMEJournal

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inthesnottitesandsubaerialbiofilms(8%and6%, respectively).Unexpectedly,theaerialcommunities appearedmorediverse(SupplementaryTableS1) andlessclearlydominatedbyknownmethanotrophicormethylotrophictaxathanthesubmersed biofilmsandmixedcavewater. The Planctomycetes inaerialbiofilmsweremostly affiliatedtothe Pir4 lineageand Planctomyces spp. ( Planctomycetaceae ),aswellasthe Phycisphaeraceae .TherecentlydiscoveredNC10candidate phylum,includingthedenitrifying,methanotrophic Methylomirabilis spp.(Ettwig etal. ,2010),was detectedonlyinsubmersedbiofilms.The Bacteroidetes, mostlypresentinsubaerialbiofilmsand snottites,includedanotablepopulationof Arenibacter spp. Summinguptheabundanceofthemostprominent knownmethanotrophicandmethylotrophictaxa (Figure5),subaerialbiofilmsandsnottitescontained significantlyfewerrespectivelineagesthanthe submersedbiofilmsandmixedcavewater( t =3.9, df=2.7, P o 0.05).BesidesknownC1-oxidizers,other taxaofclearfunctionalconnotationwerealso detected,suchas Sulfuritalea ( Betaproteobacteria ), Sulfurimonas ( Epsilonproteobacteria )and Sulfuricella spp.( Betaproteobacteria )inthemineralspring water,allwellknowntobecapableofsulfur oxidation.Moreover,severalputativelyironoxidizingtaxasuchas Siderooxydans , Ferriphaselus and Gallionella spp.(all Gallionellaceae ),aswellas closerelativesofthetheiron-reducing Ferribacterium spp.( Rhodocyclaceae ),werefoundwithdistinct distributionbetweenspringandmixedcavewater (Figure4).DiscussionMethaneasthedriverofbiofilmformationHereweprovidefirstinsightsintothebiogeochemistryandmicrobiologyofapeculiarbiofilmsystem discoveredinprealpinesouthernGermany.We showthatdeepformationwaterentersthespring cavetogetherwithappreciableamountsofmethane, asindicatedbytheelevatedmixingratiosofmethane inthewaterandatmosphereofthecave.Carbon isotopesignaturesclearlyidentifiedthemethaneas thermogenicinorigin(Aelion etal. ,2009),consistent withthewell-establishedpresenceoffossildeposits andgasreservoirsinthisregionofthesubalpine Molasse(Hiltmann etal. ,1999;Etiope,2009).In Sulzbrunn,theseepinggascanbespeculatedto actuallyliftupthedeepbrinymineralwaterthat ascendsintothecave.Althoughseepinggasbubbles havebeenreportedtoinduceporewaterflow velocitiesofuptoseveralmetersperdayincoastal seeps(O'Hara etal. ,1995),furtherinvestigationswill benecessarytodelineatethehydrogeologicalsetting inSulzbrunn. Endmembermixingcalculationsbasedonwater isotopesshowedthattheinfluxofdeepformation watercontributedroughly40 – 50%tothespring waterinSulzbrunn.Itislikelythatthismixingof distinctwaterinputs,atleastinpart,contributesto thedefinitionoftheuniquebiogeochemicalsystem inthecave.Despitethedetectionoflowamountsof oxygendirectlyinthemineralspringwater(Table1), weassumetheupwellingformationwatertobe anoxic,andoxygenexposureormixingwithmore aeratedsurfacewatertotakeplaceonlyinthelast metersbeforeenteringthecave.Werethemineral springwateraerated,wewouldhaveexpectedto detectahighabundanceofaerobicmethanotrophsin thesesamples,whichwasnotthecase.Apartfrom methane,inputsofDOCintothecaveviathe differentwaterfluxesseemednegligible.Still, comparablylowamountsofDOCinseepingsurface waterhavebeenshowntosupportappreciable populationsofheterotrophicbacteriaincaves (Ortiz etal. ,2014).Also,wecannotexcludepotential seasonalityinDOCinputsfromthesurface,which mighthavebeenmissedduringourtimepointsof sampling. Theunambiguous 13Csignatureidentifiedthermogenicmethanetobethemaindriverofbiofilm formation,especiallyforthesubmersedbiofilms.As shownabove,thishadthehighestmethaneoxidation