Comparative Analysis of Methane-Oxidizing Archaea and Sulfate

13 Pages·2001·0.59 MB·English

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Apr.2001,p.1922–1934 Vol.67,No.4
0099-2240/01/$04.0010 DOI:10.1128/AEM.67.4.1922–1934.2001
Copyright©2001,AmericanSocietyforMicrobiology.AllRightsReserved.
Comparative Analysis of Methane-Oxidizing Archaea and
Sulfate-Reducing Bacteria in Anoxic Marine Sediments
V.J.ORPHAN,1*K.-U.HINRICHS,2W.USSLERIII,1C.K.PAULL,1L.T.TAYLOR,1
S.P.SYLVA,2J.M.HAYES,2ANDE.F.DELONG1*
MontereyBayAquariumResearchInstitute,MossLanding,California95039,1andDepartmentofGeology
andGeophysics,WoodsHoleOceanographicInstitution,WoodsHole,Massachusetts025432
Received10October2000/Accepted2February2001
Theoxidationofmethaneinanoxicmarinesedimentsisthoughttobemediatedbyaconsortiumofmeth-
ane-consumingarchaeaandsulfate-reducingbacteria.Inthisstudy,wecomparedresultsofrRNAgene(rDNA)
surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several
differentsitesontheCaliforniancontinentalmargin.Twodistinctarchaeallineages(ANME-1andANME-2),
peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine
sediments.Thesamesedimentscontainedabundant13C-depletedarchaeallipids,indicatingthatoneorboth
ofthesearchaealgroupsaremembersofanaerobicmethane-oxidizingconsortia.13C-depletedlipidsandthe
signature16SrDNAsforthesearchaealgroupswereabsentinnearbycontrolsediments.Concurrentsurveys
ofbacterialrDNAsrevealedapredominanceofd-proteobacteria,inparticular,closerelativesofDesulfosarcina
variabilis.Biomarkeranalysesofthesamesedimentsshowedbacterialfattyacidswithstrong13Cdepletionthat
arelikelyproductsofthesesulfate-reducingbacteria.Consistentwiththeseobservations,whole-cellfluorescent
in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and
Desulfococcus species. Additionally, the presence of abundant 13C-depleted ether lipids, presumed to be of
bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the partici-
pation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and
ANME-2consortiaappeartoparticipateintheanaerobicoxidationofmethaneinmarinesediments,ourdata
suggestthatotherbacteriaandarchaeaarealsoinvolvedinmethaneoxidationintheseenvironments.
Microbiallymediatedoxidationofmethaneinanoxicmarine Therecentdiscoveriesofmethane-derived,isotopicallylight
systemsisagloballysignificantprocess,withupto90%ofthe archaeal lipids in seep-associated sediments and carbonates
oceanicmethaneproductionrecycledinanaerobicmarinesed- provided compelling chemotaxonomic evidence for the direct
iments(35).Anaerobicconsumptionofmethaneisgeochemi- involvement of archaea in anaerobic methane utilization (11,
callyandbiologicallyimportant,sinceitsignificantlydecreases 18,19,30,42).Hinrichsetal.(18)identifiedisotopiallydepleted
thefluxofmethanefrommarinesedimentstotheatmosphere. lipid biomarkers and archaeal 16S rRNA genes (rDNAs) oc-
The process transforms terminally reduced carbon into forms curring together in cold seep sediment samples from the Eel
thataremorereadilyaccessibletoalargergroupofmicroor- RiverBasin,whereAOMisthoughttoactivelyoccur.Results
ganisms in anoxic sediments. Localized chemosynthetic com- of this study corroborated the involvement of methanogenic
munitiesbenefitfromlargequantitiesofhydrogensulfide(2), lineages in AOM, identifying two potential archaeal groups
generatedasaby-productoftheanaerobicoxidationofmeth- related to the aceticlastic Methanosarcinales (ANME-1 and
ane.Geochemicalevidencesupportinganaerobicoxidationof ANME-2) as likely candidates for the methane-oxidizing ar-
methane (AOM) is well documented in the literature and is chaeainanoxicmarinesediments.
based on stable isotopic signatures (7), pore water chemical Further studies of Eel River Basin seep sediments and ad-
profiles (5, 23), inhibitor studies (17, 21), and sample incuba- ditionalseepsitesinSantaBarbaraBasinconfirmedthepres-
tionswithradiotracers(21,23).Theresultsofthesestudiesled enceofextremelydepletedarchaeallipids,inadditiontoiden-
to the hypothesis that AOM is mediated by a consortium tifying isotopically depleted bacterial fatty acids and glycerol
consisting of a methanogen operating in reverse (producing ethers,mostlikelyoriginatingfromtheAOMsyntrophicpart-
hydrogenandcarbondioxidefrommethane)andahydrogen- ners (19). Similar 13C-depleted microbial lipids were recently
scavenging,sulfate-reducingpartner(21).Despitetheindirect observed in hydrate-associated sediments from the Cascadia
evidence supporting microbially mediated AOM, identifying Margin(4,11)andMediterraneanmudvolcanoes(30),aswell
theindividualconsortiummembersandtheactualmechanism as in surface sediments and seep carbonates from the Black
involvedhasbeendifficult.
Sea(41).Theobservationofbotharchaealandbacteriallipids
thatarehighly13Cdepletedsuggestsaclosecouplingofanda
transfer of carbon between these two groups, providing addi-
*Correspondingauthor.MailingaddressforV.J.OrphanandE.F. tional evidence for a syntrophic association of archaea and
Delong:MontereyBayAquariumResearchInstitute,7700Sandholdt bacteria(19).
Rd.,P.O.Box628,MossLanding,CA95039.Phone,fax,ande-mail
In this study, we conducted cultivation-independent 16S
forV.J.Orphan:[phone],[phone],andorphan@mbari
.org,respectively.Phone,fax,ande-mailforE.F.Delong:(831)775- rDNA surveys on a variety of samples from different seep
1843,[phone],[email protected],respectively. environments,inwhichtheactivitiesofanaerobicmethanotro-
1922
VOL.67,2001 ANAEROBIC METHANE-OXIDIZING CONSORTIA 1923
TABLE 1. SummaryofsamplesusedintheconstructionofSSUrDNAlibrariesandtheirphysicalandchemicaldescriptions
Samplea Depth Latitude, Physicalorchemical SO4 H2S rDNAlibrary PCR No.ofclones
(cm) longitude(°) description (mM) (mM) designation primers screened
Eel-Hpc4 0–4 40.47,2124.35 Bacterialmat NAe NA Eel-TE B27f-U1492r 52
Eel-Hpc4 20–22 40.47,2124.35 Bacterialmat NA NA Eel-BE B27f-U1492r 121
Eel-pc36 4–7 40.48,2124.36 Clampatch,highCH 20 1d Eel-36e B27f-U1492r 18
Eel-pc36 4–7 40.48,2124.36 Clampatch,highCH4 20 1 Eel-36a A20f-U1492r 121
SB-pc24 14–16 34.10,2119.99 Calyptogenaclampat4ch 5.9b 11c SB-24e B27f-U1492r 91
SB-pc24 14–16 34.10,2119.99 Calyptogenaclampatch 5.9 11 SB-24a A20f-U1492r 90
SB-pc17 6–8 34.25,2119.97 Bacterialmat 11.5 10 SB-17a A20f-U1492r 77
SB-pc7 12–14 34.27,2119.96 Referencecore,background 28 0 SB-7a A20f-U1492r 25
aSB,SantaBarbaraBasin;Eel,EelRiverBasin.Waterdepthrangedbetween519and556mforboththeSantaBarbaraandEelRiverbasinsites.
