Fine-scale community structure analysis of ANME in Nyegga sediments with high and low methane ...

13 Pages·2012·1.84 MB·English

by  Ida Helene Steen

ORIGINALRESEARCHARTICLE
published:15June2012
doi:10.3389/fmicb.2012.00216
Fine-scale community structure analysis of ANME in
Nyegga sediments with high and low methane flux
IreneRoalkvam1,HåkonDahle1,YifengChen2,SteffenLethJørgensen1,HaflidiHaflidason3 and
IdaHeleneSteen1*
1CenterforGeobiology,DepartmentofBiology,UniversityofBergen,Bergen,Norway
2GuangzhouInstituteofGeochemistry,ChineseAcademyofSciences,Guangzhou,China
3DepartmentofEarthScience,UniversityofBergen,Bergen,Norway
Editedby: To obtain knowledge on how regional variations in methane seepage rates influence the
PeterDunfield,UniversityofCalgary, stratification,abundance,anddiversityofanaerobicmethanotrophs(ANME),weanalyzed
Canada
theverticalmicrobialstratificationinagravitycorefromamethanemicro-seepingareaat
Reviewedby:
Nyeggabyusing454-pyrosequencingof16SrRNAgenetaggedampliconsandquantitative
PeterDunfield,UniversityofCalgary,
Canada PCR.ThesedatawerecomparedwithpreviouslyobtaineddatafromthemoreactiveG11
MartinKrüger,FederalInstitutefor pockmark,characterizedbyhighermethaneflux.Adowncorestratificationandhighrela-
GeosciencesandNaturalResources tiveabundanceofANMEwereobservedinbothcores,withtransitionfromanANME-2a/b
(BGR),Germany
dominated community in low-sulfide and low methane horizons to ANME-1 dominance
Nils-KaareBirkeland,Universityof
Bergen,Norway in horizons near the sulfate-methane transition zone.The stratification was over a wider
CraigLeeMoyer,Western spatialregionandatgreaterdepthinthecorewithlowermethaneflux,andthetotal16S
WashingtonUniversity,USA rRNA copy numbers were two orders of magnitude lower than in the sediments at G11
JinjunKan,StroudWaterResearch
pockmark. A fine-scale view into the ANME communities at each location was achieved
Center,USA
throughoperationaltaxonomicalunits(OTU)clusteringofANME-affiliatedsequences.The
*Correspondence:
IdaHeleneSteen,Centerfor majorityofANME-1sequencesfrombothsamplingsitesclusteredwithinoneOTU,while
Geobiology,DepartmentofBiology, ANME-2a/b sequences were represented in unique OTUs. We suggest that free-living
UniversityofBergen,P.O.Box7800, ANME-1isthemostabundanttaxoninNyeggacoldseeps,andalsothemainconsumerof
N-5020Bergen,Norway.
methane.TheobservationofspecificANME-2a/bOTUsateachlocationcouldreflectthat
e-mail:[email protected]
organismswithinthiscladeareadaptedtodifferentgeochemicalsettings,perhapsdueto
differencesinmethaneaffinity.GiventhattheANME-2a/bpopulationcouldbesustained
inlessactiveseepageareas,thissubgroupcouldbepotentialseedpopulationsinnewly
developedmethane-enrichedenvironments.
Keywords:ANME,pyrosequencing,AOM,communitystructure,Nyegga,coldseep,stratification
INTRODUCTION andthepresentunderstandingisthatnospecificANMEcladeis
Anaerobicmethanotrophs(ANME)playavitalroleintheglobal consideredcharacteristicfortheSMTZ.