ratesandmethanotroph/methylotrophabundance. TheCH4consumptionratesofupto25 molgbiofilm FW 1day 1were 4 4ordersofmagnitudehigherthan ratesrecentlyreportedforthewatercolumnabove methaneseepsinLakeConstance(Bornemann etal. , 2016)andinasimilarhighrangeasreportedfor othermethane-ventinggeothermalsites(Gagliano etal. ,2016;Lennon etal. ,2017).Upscalingthisfor biofilmmassestimatesinthecave,apotential methaneturnoverof~1.6molday 1(~35.8l CH4day 1)canbeextrapolatedforthesubmersed biofilmsalone.Methaneoxidationrateswereunfortunatelynotdeterminedforthemixedcavewaterin thisstudy.Thehighabundanceofmethanotrophsin thewater(Figure5)andthereducedoxygen concentrationscomparedwithsurfaceseepagewater 80 60 40 20 100 0Methylomicrobium MethylosomaSnottitesSubaerialCrenothrix Methylobacter Methylovulvum Methylotenera MethylomirabilisSubmersedMethylomonasMineral spring Mixed cave waterAbundance of methylotrophic OTUs [%] Figure5 Summedrelativeabundanceofwell-knownmethanotrophicandmethylotrophictaxarecoveredfrombiofilmandwater samples.Shownaremembersofthe Betaand Gammaproteobacteria andtheNC10phylum.ColorcodingisidenticaltoFigure4. Methanotrophicmicrobialmegacities CKarwautz etal95 TheISMEJournal

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(Table1)suggestedthatatleastpartofthemethane oxidationcouldalsohavebeenallocatedtothewater bodyitself.However,themuchlowertotalbacterial abundancesinthewater(SupplementaryFigureS1) combinedwithasteadydischargeandthuspurging ofloticpopulationscouldstillresultinbiofilms beingthemostrelevantfortotalmethaneturnover. Incontrasttosubmersedbiofilms,the 13C signatureofsubaerialbiofilmsandsnottitesless directlypointedtowardthermogenicmethaneas beingtheirmaincarboninput.However,theisotopic signaturesofCH4andCO2inthecaveatmosphere werebothsubstantiallyheavierthandirectlyin seepinggas(Figure2),suggestingstrongstable isotopefractionation(Preuss etal. ,2013)tooccur duringoxidationbetweenthecompartments.The placementoftheaerialbiofilmsatapproximately 31 ‰ 13Cinbetweenthesignatureofbothgaseous endmembersinthecaveatmosphereindicatedan equalimportanceofbothmethanotrophyandautotrophicorheterotrophicCO2fixationforbiofilm buildup,aspreviouslyinferredforotherbiofilmsin caves(Sarbu etal. ,1996;Chen etal. ,2009).Nevertheless,thelowermethaneoxidationratesandlower abundanceofpotentiallyC1-oxidizingmicrobesboth seemtopointtowardapossibleroleofelectron donorsotherthanmethaneinaerialbiofilms.This willbediscussedfurtherdown. Theinterpretationofobservednitrogenisotope signaturesinbiofilmswasnotpossibleduetothe lackofdefinedinputsignals.Thedepleted 15N isotopevaluesofthesubmersedbiofilms( 11 ‰ ) werecomparabletovaluesfoundinMovileCave biofilms(Sarbu etal. ,1996).Themuchhighervalues ofaerialbiofilmssuggesteddistinctinputs,possibly connectedtotheknowncapacityofmanymethanotrophstofixatmosphericdinitrogen(Knief,2015). Nevertheless,themajorsourcesandroutesof nitrogencyclinginthecavesystemremaintobe specificallyelucidated.BiofilmcommunitycompositionTheformationofpenduloussnottitesandother macroscopicbiofilmstructuresincaveshasbeen observedbeforebutmostlyunderacidicorotherwise extremeconditions.Incomparisontothemassive biofilmstructures(pH~7.5)nowreportedforthe Sulzbrunncave,previouslydiscoveredsnottites appearedmuchthinnerinshape,werelessdensely distributedandformedloweramountsofextracellularpolymericsubstances(Bond etal. ,2000; Holmes etal. ,2001;Northup etal. ,2003;Jones etal. ,2010;Ziegler etal. ,2013).Togetherwiththe richdiversityofmethylotrophsandotherbacterial lineagesnowdiscoveredintheSulzbrunnbiofilms, thispointstowarddistinctbiogeochemicaland