bSO22valuesforSantaBarbaraBasinsampleswereprovidedbyJonMartin(personalcommunication).
4
cHSvaluesforSantaBarbaraBasinsampleswereprovidedbyJimBarry(personalcommunication).
d12,HSdetectedbutnotquantified.
2
eNA,notavailable.
phic microbes are indicated by the presence of 13C-depleted toanHPmodel6890GCandequippedwithacolumnidenticaltothatdescribed
biomarkers.Wesurveyedandcomparedbacterialandarchaeal above.ColumntemperaturesofbothGCsystemswereprogrammedfrom40°C
(1minunderisothermalconditions)to130°Catarateof20°/minandthento
groupspresentatgeographicallydistantmethaneseepsites,as
320°C(60minunderisothermalconditions)at4°C/min.Alcoholswereanalyzed
well as in control sediments. Whole-cell fluorescent in situ
as their trimethylsilyl ethers after reaction with N,O-bis(trimethylsilyl)-
hybridizationexperimentswerealsoconductedtoconfirmthe trifluoroacetamidereagent(plus1%trimethylchlorosilane)inDCM.Reported
identitiesofAOMconsortiummembersatthesesites,extend- d-13Cvaluesaremeansofresultsofatleasttwoanalysesandhavebeencorrected
ingpreliminaryobservationsofapreviousstudy(4). forthepresenceofcarbonatomsaddedduringderivatization.Differencesbe-
tweenindividualanalysesweregenerallylessthan1‰.
Nucleic acid extraction. For 16S rDNA analysis, total nucleic acids were
MATERIALSANDMETHODS extractedfromsedimentsamples,witheachsampleconsistingofa3-cmdepth
interval.CelllysisandDNAextractionfrom0.5g(wetweight)ofsedimentwere
Sitedescriptionandsampling.SedimentsampleswereobtainedfromtheEel
conductedusingaBio101(Vista,Calif.)Fastprepbeadbeatingmachine(Bio
RiverBasinandSantaBarbaraBasinatawaterdepthofapproximately500m
101)andaFastsoilprepkit(MoBioInc.,SanDiego,Calif).Theprotocolforthe
bymeansoftheremotelyoperatedvehicleVentana.Sampleswerecollectedwith
MoBiokitwasmodifiedbyinitiallybeadbeatingthesampleusingtheFastprep
push cores or hydraulic piston cores and subsequently stored in a nitrogen
atmosphereat4°Cuntilprocessedonshore;0.5to6haftercollection.Sample, machine(speed4.5for20s),followedbytwo5-minincubationsat70°C.The
location,areadescription,andSO22andHSlevelsaresummarizedinTable1. remainderoftheextractionprocedurewascarriedoutaccordingtothemanu-
4 2 facturer’s instructions. This procedure typically produced large-molecular-size
Sedimentsamplesfrompushcoreswereextrudedupwardsin3-cm-thicksections DNA(.20kb).Nucleicacidsfromthreeindependentextractionswerepooled
underanitrogenatmosphere.Sampleswerethensubdividedforinsituhybrid-
andpurifiedusingasmall-scaleCsCldensitygradientaspreviouslydescribed(9).
ization(0.5g[wetweight]ofsedimentplus1mlof50%ethanol–2%NaCl[1:1]),
16SrDNAlibraryconstructionandscreening.Small-subunit(SSU)rDNAs
lipidandnucleicacidanalyses,(approximately15gofsedimentfrozeninliquid
wereamplifiedbyPCRwithpurifiedDNAsamplesfromSantaBarbaraandEel
nitrogen),andporewaterchemistry(remainingsedimentsample).
River basin cold seep sediments. PCR mixtures (50 ml) contained a 0.2 mM
Porewaterchemistry.Porewatersweresqueezedfrom3-cm-thickcoreinter-
concentrationofeitherbacterium-specific(27fand1492r)orarchaeon-specific
valsusingapressurizedgassedimentsqueezer(34)andcollectedintoattached
(20fand1492r)primers.Reactionmixturesalsocontained5mlofPCRbuffer
air-tight60-mldisposablesyringes(BectonDickinson,MountainView,Calif.).
(containing2mMMgCl),2.5mMeachdeoxynucleotidetriphosphate,and0.025
SulfateconcentrationsinporewaterweremeasuredusingaDIONEXDX-120 2
UofTaqpolymerase(Promega,Madison,Wis.).
ionchromatographequippedwithanAS-9HCcolumn.Sulfatewaselutedusing
0.028MNaCO ataflowrateof1.0mlmin21.Sampleswerediluted1:100 PCRconditionsforarchaeallibraries(Eel-36a,SB-24a,SB-17a,andSB-7a).
(vol/vol)ind2eion3izedwaterpriortoanalysis.Measuredvalueswerestandardized Archaeal16SrDNAsfromtheCsCl-purifiedDNAswereamplifiedfor30cycles
against IAPSO standard seawater (28.9 mM SO22). Dissolved methane was (1.5minofdenaturationat94°C,30sofannealingat55°C,and7minofelon-
extractedfromporewatersbyaddinganequalv4olumeofnitrogengastothe gationat72°C)usingarchaeon-specificprimers(A20f,59-TTCCGGTTGATCC
syringeandthenshakingfor2minpriortomeasurementwithagaschromato- YGCCRG-39;U1492r,59-GGTTACCTTGTTACGACTT-39).Theexceptionin
graph (GC) equipped with a flame ionization detector. Pore waters used for thisprocedurewasarchaeallibraryEel-36a,whichwasconstructedundersimilar
analysis of dissolved hydrogen sulfide were obtained by centrifugation under PCRconditionsbutamplifiedforonly20cyclestominimizebiasassociatedwith
oxygen-freeconditions(2)andmeasuredusingacolorimetricassay(8). highcyclenumbers(38).