carboncyclebudget,actingasmethanesinksinmarinesystems. Although the SMTZ is often dominated by members of the
Through anaerobic oxidation of methane (AOM) they are esti- ANMEclade,otherarchaealtaxabesidesANMEhavebeenfound
mated to consume >90% of the 85–300Tg CH annually pro- tobeenrichedwithintheSMTZ.UnculturedArchaealikeMarine
4
duced,andtherebycontributetoastrongreductionof methane Benthic Group B (MBG-B) [also named Deep Sea Archaeal
emission to the atmosphere (Knittel and Boetius, 2009). Their Group (DSAG)], Miscellaneous Crenarchaeotic group (MCG),
main niche in marine sediments is the sulfate-methane transi- andMarineBenthicGroupD(MBG-D)areamongthemostabun-
tionzone(SMTZ)whichisformedwhenmethanefromsubsur- danttaxainsystemslikethePerumargin,Aarhusbay,Benguela
face reservoirs meets sulfate penetrating from the water column UpwellingSystem,andSantaBarbaraBasin(SørensenandTeske,
throughadvection(Berelsonetal.,2005;Treudeetal.,2005b;Knit- 2006; Schafer et al., 2007; Harrison et al., 2009; Webster et al.,
telandBoetius,2009).Thelocationof theSMTZrangesfroma 2011). No ANME population was identified in sediments from
fewdecimeterstoseveralhundredmetersbelowtheseafloor,and the Peru margin and Cascadian margin in the study by Inagaki
is influenced by the local geological settings such as the depth et al. (2006), despite the presence of gas hydrates located close
of the methane production zone, the flux of methane, and sul- totheSMTZ(presentat50–110mbsf andbelow),whileANME
fate through the sediment column and their consumption rates wasfoundamongthelow-abundancetaxaintheotherlocations
(Knittel and Boetius, 2009). Sequences from all the currently mentioned above. The highly abundant DSAG in Peru margin
defined ANME clades, ANME-1, ANME-2, and ANME-3, have sedimentshasbeensuggestedtobeinvolvedintheconsumption
been retrieved from SMTZs from areas like the Gulf of Mex- of methaneorinsulfatereduction(D’Hondtetal.,2004;Biddle
ico (Lloyd et al.,2006),Skagerrak (Parkes et al.,2007),Haakon et al., 2006; Inagaki et al., 2006), although the metabolic capa-
MosbyMudVolcano(HMMV;Niemannetal.,2006)respectively, bilityofthistaxonremainsunsolved.Furthermore,themethane
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Roalkvametal. ANME-stratificationinfluencedbymethaneflux
fluidfluxintotheSMTZinPerumarginhasbeenestimatedto1.6– et al.,2006;Chen et al.,2010;Hustoft et al.,2010;Ivanov et al.,
8.8mmol/m2year(Biddleetal.,2006),whichisconsiderablylower 2010;Plaza-Faverolaetal.,2010,2011;Vaularetal.,2010;Reiche
thanthefluxinANME-dominatedareaslikeHydrateRidge(11– etal.,2011).Recent2D/3Dseismicandmultibeammappingofthe
33×103mmol/m2year; Torres et al.,2002),the Gulf of Mexico Nyeggaareahasrevealedanareawithahighdensityofpockmark
(500–2300mmol/m2year;Lloydetal.,2010),andEckernfördeBay structures, many with underlying gas blanking areas extending
(240–690mmol/m2year;Treudeetal.,2005a).Hence,themicro- downtoapronouncedbottomsimulatingreflector(BSR)at250–
bialcommunitycompositionandabundanceofvariousarchaeal 300mdepthbelowseafloor(mbsf;Bünzetal.,2003;Hustoftetal.,
taxainmarinesedimentscouldberelatedtothesupplyofmethane 2007,2009,2010;Hjelstuenetal.,2010;Plaza-Faverolaetal.,2010;
throughthesystemovertime,andwhethertheavailablemethaneis Reiche et al., 2011). During a cruise with R/V G.O.Sars to the
sufficienttosustainanANME-dominatedcommunityoverother NyeggaareainAugustof2008,the3mlonggravitycoreGS08-155-