ecophysiologicaldriversofbiofilmformationinthe differentsystems. Wesuggesttheidentifiedbiofilmcompartmentsto beafunctionofmethaneinflux,watersubmersion andoxygenandnutrientsupplywithinthecave.The highabundanceofmethanotrophsandmethylotrophsofupto~45%insubmersedbiofilmsand between10and~20%intheotherbiofilmcompartmentswasconsistentwiththeimportanceof methaneasthedriverofbiofilmformation.An abundanceofaerobicmethanotrophsofupto40% hasbeenreportedpreviouslyforaterrestrial methaneseep(Gagliano etal. ,2016).However, centralquestionsremainhowcarbonandenergy flowsaresharedbetweenthemethanotrophs,other methylotrophsandthediversenon-methylotrophic lineagesdiscoveredintheSulzbrunnbiofilms. Distinguishingbetweenobligateandfacultative methanotrophsrequiresgenomicandproteomic information,whichisnotyetavailableforthe investigatedsystem.However,theecophysiologyof someofthemicrobesdetectedcanbecautiously extrapolatedfromtheliterature.Forexample, Methylobacter spp.aregenerallyconsideredas obligatemethanotrophs(Knief,2015),while Methylotenera spp.andother Methylophilaceae aremostly knownasobligatenon-methane-utilizingmethylotrophs(Kalyuzhnaya etal. ,2012).Theco-occurrence ofthesemethanotrophsandmethylotrophs,especiallyinthesubmersedbiofilms,suggestmethanefueledcooperation(Chen etal. ,2009).Membersof the Methylococcaceae and Methylophilaceae have beenpreviouslyreportedtotrophicallyinteractin methane-fueledsystemsunderoxicandmicrooxic conditions(Beck etal. ,2013;Oshkin etal. ,2015). Membersofthe Methanococcaceae havebeenshown toshuntcarbontodiversenon-methylotrophic communitymembersinmicrobialmatssituatedat marinehydrocarbonseeps(Paul etal. ,2017).Thus, intheSulzbrunnbiofilms,complexcommunities andinteractionnetworkscanbeconsideredtodrive methaneoxidationratherthansinglemicrobial species. Methanotrophsarealsowellknownasproducers ofabundantextracellularpolysaccharides(Linton etal. ,1986;Strong etal. ,2015).Manyofthem possesstheribulosemonophosphatepathwaytofix methyl-group-derivedcarbon.Theproductionof extracellularpolysaccharidesfrommethanolis balancedintermsofadenosinetriphosphateand reducingequivalents(Linton etal. ,1986).The observedexopolysaccharideproductionisconceivableasanenergy-spillingreaction,preventingthe buildupoftoxicformaldehydeunderexcess methanesupply,andprovidingmethane-derived reducedcarbontootherheterotrophicmembersof thebiofilmcommunity.Somemethanotrophshave beenshowntofermentmethaneandreleaselarge amountsofreducedcarbonunderoxygen-limited conditions(Kalyuzhnaya etal. ,2013).Growth limitationbylimitednitrogenorphosphorous supply,assuggestedespeciallyfortheaerialbiofilms byhighC:N:Pratios,wouldsupportthisscenario. Althoughmanymethanotrophsarecapableoffixing dinitrogen(Knief,2015),Plimitationwillnotbe Methanotrophicmicrobialmegacities CKarwautz etal96 TheISMEJournal

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readilycomplementedinaerialbiofilms.TheC:P (~4700)andN:P(~80)ratiosobservedinbiofilmsare clearlymuchhigherthancanonicalRedfieldratios orthanC:N:PratiossuggestedtoindicatebacterialP limitation(Vrede etal. ,2002).Therefore,wepropose thatthemassiveextracellularmatrixformedinthe Sulzbrunnbiofilmsserves,atleastinpart,asan electronsinkfornutrient-limitedmethylotrophs. Besideswell-knownproteobacterialmethanotrophsandmethylotrophs,membersofthecandidate genus Methylomirabilis werealsodetectedbut appearedrestrictedtosubmersedbiofilmsinSulzbrunn.Theseareknownasnitrate-dependentanaerobicmethaneoxidizersproposedtointraaerobicallyoxidizemethaneunderNOdismutation (Ettwig