Lipid analysis. Sediment samples (;10 g [wet weight]) were sterilized by PCRconditionsforbacteriallibraries(Eel-36e,Eel-TE,Eel-BE,andSB-24e).
ultrasonication(ultrasonicbath)in20to40mlofamixtureofdichloromethane Bacterial16SrDNAswerePCRamplifiedforeither30cycles(Eel-TE,Eel-BE,
(DCM)andmethane(9:1,vol/vol)for;30min.Afterevaporationoftheresidual andSB-PC24)or20cycles(Eel-PC36)usingabacterium-specificforwardprimer
solvent,thesedimentswereovendriedat;50°C.Experimentshaveshownthat (B27f,59-AGAGTTTGATCCTGGCTCAG-39)andauniversalreverseprimer
oven drying largely suppresses degradation of analytes by microbial activities (U1492r,59-GGTTACCTTGTTACGACTT-39).PCRconditionswerethesame
(K.-U.Hinrichs,unpublisheddata).Freelipidswereextractedfromdriedand asstatedaboveforthearchaeallibraryconstruction.
homogenizedsedimentsusingaDIONEXAcceleratedSolventExtraction200 Cloningandsequencing.Ampliconswerepooledfromthreereactionsand
system at 100°C and 1,000 lb/in2, with DCM-methanol (90:1, vol/vol) as the cleanedusingaQiaquickPCRpurificationkit(Qiagen,Valencia,Calif.)forall
solvent.Extractswereseparatedintofourfractionsofincreasingpolaritiesusing 16SrDNAlibraries.CleanedproductswerethenclonedwithaTAcloningvector
SUPELCO LC-NH glass cartridges (500 mg of sorbent) and a sequence of kit according to the instructions of the manufacturer (Invitrogen, Carlsbad,
2
solventmixturesofincreasingpolarities(hydrocarbons24mlofn-hexane,ke- Calif.).Screeningforthelibrarieswasconductedbyrestrictionfragmentlength
tonesandesters26mlofn-hexane–DCM[3:1],alcohols25mlofDCM-acetone polymorphism analysis on M13F- and M13R-amplified products using either
[9:1],andcarboxylicacids22%formicacidinDCM).Individualcompounds HaeIIIorRsaI(Promega).M13-amplifiedPCRproductswereinitiallydiluted
werequantifiedandidentifiedusingaHewlett-Packard(HP)model6890GC 1:20inPCRbuffer.Fivemicrolitersofthedilutedproductwasthenusedinthe
equippedwithaJ&WmodelDB-5(60-mlength,0.32-mminnerdiameter,and restrictiondigestcontaining0.5mlofenzymeand2mlofbufferina20-mltotal
0.25-mm film thickness) capillary column and coupled to an HP model 5973 volume,accordingtothemanufacturer’sinstructions.Uniquecloneswereiden-
mass-selectivedetector.Stablecarbonisotopiccompositionsofindividualcom- tifiedandplasmidswerepurifiedeitherwithaWizardgenomicDNApurification
poundsweredeterminedusingaFinniganDeltaPlusmassspectrometercoupled kit(Promega)orbyelectroelutionbyanautomatedminiprepprotocol(McCon-
1924 ORPHAN ET AL. APPL.ENVIRON.MICROBIOL.
FIG. 1. PorewaterprofilesofSO22(})andCH (F)forselectEelRiverBasinseepsamples.(A)Profileforaseepcoreusedinthegeneration
4 4
oftheclonelibrariesEel-36aandEel-36e;(B)chemicalprofileforaseepcoreinwhichANME-2andDesulfosarcinalesaggregatesweredetected
byFISH(depth,3to6cm)(Fig.5).
nell, La Jolla, Calif.). Cleaned plasmid preparations were quantified and se- aggregateswerephotographedusingaSpotSP100cooleddigitalcolorcharge-
quencedusingaThermoSequenaseFluorescentLabeledPrimerCycleSequenc- coupled-devicecamera(DiagnosticInstruments,Inc.,SterlingHeights,Mich.).
ingkit(Amersham,Braunschweig,Germany)andanautomatedmodel4000Lor CapturedimageswereoverlaidinAdobePhotoshop4.0.
4200DNAsequencer(LI-CORBioTech,Lincoln,Nebr.).Double-strandedse- Nucleotidesequenceaccessionnumbers.TherDNAsequencesweresubmit-
quencingwascompletedusingasuiteofprimerstargeting16SrDNA. ted to GenBank and have been assigned the following accession numbers:
Phylogeneticanalysis.SequencesfromclonesweresubmittedtoGenBankfor AF354126toAF354167.
preliminary analysis using the BLAST program of the Ribosomal Database
Project to identify putative close phylogenetic relatives (27). Sequences were
RESULTS
alignedtotheirnearestneighborwiththeautomatedalignmenttooloftheARB
programpackage(O.StrunkandW.Ludwig[ed.],ARB:asoftwareenvironment
Chemicalprofilingofcoldseepsediments.Twoactivemeth-
forsequencedata.1999.[http//www.mikro.biologie.tu-muenchen.de]).Phyloge-
netictreesweregeneratedusingtheSEQBOOT,DNADIST,andNEIGHBOR ane seep sites within the Eel River Basin were targeted for
programsofthePHYLIPversion3.5software(12).TheKimuratwo-parameter extensive chemical and microbial analysis in August 1999.
modelwasusedtoestimateevolutionarydistance,and1,000bootstrapanalyses Depthprofilesfor14pushcoreswithanaveragelengthof15
wereperformedtoassignconfidencelevelstothenodesinthetrees. cmwereanalyzedforconcentrationsofSO 22,CH ,andCO .
Fluorescentinsituhybridization(FISH).SelectedEelRiverBasinsediments, 4 4 2
Nineofthe14seep-associatedcoreshadprofilescharacteristic
displayingchemicalporewaterprofilesdiagnosticofactiveAOM(Fig.1)and
containing13C-depletedbiomarkers,werescreenedforthepresenceofarchaeal- of active anaerobic methane oxidization, with shallow sulfate
bacterialaggregations.Sedimentsamples(0.5cm3)storedin2%NaCl–ethanol depletiondepthsthatreachedundetectablelevelsofSO 22at
4
(1:1)werediluted(1:10)inphosphate-bufferedsalineandtreatedwith15sof depths of ,15 cm. Pore water chemistry features, typical for
mild sonication at 32 A (Sonics and Materials Inc., Danbury, Conn.). (Prior
continentalmarginsedimentswithabundantmethane(i.e.,the
fixationwithformaldehydewasnotabsolutelyrequiredforsuccessfulhybridiza-
tionwiththeoligonucleotideprobes.)Dilutedsampleswerecentrifugedbriefly intersectionofmethaneandsulfateminimainaso-calledtran-
for5sat5,000rpmtopelletlargesedimentparticles,and50to70mlofthe sitionzone)(23,28),wererarelyobservedinseepsedimentsin
supernatant was filtered onto a 0.2-mm-pore-size GTTP polycarbonate filter. the Eel River Basin. Instead, high concentrations of methane
Filters were then treated for 2 min with a phosphate-buffered saline–ethanol
andsulfateoftenoccurredtogetherintheupper10cm(Fig.1).