unculturedArchaea. 15GC(referredtoas15GChereafter)wasretrievedfromtheCN03
FortheG11pockmarkatNyegga(thesouthernVøringPlateau, area(64˚45.274(cid:48)N05˚04.088(cid:48)E)at725mwaterdepth.Theambi-
offshore Mid-Norway) located at water depth of 730m the entseawatertemperaturewasmeasuredwithCTDtobebetween
methane flux rate is estimated to 300–500mmol/m2year (Chen −0.6 and −0.7˚C. After retrieval of 15GC, one half of the core
et al., 2010). New high-throughput pyrosequencing technolo- wasimmediatelysampledfordetailedmicrobialdiversitystudies
gies allow a deeper sampling of the ecosystems of interest, and andgeochemicalmeasurementswhiletheotherhalfwasstoredat
approachesinvolvingbarcodesoruniqueDNAsequenceidenti- 4˚C as an archive for non-destructive MST and XRF core scan-
fiershavebeendevelopedformultiplexsequencing(Huberetal., nerstudiesinlaboratoriesonland.Rhizonsamplerswereusedto
2007;Parameswaranetal.,2007;Hamadyetal.,2008).Thestrati- extractpore-waterfromeighthorizonsthroughoutthecore;at24,
ficationofmicroorganismsinsedimentsfromtheG11pockmark 57,89,129,171,244,258,and290cmbelowseafloor(cmbsf).The
wasrecentlyanalyzedbyusingFISH,quantitativePCR,and16S subsampleswerepreservedinglassvialsandkeptcooluntilthey
rRNA gene amplicon libraries of several subsamples (Roalkvam wereanalyzedaccordingtotheapproachinChenetal.(2010)in
etal.,2011).ThesedimentcorewassampledinsidetheG11pock- ordertodeterminetheconcentrationofdissolvedsulfate(SO2−)
4
markinapingostructure,whichischaracterizedbyanelevated andtotaldissolvedhydrogensulfide(ΣH S).SubsamplesforDNA
2
seafloorduetothelocalaccumulationofgashydratesbelow(Hov- extractionwereasepticallyretrievedat10,30,50,80,100,120,140,
landandSvensen,2006),wherethemethanefluxisrelativelyhigh. 160,180,200,220,240,255,270,and300cmbsf(±0.5cm)byusing
The horizons in the shallower parts of the core [0–3cm below sterile1mLtipcutplasticsyringesbeforetheyweresnap-frozen
seafloor (cmbsf)] were dominated by aerobic methanotrophs inliquidN andstoredat−80˚C.
2
within Gammaproteobacteria, and sulfur oxidizing taxa within
Epsilonproteobacteria.Atdepthsbelow4–5cmbsf,astratification ESTIMATIONOFMETHANEFLUX
of ANME clades was observed with transitions between hori- Sulfategradientsmaybeusedtoestimatetheinsitumethaneflux
zons dominated by ANME-2a/b,ANME-1, and ANME-2c with (Borowskietal.,1996).Thesulfatediffusivefluxisobtainedfrom
increasingdepth. the linear zone in the concentration profile,i.e.,from the depth
Here,weused454-pyrosequencingof16SrRNAtaggedPCR- range in which there is no production or consumption. Fick’s
amplicons combined with quantitative PCR to investigate the firstlaw(KromandBerner,1980)wasusedtocalculatethesul-
stratification of the microbial communities in a Nyegga sedi- fatediffusivefluxinthecore,asdescribedbyChenetal.(2010).
ment with a relatively low methane flux 80mmol/m2year and As the consumption of sulfate and methane has the stoichiom-
deep SMTZ at 205–255cmbsf (Chen et al.,2011). Furthermore, etry 1:1 duringAOM (Boetius et al.,2000),the sulfate diffusive
wecomparethemicrobialdiversityanddominatingANMEphy- fluxisequivalenttothemethaneflux.Thefluxwasestimatedto
lotypesinsedimentswithlowmethanefluxwiththosepresentin ∼80mmol/m2year in 15GC, when a core porosity of 63% was
sedimentswithhighermethanefluxfromapingostructurewithin used(Chenetal.,2011).