etal. ,2010).Theirdetectionpointstoward thepossibleoccurrenceofanaerobicmethaneoxidationinspecificmicronichesoftheSulzbrunn biofilms.Furthermore,wearecurrentlyinvestigating whether Archaea couldalsopossiblybeinvolvedin methanecyclinginthesystem.Preliminarydata suggestthatalowabundanceoflargelyuncultured archaeallineagescanbefoundinthesubmersed biofilmsandwatersamplesbutnotinaerialbiofilms. Furthermore,theabundantdetectionofputatively sulfur-oxidizing(Watanabe etal. ,2014)andironoxidizing(Emerson etal. ,2013),aswellasironreducing(Cummings etal. ,1999),membersofthe Betaproteobacteria ,especiallyinspringandmixed cavewaters,pointstowardactivesulfurandiron cyclinginthecave.However,theseprocesses,as wellastheirpossiblelinktocarboncycling,could notbefurthertracedinthepresentstudybutwillbe thesubjectoffuturework.PossibleroleofiodineThevisualizationofthebiofilmmatrixrevealed uniquestructuralfeaturesofthebiofilms.Although largeglobularstructureshavebeenpreviously reportedforbiofilmsintechnicalsystems(Okabe etal. ,1999;Weissbrodt etal. ,2013),acomparably massiveembeddingofsingleorsmallnumbersof cellsincapsulesandnetworksofglycoconjugates,to thebestofourknowledge,hasnotbeenobserved.It istemptingtospeculatethatbesidesapossiblerole asanelectronsink,thebiofilmmatrixcouldalso serveasprotectivebarrieragainstharmfulagents possiblypresentintheSulzbrunnsystem.The conceptofbiofilmsasadiffusivebarrieragainst antimicrobialsiswellestablished(Flemming etal. , 2016).Intheiodine-richwatersandbiofilmsofthe Sulzbrunncave,thepossibilityofbactericidal activityofiodinespeciesshouldbediscussed. Iodineiswellknownasadisinfectant,but interestingly,themechanismsofitstoxicityarestill notfullyelucidated,probablyowingtoitscomplex chemistry(Küpper etal. ,2011).Elementaliodine(I2) isnotstableinaqueoussolution,whereitreadily hydrolysestoiodide(I),hypoiodousacid(HOI)and severalotheriodinespecies(Gottardi,1999).Under elevatedpH,theformationofiodate(IO3 )by chemicaldisproportionationisalsopossible.Iodide andiodateareconsideredasnon-toxic,while elementaliodine,hypoiodousacidandtriiodide (I3 )aresuggestedasbactericidaloxidizingagents (Gottardi,1999).Owingtoitscomplexbehaviorasa solute,comprehensiveiodinespeciationischallengingandhasnotyetbeenaccomplishedfordifferent Sulzbrunnsamples. Althoughweassumethatmostofthetotaliodine emergingwiththereducedmineralwaterwas iodide,thiscouldundergoanumberofmicrobially drivenoxidationandvolatilizationreactionsinthe cave.Theoxidationofiodidetoelementaliodinein thepresenceofpolysaccharideshasbeenshownfor Pseudomonasiodooxidans (GozlanandMargalith, 1974)anddistinct Alphaproteobacteria (Amachi etal. ,2005),whichwereevenstimulatedunder highiodideconcentrations(Arakawa etal. ,2012). An Arenibacter sp.( Bacteroidetes )hasbeenreported toaccumulateiodineduringthatprocess(Ito etal. , 2016).Moreover,variousisolatesfromiodine-rich habitats,including Erythrobacter , Pseudomonas and Rhizobium spp.,havebeenshowntomethylate iodide,thusvolatilizingitashighlyreactiveiodomethane(CH3I)(Amachi etal. ,2005;Fujimori etal. , 2012).Andfinally, Pseudomonas sp.SCThasbeen showncapableofanaerobicgrowthwithiodateas soleelectronacceptororwhilesimultaneously reducingnitrate(Amachi etal. ,2007). Theabundantdetectionofalloftheabovegenera intheSulzbrunnbiofilms,aswellasthehigher iodineconcentrationsfoundinthesnottites (Table2),implythatvolatilizationprocessesmay actuallyhavebeenongoinginthecave.The volatilizationasiodomethaneandsubsequentoxidationbymethylhalideoxidizersinaerialbiofilms (McDonald etal. ,2002)seemsplausibleandcould establishalinkbetweenthecyclingofmethaneand