(1:1)solutionanddriedbeforehybridization.Hybridizationandwashbuffers
Some seep cores contained high concentrations of methane
weresynthesizedaccordingtothemethodofGlo¨ckneretal.(15)using30%
formamideinthehybridizationbufferand80mMNaClinthewashsolution. (upto6.6mM),butlittletonosulfatedepletionwasobserved.
OligonucleotideprobeswerelabeledwitheitherCy3orfluoresceinisothiocya- This is most likely reflective of non-steady-state conditions
natefluorochromes(GensetS.A.,Paris,France).Oligonucleotideprobestarget- causedbydynamicfluidandgastransportinactiveseepzones
ingthebacterialDesulfosarcinales-Desulfococcusgroup(DSS658,TCCACTTC
(43).Independentoftheporewatersulfateconcentration,the
CCTCTCCCAT)andtheseep-specificarchaealANME-2group(EelMSMX932,
AGCTCCACCCGTTGTAGT)havebeenpreviouslyreported(4,32).General majority of the seep cores profiled (seven out of nine) also
archaeal and bacterial probes, AR915 and EUB338, were used as previously contained characteristic archaeal and bacterial lipid biomark-
described(1).Hybridizationwasconductedat46°Cfor2handfollowedbya ersindicativeofAOMconsortia(18,19,30).
wash at 48°C for 15 min. Washed filters were stained with a dilute 4969-dia-
Santa Barbara Basin seep sediments, including seep cores
midino-2-phenylindole(DAPI)solution(5mg/ml)for1minandexaminedunder
epifluorescencemicroscopywithanAxiophot2microscopeusinga1003Pla- SB-pc24andSB-pc17usedin16SrDNAlibraryconstruction,
nAPO objective (Zeiss, Thornwood, N.Y.). Archaeal-bacterial dually stained displayed high hydrogen sulfide concentrations (up to 11.5
VOL.67,2001 ANAEROBIC METHANE-OXIDIZING CONSORTIA 1925
TABLE 2. Concentrationsandd-13Clevelsofarchaealandbacteriallipidsandd-13ClevelsoftotalorganiccarbonandcarbonateofEel-pc36
Depthinterval Archaeol sn-a2r-cHhyaderoolxy- Crocetane cisM-vA7G-1E6:1 d-13C(‰)of d-13C(‰)
totalorganic
(cm) d-13C(‰) Concn(mg/g) d-13C(‰) Concn(mg/g) d-13C(‰) Concn(mg/g) d-13C(‰) Concn(mg/g) carbon ofCO322
0–3 2101.1 1.3 2105.2 3.8 NDa ND 276.7 1.00 228.8 218.0
3–6 2100.6 1.3 2105.8 2.7 291.6 0.3 277.6 1.16 228.9 224.0
6–9 2102.6 1.5 2105.5 2.3 ND ND 280.8 0.95 229.7 232.5
9–12 2102.1 1.1 2105.7 1.0 ND ND 286.5 1.03 228.5 229.9
aND,notdetermined.
mM) and low sulfate concentrations (,5 mM) at 10 cm of also reflected in the isotopic values of authigenic carbonates
sediment depth. Nonseep reference cores from both environ- extracted from the same core, with d-13C values being signifi-
ments showed minimal sulfate depletion throughout the core cantlylowerinmoredeeplyburiedsamples(Table2).
andundetectablelevelsofmethane,hydrogensulfide,andiso- Phylogenetic diversity of archaea. Culture-independent
topicallydepletedlipidbiomarkers(Table1;datanotshown). analysis of archaeal assemblages within seep sediments con-
Chemotaxonomic analysis of archaeal diversity. Chemot- taining isotopically depleted archaeal lipid biomarkers revealed
axonomic analysis of seep-associated sedimentary lipids from five distinct archaeal phylogenetic clades affiliated with both
theEelRiverandSantaBarbarabasinsrevealedhighconcen- theEuryarchaeotaandCrenarchaeota(Table3).Ofthese,two
trations of diverse, extremely d-13C-depleted archaeal lipids, groupsperipherallyrelatedtotheMethanosarcinales,ANME-2
themostprominentbeingarchaeol,sn-2-hydroxyarchaeol,sat- andANME-1,representedasignificantproportionofthetotal
urated and unsaturated 2,6,10,15,19-pentamethylicosane (PMI), rDNAclonesinmethane-ladensediments.
andcrocetane.Depthprofilesofsedimentarylipidbiomarkers 16S rDNAs from the ANME-2 clade (85 to 89% similar to
in Eel River Basin seep core Eel-pc36 revealed abundant, Methylococcoidesmethylutens)weremostfrequentlyrecovered,
13C-depletedarchaealandbacteriallipidsthroughoutthecore representing21to71%ofthetotalclonesintwoofthethree
(Table 2). The archaeal lipids archaeol and sn-2-hydroxy- archaeallibrariespreparedfromEelRiverandSantaBarbara
archaeoldisplayedrelativelyconstantd-13Cvaluesoflessthan basin sites (Table 3). Phylotypes grouping within ANME-2
2100‰ at all depths, suggestive of constant fractionation by from the Eel River and Santa Barbara basins formed three
archaeal groups under conditions of excess methane avail- distinctclustersofhighlyrelatedsequences(.97%similarity)
ability.Incontrasttotheisotopicvaluesofthearchaeallipid (designated subgroups a, b, and c), with maximum overall
fraction,profilesofbacteriallipids(fattyacidsandsn-1-mono- sequencesimilarityof87%forthisgroup(Fig.2).Thediversity
alkylglycerolethers[sn-1-MAGE])inthecoreweremorevari- of ANME-2 phylotypes detected within single samples was
able, showing decreasing d-13C values with increasing depth, high, with representative sequences falling into two or more
likely due to the decreasing d-13C of pore water substrates. subgroups. The exception to this was library SB-17a, which
Evidence for an isotopically depleted carbon pool at depth is contained phylotypes clustering only within subgroup c. The
TABLE 3. Majorarchaeallineagesrepresentedin16SrDNAlibrariesa
Presenceinorabsencefromindicatedsample(%oftotalorganisms)in16SrDNAlibrary:
Eel-TAb Eel-BAb Eel-36a SB-24a SB-17a SB-7a pISA(39) GBc MT/C(29) APA(46) JTB(26)
EelRiverBasin SantaBarbaraBasin
Archaea Eel-Hpc4(0–4cm) d(20–22cm)Eel-Hpc4 dEel-pc36(4–7cm) d(13–16cm)SB-pc24 SB-pc17(4–7cm) SB-pc7(4–7cm) Hydrothermalventsedi-mentsfromJapan SeepsedimentsfromGuaymasBasin Coastalsaltmarsh,Essex,UnitedKingdom Deep-seasedimentsfromtheAtlantic ColdseepsedimentsfromtheJapanTrench
ANME-1 1(55) 1(84) 1(7.5) 2 1(18) 2 1 1 2 2 2
ANME-2 1 1(16) 1(71) 1(67) 1(22) 2 2 1 1 2 2
Methanosarcinales 2 2 1(6) 1(9) 2 2 2 2 1 2 2
Crenarchaeota 2 2 1(2) 1(2) 1(4) 2 1 2 2 1 1
Thermoplasmales 1 2 1(2) 1(3) 1(41) 1 1 2 1 1 1
a1,sequencetypedetected;2,notdetected.Numbersinparenthesesbeside16SrDNAlibrarydesignations(pISA,GB,MT/C,APA,andJTB)arereference
numbers.