theG11pockmark(Roalkvametal.,2011).Overall,wedemon-
strate that the local fluid flow regimes influence the abundance DNAEXTRACTIONAND16SrRNAGENEAMPLICONLIBRARY
anddiversityof microbialpopulationsincoldseepsedimentsat PREPARATION
Nyegga,andpossiblyincoldseepsedimentsingeneral. TotalgenomicDNAwasextractedfrom∼0.5gofsedimentfrom
allsubsamplesusingFastDNASpinkitforsoil(MPBiomedicals),
MATERIALSANDMETHODS and was subsequently quantified by A260/A280 ratio measure-
SITEDESCRIPTIONANDSAMPLING ments, as described in Roalkvam et al. (2011). The pipeline of
TheNyeggaareaislocatedontheupperMid-Norwegiancontinen- 16SrRNAgeneampliconlibrarypreparation,sequencefiltering,
talslope,atthenortheastflankof theStoreggaSlide(Figure1), and taxonomical classification of amplicons is described else-
and is characterized by a high density of pockmarks and fluid where(Lanzénetal.,2011;Roalkvametal.,2011).Inshort,DNA
seepagestructures(Evansetal.,1996;Hustoftetal.,2010;Reiche from seven subsamples in 15GC (10, 30, 80, 120, 180, 240, and
etal.,2011).Theareahasbeenafieldformultidisciplinarystudies 270cmbsf)wasappliedinatwostepPCRinordertogeneratea16S
ongashydrates,authigeniccarbonates,fluidflow,andpore-water rRNAgeneampliconlibraryforeachhorizonwherebothPCR’s
geochemistry the last decade with a special focus on the active followedapreviouslydescribedprotocol(Roalkvametal.,2011).
micro-seepingareaaroundpockmarksG11andCN03(alsocalled InordertoevaluatetheaccuracyofthePCRandthesequencing
CNE03;Hovlandetal.,2005;HovlandandSvensen,2006;Mazzini step,subsample270cmbsffrom15GCwasanalyzedintriplicates.
FrontiersinMicrobiology|ExtremeMicrobiology June2012|Volume3|Article216|2
Roalkvametal. ANME-stratificationinfluencedbymethaneflux
FIGURE1|OverviewmapoftheNorwegianSeaandthesurrounding LocationoftheCN03targetsiteandthepockmarkG11attheNyeggaarea
landareaswiththelocationoftheNyeggastudyareaandtheHMMV (B).Ahigh-resolutionTOPASprofile(LineGS07-148-126)acrosstheCN03gas
(HåkonMosbyMudVolcano).Thepositionofthemainpathwayofthewarm seepingareawithinsertedthelocationofthestudiedcore
surfacecurrentNWAC(NorwegianWaterAtlanticCurrent)isoutlined(A). GS08-155-15GC(C).
TemplateDNAfromallsubsampleswereamplifiedintriplicates QUALITYFILTERINGOF16SAMPLICONSEQUENCESANDTAXONOMIC
usingtheprimersUn787f(5(cid:48)-ATTAGATACCCNGGTAG;Roesch CLASSIFICATION
etal.,2007)andUn1392r(5(cid:48)-ACGGGCGGTGWGTRC;modified Quality filtering and noise removal of pyrosequencing reads of
fromLaneetal.,1985).Thetriplicateswerepooled,andimpuri- ampliconswerecarriedoutusingAmpliconNoise(Quinceetal.,
tieswereremovedusingMinElute®PCRpurificationkit(Qiagen). 2011) as described in Roalkvam et al. (2011). In summary,
PurifiedampliconswereusedastemplateinasecondPCRwhere noise, and errors introduced during PCR and pyrosequencing
theabovementionedprimersweremodifiedtospecificationsin are corrected during four steps: filtering, flowgram clustering,
Lib-L chemistry: the GS FLX Titanium Primer A sequence and sequence-clustering,andchimeraremoval.Thefilteredsequences
a specific MID sequence of 10bp for each sample was included werealignedtoareferencedatabasepreparedfromSilvaSSURef
in forward primer Un787f, while the GS FLX Titanium Primer release102(Lanzénetal.,2011)usingblastn(defaultparameters).