iodineinthesystem.Itcanbecautiouslyspeculated thatiodidereleaseduponiodomethaneoxidationby methylotrophsinsnottitescouldthenbeoxidizedto iodinebyothercommunitymembers,thuspossibly contributingtoiodinestressandglycoconjugate productioninbiofilms.Asafirststeptofollow-up onthis,wehavetriedtoquantifyiodomethaneinthe caveatmospherebygaschromatography.Although wewereabletodetectituponseveraloccasions (datanotshown),aconsistentandreproducible quantificationwasnotaccomplishedsofar,possibly duetothehighlyreactivenatureofthis methylatingagent.ConclusionsHeretheSulzbrunnspringcaveisdescribedasa uniquehabitatformicrobialbiofilmgrowth. Althoughthecaveissituatedjustseveralmeters belowthesurface,microbialcommunitieslargely independentfromsurfacecarbonandenergyinputs Methanotrophicmicrobialmegacities CKarwautz etal97 TheISMEJournal

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werediscovered.Incontrasttoourinitialexpectation,biofilmmicrobiotaweresurprisinglydiverse, withahostofpopulationscloselyrelatedtowellknownmethanotrophs,methylotrophsandalso potentiallyiodine-cyclingbacteria.Thesefindings providefurtherevidencefortherelevanceof subterraneanmethanesinksbymicrobes (McDonough etal. ,2016;Lennon etal. ,2017).We proposethatthemassiveextracellularpolymeric substanceproductionobservedcouldserveasan electronsinkforthenutrient-limitedandtherefore growth-limitedmethylotrophs.Althoughthese firstinsightsintoanapparentlyuniquesubsurface biofilmsystemareveryintriguing,manyopen researchquestionsremain.Theapplicationof13C-labeledmethaneandmethylotrophicsubstrates incombinationwithnucleicacid-basedstableisotope probingiscurrentlyongoingandwillhelptofurther unravelthecomplexpatternsofcarbonandenergy sharingtobeexpectedwithinthebiofilms.Future researchshouldalsoaddressthespatialorganization andmetagenomicrepertoireofbiofilmmicrobiota,as wellasthepossibleroleof Archaea , Protozoa ,phage andfauna,inthefoodwebofthisecosystem apparentlydominatedbyprokaryotes.ConflictofInterestTheauthorsdeclarenoconflictofinterest.AcknowledgementsWethankFranzHösle,Sulzbrunnforsiteaccessand samplingsupport.RolandEichhornoftheLfUinHofis acknowledgedforhiscontinuedsupportofoursite investigationandforscientificdiscussion.Wethank ChristineStumpp,PetraSeibel,HaraldLowagandArmin Meyer(HelmholtzMünchen)fortheanalysisofstable isotopesandBernhardMichalke(HelmholtzMünchen)for inductivelycoupledplasmaanalytics.Technicalsupport ofKathrinHörmanninsequencing,NinaWeberinflow cytometry(bothatHelmholtzMünchen)andofUte Kuhlickeinconfocallaserscanningmicroscopy(UFZ Magdeburg)isalsogreatlyacknowledged.LaurenBradford (HelmholtzMünchen)isthankedforexpertlanguage editing.Thisresearchwassupportedbyfundsofthe Helmholtz-ZentrumMünchenandtheHelmholtzCenter forEnvironmentalResearch – UFZ.ReferencesAelionCM,HöhenerP,HunkelerD,AravenaR.(2009). 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ISMEJ 7 : 1725 – 1737. ThisworkislicensedunderaCreative CommonsAttribution-NonCommercialShareAlike4.0InternationalLicense.Theimagesor otherthirdpartymaterialinthisarticleareincluded inthearticle ’ sCreativeCommonslicense,unless indicatedotherwiseinthecreditline;ifthematerial isnotincludedundertheCreativeCommonslicense, userswillneedtoobtainpermissionfromthelicense holdertoreproducethematerial.Toviewacopy ofthislicense,visithttp://creativecommons.org/ licenses/by-nc-sa/4.0/©TheAuthor(s)2018SupplementaryInformationaccompaniesthispaperonThe ISMEJournalwebsite(http://www.nature.com/ismej) Methanotrophicmicrobialmegacities CKarwautz etal100 TheISMEJournal


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