bDatawerepreviouslyreportedinreference18.
cDataarefromAndreasTeske(personalcommunication).
dd-13C-depletedarchaeallipidbiomarkersdetectedinmethaneseepsedimentsamplesincludedarchaeol,sn-2-hydroxyarchaeol,dihydrophytol,unsaturatedPMI,
andcrocetane.ArchaealbiomarkerswerenotdetectedinsampleSB-17aorinnonseepcontrolsampleSB-7a.CrocetanewasnotdetectedinsampleEel-BA.d-13C
valuesoftheselipidsrangedfrom281to2129‰.
1926 ORPHAN ET AL. APPL.ENVIRON.MICROBIOL.
FIG. 2. Phylogenetictreeshowingrelationshipsof16SrDNAarchaealclonesequencesfromSantaBarbaraBasinandEelRiverBasinseep
sites(inboldface)toselectedculturedandenvironmentaleuryarchaeotalsequencesinthedatabase.BoldfaceEelRiverBasinclonescontaining
accessionnumberswerepreviouslyreportedinreference18.Environmentalsequencesincluded2MTand2Cfromanoxicsaltmarshsediments
(29),CRAfromdeepseasediments(46),pISAfromhydrothermalventsediments(39).Thetreewasgeneratedbyneighbor-joininganalysisand
correctedwithamaskthatincludedonly60%oftheconservedregions.Onethousandbootstrapanalyseswereperformed,andpercentagesgreater
than50%arereported.Thebarrepresentsa10%estimatedsequencedivergence.environ.,environmental.
SB-17a rDNA clone library was atypical compared to other spectively.LiketheANME-2group,theANME-1groupcon-
seep libraries overall, with the majority of phylotypes being tainsasignificantamountofintragroupdiversity,withthemost
associatedwithrelativesoftheThermoplasmales(Table3). distant sequences in this clade sharing 90% similarity to each
The putative methane-oxidizing archaeal group ANME-1, other. However, in contrast to the greater ANME-1 diversity
recently described from Eel River Basin methane seep sedi- previously reported from a single Eel River Basin cold seep,
ments, was repeatedly detected at methane seep sites in the fewerANME-1-relatedsequenceswererecoveredinthisstudy
Eel River and Santa Barbara basins. ANME-1-related phylo- andtherewaslessdiversitywithinsinglesamples(Fig.2).The
types represented 7.5 and 18% of the total archaeal clones ANME-1phylotypesrecentlyreportedfrommethane-rich,hy-
screened in the 16S rDNA libraries Eel-36a and SB-17a, re- drothermallyactivesettingsformedaseparateclade(39)and
VOL.67,2001 ANAEROBIC METHANE-OXIDIZING CONSORTIA 1927
FIG. 3. ReconstructedionchromatogramofthealcoholfractionfromsampleEel-pc36(6to9cm).Labeledpeaksdesignatecompoundswith
isotopiccompositionsindicatingapartialorexclusivederivationfromanaerobicmethanotrophicmicrobes.p,C toC acyclicalcoholsfrom
14 17
bacteria; #, sn-1-MAGE with ether-linked C to C acyclic alcohol moieties from bacteria. The numbers beside “DAGE” designate carbon
14 18
numbersofether-linkedalkylmoietiesexpressedasnumbers,e.g.,DAGE-15/15isdietherwithtwoC -alkylmoieties.phy-gly,sn-1-monophyta-
15
nylglycerolether(archaea);AR,archaeol(archaea);OH-AR,sn-2-hydroxyarchaeol(archaea);STD,standard.
weremorecloselyrelatedtoeachotherthantotheANME-1 recovered from other marine sedimentary environments (46,
coldseepsequences(.96%)(Fig.2). 47).
Two other methanogen-like sequences, closely related to Lipidchemotaxonomyofbacteria.Twoclassesoflipidswith
culturedmembersoftheMethanosarcinales,madeuplessthan significant contributions from bacterial, syntrophic partners
10%oftheSB-24aandEEL-36alibraries(Table3).Phylotype participatingintheAOMconsortiumweregenerallypresentat
SB-24a1C2, representing 9% of the clones from the Santa seep sites in the Eel River and Santa Barbara basins. These
BarbaraBasinlibrary,ishighlysimilar(98.8%similarity)tothe compounds are fatty acids ranging from C to C in carbon
14 18
obligate methylotroph Methanococcoides burtonii, previously number and monoalkylglycerolethers (MAGE) and dialkylg-
isolatedfromanantarcticlake(13).EelRiverBasinsequence lycerolethers(DAGE)withnonisoprenoidalalkylmoietiesand
types,representedbyEel-36a2H11,werealsocloselyrelatedto chainlengthsfromC toC .Theparticipationoforganisms
14 18
the methylotrophic Methanococcoides spp. (.95% similarity) thatproduceMAGEandDAGEinAOMisindicatedbyd-13C
andmadeup6%oftheEel-36alibrary. values that require more or less exclusive utilization of meth-
RemainingeuryarchaeotalsequencesweresimilartorDNAs ane-derived carbon for biosynthesis (approximately 2100‰
previouslyrecoveredfrommarinesediments(29)thataredis- for most MAGE and selected fatty acids [19]). In addition to
tantlyrelatedtotheThermoplasmales(Fig.2).Relativesofthe the MAGE and fatty acids, the sedimentary alcohols showed
Thermoplasmaleswereaminorproportionofthetotalclones, very similar structural and isotopic features, suggesting that
representing,3%oforganismsinallseeplibrariesexceptthe certainsyntrophicbacterialmembersoftheAOMconsortium
Santa Barbara Basin library SB-17a. 13C-depleted archaeon- synthesizethemaswell.Fattyalcoholsareknownproductsof
specific biomarkers were below detection levels in SB-pc17, some bacteria, but no systematic surveys, i.e., comparable to
which in addition to the high proportion of Thermoplasmales thoseoffattyacids,havebeenundertakenonalcoholcontents
phylotypes may indicate low methane flux at this site. Se- in microorganisms. Very similar relative amounts of MAGE
quencesrelatedtotheThermoplasmalesrepresented100%of and fatty alcohols with carbon isotopic compositions ranging
the clones analyzed in library SB-7a, constructed from sedi- from2100to260‰occurcommonlyinsedimentswithactive
ments from a control site in Santa Barbara Basin, where nei- AOM (e.g., a representative chromatogram is illustrated in
therANME-2orANME-1clones,sulfatedepletion,highsul- Fig.3).