B sequence was included in reverse primer Un1392r. The final Sequenceswithabit-scoreabove150wereassigntotheirequiv-
amplicons were purified as described above and the concentra- alenttaxainthemodifiedSilvaTaxonomydescribedabovebased
tionwasdeterminedbySYBR-Greenquantification,asdescribed onthetaxonomyofthebestblastnbitscoreswithina10%range,
inRoalkvametal.(2011).Priortothepyrosequencingallsamples usingMEGANversion3.7(Husonetal.,2007).Finally,theassign-
were pooled, and a final purification using Agencourt AMPure mentswereexportedandweighedaccordingtoitscluster’scopy
XP(BeckmanCoultergenomics)wasappliedtoensureremoval number.
of all impurities. The GS FLX instrument (Roche) at the Nor- Theampliconlibraryfrom270cmbsf wasmadeintriplicates
wegian Sequencing Centre was used with 450bp chemistry for priortosequencingtotesttheprecisionandreproducibilityofour
454-pyrosequencingofallamplicons.Therawsff-filesof16Stag- primersandthepipelineforampliconconstructionandfiltering.
encodedampliconsfromallsubsamplesingravitycore15GCfrom Therelativeabundanceofthetaxalistedatalltaxonomicallevels
NyeggahavebeensubmittedtotheSequenceReadArchiveunder was compared between the parallel samples, all showing nearly
theaccessionnumberSRA026733. the same relative taxa distribution (maximum deviation at any
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Roalkvametal. ANME-stratificationinfluencedbymethaneflux
giventaxonomiclevelwas0.014%).Thetriplicatesweretherefore standardcurveandgenomicDNAfromE.coliwasusedasnega-
merged and treated as one subsample, comprising 61268 reads, tivecontrol.Foreachsubsample,themeannumberof16SrRNA
duetothelowdeviationinrelativeabundance. genecopies/gsedimentandcorrespondingstandarddeviationwas
usedasenumerationofBacteriaorArchaeainthecore.
OPERATIONALTAXONOMICUNITASSIGNMENTANDDIVERSITYINDEX
ESTIMATIONS
RESULTS
TocomparethemicrobialcommunitiesinGC15fromtheCN03
GEOCHEMISTRY
area with those in the more active area within the pingo struc- Theconcentrationofdissolvedsulfate(SO2−)andtotaldissolved
tures at G11 pockmark (Roalkvam et al., 2011) on operational 4
hydrogensulfide(ΣH S)inpore-waterfromcore15GCfromthe
taxonomical units (OTU)-level, the individual 16S rRNA gene 2
CN03 area was determined. The results show a linear decrease
tagged amplicon files were merged and grouped into OTUs intheconcentrationofSO2−withdepth,rangingfrom27.2mM
using theAmpliconNoise software and its incorporated features 4
at24cmbsfto2.7mMat290cmbsf,concomitantwithagradual
(Quince et al., 2011). In AmpliconNoise, a quick pre-clustering
increaseintheconcentrationofΣH Sfrom1.2to6.28mMforthe
offlow-gramswasperformedpriortothepair-wisealignmentof 2
samedepthinterval,respectively(Figure2).However,thehighest
sequencesusingtheNeedleman–Wunschalgorithm(Needleman
concentrationofΣH Swasmeasuredto13.38mMat244cmbsf.
andWunsch,1970),followedbyahierarchicalmaximumlinkage 2
Themethanefluxwasestimatedto∼80mmol/m2yearin15GC,
clusteringwithof97%sequenceidentity.Inordertoassignataxon
basedonthelinearzoneinthesulfateconcentrationprofile.The
toeachcluster,onerepresentativesequencefromeachOTUwas
SMTZin15GCwasestimatedtobeat∼205–255cmbsf,whichis
selectedandalignedtotheSilvaTaxonomyusingMEGAN.The
supportedbythepeakat244cmbsfintheδ13C measurements
sequencesrepresentingeachOTUwerealignedtotheSilvaSSURef DIC
(Chenetal.,2011).Consumptionofsulfateismainlyascribedto
release104inArb,andatotalof23sequenceswereexcludeddue
AOM(85%)andoxidationofotherorganicmaterial(15%;Chen
toshortlength(<220bp)orchimeras,leaving3299sequences.