fidelevels,nor13C-depletedlipidbiomarkerswereobserved. Phylogenetic diversity of d-proteobacteria. Table 4 shows
Like the Thermoplasmales-related phylotypes, crenarchaeo- themajord-proteobacteriallineagesdetectedinfourcoldseep
tal sequences were present in both Eel River and Santa Bar- sediment samples that contain 13C-depleted bacterial lipids.
bara basin sites and comprised a relatively low percentage Analysis of 16S rDNA sequences recovered from the same
(,4%)ofclonesinthemethaneseeplibraries(Table3).Cre- sedimentsamplesrevealedapredominanceofd-proteobacte-
narchaeotal sequences from both sites were highly similar to rial phylotypes, represented by six distinct lineages. The ma-
each other (98.9%) and were most closely related to rDNAs jorityoftheseeplibrariescontainedrepresentativesfromtwo
1928 ORPHAN ET AL. APPL.ENVIRON.MICROBIOL.
TABLE 4. Major16SrDNAphylotypesassociatedwithsulfate-reducingbacteria
Presenceinorabsencefromindicatedsamplein16SrDNAlibrary(reference):
Eel-TE Eel-BE Eel-36ea SB-24ea ODPB(3) GCAb JTB(26) TAYNAYA(6) sva(47)
m nts
d-Proteobacteria Eel-Hpc4(0–4cm) Eel-Hpc4(22cm) Eel-pc36(4–7cm) SB-pc24(13–16cm) GashydratesedimentfrotheCascadiaMargin Hydrocarbonseepsedime JapanTrenchcoldseepsediments Antarcticfjordsediments ShelfsedimentfromtheArcticOcean
Desulfosarcina 1 1 1 1 1 1 1 1 1
SeepEel-1 1 1 2 2 2 1 2 2 2
Desulfobulbus 1 2 1 2 1 2 2 2 1
SeepEel-2 1 1 2 2 1 2 2 2 2
SeepEel-3 1 2 2 1 2 2 2 2 1
Myxobacterium 2 2 1 2 2 2 1 2 1
d-1a3CFaitntysaecdiidmseanstsaorcyiaftaetdtywaicthidssurlafantgee-drefdruocming2b5a2cttoer2ia1(0130‰,40.)Ndoetteecttheadtidn-1m3Cethleavneelssienepphseodtoimsyennthtestaimcaplllyesdienrcivlueddeldipnid-Cs1a4r:0e,anp-Cpr1o6:x1im(va:5t)e,layn2d2150‰Me.-C16:0.Levelsof
bDataarefromK.R.O’Neill(GenBankdatabaseaccessionno.AF154080toAF154106).
or three major groups within the d-proteobacteria (Table 4). andarecloselyaffiliatedwithenvironmentalclonesrecovered
Thesesequenceclusterswereeachrelatedby.81%sequence fromasulfate-reducingbenzene-mineralizingenrichment(;92%
similarity and grouped among both cultured sulfate reducers similarity)(Fig.4)(31).Inaddition,Eel-3phylotypes,detected
(i.e.,DesulfosarcinalesandDesulfobulbusspp.)andnovelclus- in both the Eel River and Santa Barbara basins were not
terssofarrepresentedonlybyenvironmentalsequencesorig- closelyrelatedtoknownd-proteobacteria,withonly76%sim-
inating from similar environments (groups Eel-1, Eel-2, and ilarity to Desulfovibrio gabonensis and 80% similarity to an
Eel-3)(Fig.4). environmentalclonerecoveredfromcoldseepsinJapan(26).
SequencesrelatedtotheDesulfosarcinales(91%similarityto Eel-3phylotypeswerehighlysimilartoeachother(99%)and
Desulfosarcina variabilis) were the most common phylotype comprised5ofthe52clonesscreenedfromlibraryEel-TE.
recovered, being detected in all four 16S rDNA libraries and Comparisonofbacterial16SrDNAdiversitybetweenSanta
representing up to 17.6% of total clones in library SB-24e. BarbaraandEelRiverbasinsites.Fourothermajorlineages
These ubiquitous rDNA sequences formed a distinct clade, ofthedomainBacteriawererepresentedin16SrDNAlibrar-
morecloselyrelatedtoeachotherthantoknownsequencesin ies from both Santa Barbara Basin (SB-24e) and Eel River
thedatabase,withgreaterthan94%similarityovera1,300-bp Basin(Eel-BE)coldseeplibraries,includingg-proteobacteria,
region. Although recovered from geographically distant sites, the Flexibacter-Bacteroides-Cytophaga division, candidate divi-
somephylotypeswithinthisgroupfrombothEelRiverBasin sion OP-9, and the Acidobacterium-Holophaga group (Table
(Eel-36e1h1)andSantaBarbaraBasin(SB-24e1c6)werenear- 5).InboththeSB-24eandEel-BElibraries,d-proteobacteria
ly identical, with only a 0.8% sequence difference detected related to sulfate-reducing bacteria were the most abundant
(Fig.4). phylotypes recovered, representing 25 and 36.4% of total
In addition to phylotypes related to the Desulfosarcinales, clones screened, respectively. In addition to sulfate-reducing
16SrDNAsaffiliatedwiththepropionate-utilizingDesulfobul- phylotypes,librarySB-24ealsocontainedalargepercentageof
bus were also recovered from two sites in Eel River Basin. e -proteobacterial phylotypes (26%) related to other sulfide-
Desulfobulbus-related clones were closely related to environ- oxidizingchemoautotrophicmicroorganisms(93.5%similarto
mentalclonesrecoveredfromarcticsediments(92%similarto Thiomicrospiradenitrificans).Relatede -proteobacterialphylo-
environmental clone sva0631) as well as methane hydrate- types were also recovered in one Eel River library, Eel-36e
associated sediments from Cascadia Margin (Fig. 4) (3, 33). (;92%similaritytoSB-24ephylotypes),similartothosefound
Additionally,phylotypesclusteringwithintheEel-2cladewere in other seep environments, including methane-rich anoxic
also peripherally related to Desulfobulbus members (;88% sediments from the Cascadia Margin, the Japan Trench, and
similaritytoDesulfobulbuspropionicus).Eel-2phylotypescom- SagamiBay(3,26,44)(Table5).