etal.,2011).AcharacteristicSMTZwasnotobservedin29ROV.In
DiversityindiceswerecalculatedbytheShannon–Weaverindex
15GC,themethaneconcentrationintheheadspaceofpore-water
(Weaver and Shannon,1949) and Rao’s quadrate entropy index
samplesrangedfrom0.012to0.38mmol/Linhorizonsabovethe
(Rao,1982)usingR(version2.13.1)withtheVeganpackageinte-
SMTZ,and gradually increased values from 0.16 to 3.6mmol/L
gratedoranin-houseRscript,respectively.Thedistancematrix
between243and290cmbsf(Vaular,2011).
needed for Rao’s quadrate entropy index was generated using
The15GCcoreconsistsof siltandclayrichsedimentswitha
PhylogenyInferencePackage(PHYLIP;Felsenstein,1989)inArb
highbulkdensity(1.9–2.3g/cm3)andlowporosity,presentedas
(Version5.0).Diversityindices,suchastheShannonindex,maybe
fractionalporosity(∼25–45%),andalowpermeability(Figure2).
influencedbysamplingsize.Tocomparediversityindicesacross
The sediment interval starting at 80–90cmbsf and ending at
pyrosequencing libraries of variable size,all sequence tags from
∼230–240cmbsf,withinthepresentSMTZ,ispiercedbypiping
allsampleswerefirstclusteredintoOTUs.Theneachlibrarywere
structuresandchemosyntheticshellsandshellfragments,indicat-
randomlysubsampledusingasubsamplingsizeequaltothesam-
ingthatthecoresitehasbeenanactivemethaneseepingareaat
plesizeofsmallestlibrary(796reads)andbykeepingtheoriginal
earlier stage. The high variability in the carbonate content,rep-
OTU assignments for each sequence tag. Reported mean values
resentedbytheXRFanalysisontheCaelement,showsthatthat
and standard deviations were calculated from indices calculated
episodic activity of biogenic production is also found in more
from1000subsamplingiterationspersample.
recenttimes,atadepthof∼30cm(Figure2).
QUANTITATIVEPCR
The number of 16S rRNA genes from bothArchaea and Bacte- QUANTITATIVEPCR
riaineachsubsampleof15GCwereenumeratedusingreal-time Both Bacteria and Archaea in 15GC were enumerated as 16S
quantitativePCR,asdescribedinRoalkvametal.(2011).Inshort, rRNA gene copies/g sediment using quantitative PCR. The rel-
genomic DNA from subsamples was quantified in duplicates, ativeabundanceof16SrRNAgenecopies/gsedimentthroughout
whereeachreaction(20µl)contained1×PowerSYBR-GreenPCR thecorewas2.05×106–1.06×107 forBacteriaand5.76×106–
MasterMix(AppliedBiosystems),1µMofeachprimer,and1ng 5.75×107 for Archaea (Figure 2). At all depths Archaea domi-
template. The 16S rRNA genes of bacterial origin were ampli- natedoverBacteria,accountingfor51.7–93.3%of all16SrRNA
fied using the primers B338f (5(cid:48)-ACTCCTACGGGAGGCAGC; genecopies.Thenumberof16SrRNAgenecopiesofarchaealori-
Amann et al., 1995) and B518r (5(cid:48)-ATTACCGCGGCTGCTGG; ginincreasedtowardtheSMTZanddecreasedbelowthiszone.In
Muyzer et al., 1993) and 40 cycles of the thermal program comparison,bacterial 16S rRNA gene copies dominated 29ROV
describedbyEinenetal.(2008).Thestandardcurvewasgener- in the horizons from the sediment surface to 7–8cmbsf, rang-
ated using DNA from Escherichia coli, and genomic DNA from ing between 6.72×106 and 9.1×108 16S rRNA gene copies/g
Archaeoglobus fulgidus was used as negative control. Similarly, sediment (Roalkvam et al., 2011). The bacterial population in
archaeal 16S rRNA genes were amplified using primers Un519f 15GCwasthusbetweentwoandthreeordersofmagnitudelower
(5(cid:48)-TTACCGCGGCKGCTG;Ovreas et al.,1997) andA907r (5(cid:48)- thanin29ROV,exceptforthedeepesthorizonin29ROVwhere
CCGTCAATTCCTTTRAGTTT; modified from Muyzer et al., thebacterialpopulationdecreasedrapidly.Finally,theincreasein
1995)and40cyclesofthethermalprogramdescribedbyRoalk- thearchaealpopulationwithincreasingdepthswasoneorderof
vametal.(2011).Thelinearizedfosmid54d9wasusedtogenerate magnitudefor15GCandthreeordersofmagnitudefor29ROV.
FrontiersinMicrobiology|ExtremeMicrobiology June2012|Volume3|Article216|4
Roalkvametal. ANME-stratificationinfluencedbymethaneflux
FIGURE2|Theanalyzesofgeologicalparametersandphysical SO2−,Enumerationofarchaealandbacterial16SrRNAgenecopies/g
4
propertiesincore15GCshow(fromlefttoright):X-rayimageofthe sedimentsbasedonquantitativePCR.LegendsforLithology:(−)
core,Lithologicallog,Grainsize,Bulkdensity,andfractionalporosity silty-clay,()piping,andbioturbation,(ζ)shell/shellfragments,((cid:7))
basedonMSTloggerunit,Ca,andS(countpersecond)basedon subsamplesforDNAextraction,(∗)subsamplesfor16SrRNAgenetagged
XRFelementcorescan,GeochemicalanalyzesofHSand ampliconlibraryconstruction.
2
In summary,the 16S rRNA gene quantifications showed two Table1|Statisticalparametersof15GC.
tothreeordersof magnitudelowerrelativecellsnumbersinthe
core from the micro-seepage CN03 area than in the active G11 Depth(cmbsf) Numberofreads NumberofOTUs
pockmark.
10 20471 455
30 18141 791
TAXONOMY 80 4859 144
Theapplicationofpyrosequencingof16SrRNAgenetaggedPCR- 120 2601 84
ampliconstoobtaindetailedknowledgeonthecommunitystruc- 180 2388 107
ture has recently been proven efficient in studying stratification 240 17054 206
of microorganisms in sediment cores at a much higher resolu- 270a 19501 131
tionthanhasbeendonepreviously(Lanzénetal.,2011;Roalkvam 270b 20367 106
etal.,2011).Thisapproachwasusedtoexaminethecommunity 270c 21400 138
structure in 15GC, from seven depth horizons (10, 30, 80, 120,
180,240,and 270cmbsf) were analyzed by 454-pyrosequencing a–cAmpliconsfromsample270cmbsfwasmadeintriplicates.
yielding 3344-26491 reads, whereof 16.4–29.0% were removed
duetopoorqualityorchimericsequences.Theremainingnum- and Planctomycetes, Chloroflexi, and Candidate divisions (JS-
berofreadsinthedatasetwerebetween2388and21400for15GC 1 and OP8; Bacteria). Hence, the number of classified reads at
subsamples(Table1). lower taxonomic levels decreased, where up to 88.6 and 89.8%
Taxonomic classification revealed a high abundance of taxa ofthecommunityremainedunclassifiedatorderorfamilylevel,
thataredeficientlydescribedbelowphylumandclasslevel,such respectively. Therefore, only reads binned at phylum and class
as uncultivated taxa within Marine group 1 (MG-1; Thaumar- level, in addition to selected groups within Methanomicrobia
chaeota);ThermoplasmataandMBG-B/DSAG(Crenarchaeota); (Euryarchaeota),wereusedfurtherinthiswork.