prised a significant percentage of clones recovered from Eel Diversephylotypesrelatedtotheg-proteobacteriawerede-
RiverBasinseepsampleEel-Hpc4(17%)andweredetectedin tectedinallfourbacteriallibraries,withmanyphylotypesbe-
both shallow (4- to 7-cm) and deep (20- to 22-cm) sediment ingrelatedtosequencesrecoveredfromarcticsediments(85to
sections (Table 4). Similar to those within Eel-2 clones, phy- 90%similarity)(33).Similartothee -proteobacterialrepresen-
lotypes within the Eel-1 clade were also recovered from both tatives, a number of g-proteobacteria phylotypes recovered
depth intervals from seep core Eel-Hpc4. Eel-1 phylotypes fromEelRiverandSantaBarbarabasinsiteswererelatedto
represented 12% of the total clones screened from library both free-living and symbiotic sulfide oxidizers (91% similari-
Eel-BE(constructedfromthe20-to22-cmsedimentinterval) ty), as well as other environmental sequences recovered from
VOL.67,2001 ANAEROBIC METHANE-OXIDIZING CONSORTIA 1929
FIG. 4. Phylogenetictreeshowingrelationshipsof16SrDNAd-proteobacterialclonesequencesfromSantaBarbaraBasinandEelRiverBasin
seeps(inboldface)toselectedculturedandenvironmentalproteobacterialsequencesinthedatabase.Environmentalsequencesincludedsvafrom
arctic sediment (47) and SB from a benzene-mineralizing enrichment culture (31). The tree was generated by neighbor-joining analysis and
correctedwithamaskthatincludedonly60%oftheconservedregions.Onethousandbootstrapanalyseswerepreformed,andpercentagesgreater
than50%arereported.Thebarrepresentsa10%estimatedsequencedivergence.
similar seep habitats (26). Additional groups represented (96.7% similarity) were also detected in the Santa Barbara
withintheg-proteobacteriaincludedSB-24ecloneshighlysim- Basinlibrarybutcomprisedonlyarelativelysmallpercentage
ilar to Halomonas variabilis (95.3% similarity) and Eel-TE of the total clones (;3%) (Table 5). Included in the seep-
clonesrelatedtotheaerobicmethane-oxidizingMethylobacter associated OP-9 cluster were environmental clones from cold
luteus (95.7% similarity) and the psychrophile Colwellia psy- seepsintheJapanTrench(26),hydrocarbon-loadedseepsed-
chroerythrus(97.7%similarity). iments (GenBank accession no. AF154106), and a benzene-
Relatives of candidate division OP-9, originally described mineralizing consortium clone SB-15 (31). Sequences related
fromhotspringenvironments,comprisedasignificantpropor- to the recently described Acidobacterium-Holophaga division
tion of the total clones screened in library Eel-BE (21%). werealsodetectedbutinlowabundance(.4%).
Sequences highly related to the Eel River OP-9 phylotypes Planctomyces spp. and Cytophagales spp.-related phylotypes
1930 ORPHAN ET AL. APPL.ENVIRON.MICROBIOL.
TABLE 5. Majorbacteriallineagesrepresentedin16SrRNAgenelibrariesfrommethaneseeps
Presenceinorabsencefromindicatedsample(%oftotalorganisms)in16SrDNAlibrarya:
SB-24e Eel-BE Eel-TEc Eel-36ec JTB(26) GCAb TAY-NAYA(6) sva(47)
nt
e
Organisms Closestrelative SantaBarbaraBasinSB-pc24(13–16cm) EelRiverBasinEel-Hpc4(20–22cm) EelRiverBasinEel-pc36(4–7cm) SantaBarbaraBasinSB-pc24(13–16cm) ColdseepsedimentfromJapan Oilseepsediments AntarcticfjordsedimfromTaynayaBay ShelfsedimentsfromtheArcticOcean
d-Proteobacteria 1(25) 1(36.4) 1 1 1 1 1 1
e -Proteobacteria S-oxidizingsymbionts 1(26) 2 2 1 1 1 2 2
g-Proteobacteria (S-oxidizingsymbionts1others) 1(4.3) 1(3.3) 1 1 1 1 1 1
a-Proteobacteria Rhizobiumspp. 2 2 2 1 1 2 1 1
Planctomyces Pirellulaspp. 1(3.3) 1(1) 1 2 2 2 2 1
FBCd Cytophagaspp. 1(3.3) 1(6.6) 1 2 1 2 1 1
Graciliutes Haloanaerobiumspp. 2 1(2.5) 2 2 2 1 2 2
Gram-positivebacteria Propionigeniumspp. 2 1(4.1) 1 2 1 1 1 1
Greennonsulfurorganisms 1(5.5) 2 2 2 2 1 2 1
Holophaga Acidobacteriumspp. 1(3.3) 1(2.5) 1 2 2 1 1 1
CandidatedivisionOP-9 1(3.3) 1(21) 2 2 2 1 1 2
CandidatedivisionOP-11 2 1(4.1) 2 2 2 2 2 2
Spirochetes 1(5.5) 2 2 2 2 2 2 2
aNumbersinparenthesesbesidelibrarydesignationsarereferencenumbers.
bHydrocarbonseep.DataarefromK.R.O’Neill(GenBankdatabaseaccessionno.AF154080toAF154106).
cBasedonapartiallibraryscreeningofEel-TE(52clones)andEel-36e(18clones).
dFBC,Flexibacter-Bacteroides-Cytophagadivision.
werecommoninSantaBarbaraandEelRiverbasinlibraries, size,rangingbetween5and30mmindiameter,anddisplayed
representing3.3and6.6%oftheclones,respectively.Environ- anirregularstainingpatternwithDAPI.
mentalsequencesrelatedtotheCytophagalesappeartohavea
widespreaddistributioninmarinesediments,detectedinboth DISCUSSION
seep-associated and other marine sedimentary environments,
including the Arctic Ocean, an antarctic fjord, and the Japan Oneofthemoreimportantbiogeochemicalprocessesinflu-
Trench (6, 26, 33). Planctomyces-related sequences were less encingcarbonturnoverincontinentalmarginenvironmentsis
frequentlydetectedinmarinesedimentdiversitysurveysthan the anaerobic oxidation of methane. Although there is con-
Cytophagales-relatedsequences.Relatedphylotypeswerepre- vincing biogeochemical evidence for archaeon and sulfate re-
viouslyreportedinarcticandPugetSoundsediments(16,33). ducer cooperative involvement in AOM, identification of the
Although frequently reported in molecular surveys of marine potential organisms involved has been reported only very re-
sediments,includingthosefromseepenvironments,sequences cently (4, 18). Combining environmental 16S rDNA surveys
clustering within the gram-positive bacteria comprised only a andwhole-cellinsituhybridizationwithchemotaxonomicsur-
small percentage (4%) of the total clones screened in this veys of 13C-depleted sedimentary lipids from geographically
study. Gram-positive bacterial phylotypes were detected only distant methane seep sites, we confirm and extend previous
in16SrDNAlibrariesfromtheEelRiverBasin. observationsofAOMconsortiainmarinesediments.Ourdata
Insituwhole-cellhybridization(FISH).Weusedpreviously indicatethewidespreadoccurrenceofspecificgroupsofmeth-
designed (4) 16S rDNA-targeted oligonucleotide probes to ane-consumingarchaeaandsulfate-reducingbacteriainvolved
visualize microbial groups that are thought to be involved in inAOM.Ourdataalsosupportpreliminaryphylogeneticevi-
the anaerobic oxidation of methane. Oligonucleotide probes dence (4, 18) identifying some of the key microbial groups
specific for members of the Desulfosarcinales and Desulfococ- mediatingthisspecializedprocessinthemethane-rich,anaer-
coides groups (DSS658) and the newly described archaeal obicenvironments.
ANME-2 group (EelMSMX932), as well as general archaeal Archaeal diversity within methane seeps. Levels of total
and bacterial probes (AR915 and EUB338), were hybridized seep-relatedarchaealSSUrDNAdiversitydetectedfromboth
withcellsfrommethaneseepsedimentsamples(4,32).Atotal Eel River Basin and Santa Barbara Basin were very similar.
of four cores (Eel-pc6 [4 to 7 cm], Eel-pc36 [4 to 7 cm], Fivemajorphylogeneticgroupswererepresentedandincluded
Eel-pc21[1to4cm],andEel-pc54[3to4cm])fromEelRiver sequences highly related to cultured Methanosarcinales, the
BasinseepscontainedcellshybridizingwitharchaealANME-2 newly described ANME-2 clade, phylotypes more distantly
and sulfate-reducing DSS658 probes. Archaeal and bacterial related to cultured members within the Methanosarcinales
cells hybridizing with the EelMSMX932 and DSS658 probes (ANME-1 group), and low-temperature relatives of the Ther-
occurred together as structured aggregates, comprised of ir- moplasmales and Crenarchaeota. Comparisons to other 16S
regularcoccoidcells1to2mmindiameter(Fig.5).Archaeal rDNAphylogeneticsurveysofsedimentaryarchaearecovered
andsulfate-reducingbacterialaggregateswerehighlyvariedin from both seep and nonseep marine habitats suggest that the
VOL.67,2001 ANAEROBIC METHANE-OXIDIZING CONSORTIA 1931
FIG. 5. Whole-cellFISHofputativemethane-oxidizingconsortiainmethaneseepsediments.(A)DAPIstainofaggregatesfroma3-to6-cm
sediment depth from Eel River Basin cold seeps. Sediment preperations were simultaneously hybridized with a fluoroscein-labeled probe for
Desulfosarcina-Desulfococcusmembers(DSS658)(B)andaCy-3labeledarchaealANME-2-groupspecificprobe(EelMSMX932)(C).(D)Overlay
ofbothCy-3ANME-2group(red)andfluorosceinDulsulfosarcinales(green)resultstoshowthearchitectureoftheaggregate.Scalebar510mm.
ANME-1 and ANME-2 groups are specific to anoxic seep- valuesandlipidconcentrationsinsedimentaryverticalprofiles
associated environments but that phylotypes related to the suggest that the composition of methane-consuming archaea
Thermoplasmales and low-temperature Crenarchaeota are more doesnotchangesignificantlywithdepth.Similar13C-depleted
broadlydistributedinmarinesediments. diphytanylglycerolethers (archaeol and sn-2-hydroxyarchaeol)
Coincidingwiththearchaealgenotypesrecovered,sedimen- have been previously detected in other methane-laden sedi-
tary biomarkers related to the archaea were also abundant, mentary environments, including seep sediments in the Med-
comprised of at least four distinct lipids with extremely low iterranean and at the Cascadia Margin, suggesting that Meth-
levelsofd-13C(downto2129‰).Twoofthemostprominent anosarcinales relatives and, more specifically, members of the
biomarkers, sn-2-hydroxyarchaeol and PMI, are specifically ANME-2groupmaybeactivemethaneconsumersinhigh-flux
abundantinmembersoftheMethanosarcinalesandwerecor- methane-richmarineenvironmentsworldwide(4,19,30).Fur-
relatedwiththerecoveryofMethanosarcinales-relatedANME- thermore,ANME-2-relatedphylotypeswerepreviouslyrecov-
2rDNAclonesfromseepsampleswithinSantaBarbaraBasin eredfromhighlyreduced,methane-richsaltmarshsediments,
(SB-pc24) and Eel River Basin (Eel-pc36). These findings suggestingthatthisgroup’sinvolvementinAOMmightextend
strongly support the hypotheses that ANME-2 members are toshallow,warmerwatercoastalflatsaswell(29).Inaddition
onesourceoftheisotopicallylightarchaeallipidsandthatthey tocontainingarchaeolderivatives,selectedsamplesfromboth
actively consume methane within a variety of methane-rich the Santa Barbara and Eel River basins contained 13C-de-
anaerobic marine environments. Boetius et al. (4) came to pletedcrocetane,previouslyattributedtoanaerobicmethane-
similar conclusions based on FISH results in methane-rich utilizingmicroorganisms(11,42).Althoughmicrobialsources
sedimentsatHydrateRidgethatalsocontainthesame,isoto- of crocetane are unknown, its structure with the tail-to-tail
pically light archaeal lipids. In addition, the uniform isotopic linkageofisopreneunitssuggestsanarchaealorigin,different