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Roalkvametal. ANME-stratificationinfluencedbymethaneflux
MICROBIALDIVERSITYINAMPLICONLIBRARYANDABUNDANCE and 61.4%,which rapidly decreased toward the SMTZ (5.2% at
Most of the detected bacterial taxa were found in the shal- 180cmbsf andfurtherto<1%indeeperpartsof thecore).The
lowerhorizons,includingphylasuchasProteobacteria,Plancto- DSAGdidnotreachashighrelativeabundanceastheMG-1,but
mycetes,Deinococcus-Thermus,andtheCandidatedivisionOP8, representedahighshareof themicrobialcommunityaboveand
all decreasing in abundance to less than 1% below horizons at within the SMTZ,comprising between 16.2 and 29.2% in hori-
80–120cmbsf (Figure3A).Similarly,theabundanceof thephy- zonsat10–240cmbsf,exceptat270cmbsf wheretheabundance
lum Chloroflexi decreased rapidly in horizons below 80cmbsf, wasreducedto3.9%(Figure3A).Inthedeeperhorizons(180–
although comprising up to 2% in some of these horizons. The 270cmbsf),wheremethaneconcentrationsupto3.6mmol/Lhas
BacteriawasdominatedbytheCandidatedivisionJS-1,whichwas been detected in the pore-water (Vaular, 2011), ANME clades
presentthroughoutthesedimentswiththehighestabundanceat affiliated with Methanomicrobia were increasingly dominant. A
10and30cmbsf accountingfor13.2and28%of thetotalnum- similar stratification of dominating ANME clades with increas-
berofreads,respectively.Ineachsedimenthorizon,uncultivated ing depth as in 29ROV was observed with a transition from an
lineages of Archaea dominated,congruent with the quantitative ANME-2a/b dominated community to an ANME-1 dominated
PCR-data(Figure2).Low-abundancearchaealtaxa,suchasMCG community.However,thestratificationwasoverwidersediment
(1.1–1.8%) and Group C3 (group 1.2; 1.2–2.6%) within Cre- depths in 15GC ranging from 120 to 270cmbsf in comparison
narchaeota and Thermoplasmata (0.3–4.2%) and Archaeoglobi to4–22cmbsf in29ROV.Thehighestabundanceof theANME-
(1.2%) within Euryarchaeota were mainly present in horizons 2a/bcladewasfoundat120and180cmbsf,with16.9and33.3%
between 10 and 80cmbsf. Different depth profiles of the most of the total reads respectively (Figure 3B), and hence compris-
abundanttaxaMG-1,DSAG,andMethanomicrobiawereobserved ing a similar share of the community as in 29ROV (Roalkvam
(Figure 3A). The shallower horizons (10–120cmbsf) above the etal.,2011).TheabundanceofANME-2a/bdecreasedgradually
SMTZ had high abundance of MG-1, ranging between 15.2 to<1%withincreasingdepth,whileANME-1increasedfrom11.1
FIGURE3|Microbialcommunitystructuresatdifferentdepthsin15GC,basedon454-pyrosequencingof16SrRNAgenetaggedamplicons.The
distributionofselectedtaxaisshownatphylum/classlevel(A)andwithintheclassMethanomicrobia(B).
FrontiersinMicrobiology|ExtremeMicrobiology June2012|Volume3|Article216|6
Roalkvametal. ANME-stratificationinfluencedbymethaneflux
to47.9%between180and240cmbsf.Afurtherincreaseto82.2%
at270cmbsf wasobserved,whichwasthehighestabundanceof
ANME-1in15GC(Figure3B).Thehighestrelativeabundanceof
ANME-1in15GC(47.9–82.2%)correspondedtotheabundance
ofANME-1in29ROV(64–89%;Roalkvametal.,2011).However,
theabundanceofANME-2cincreasedtoamaximumof 60%at
20–22cmbsfin29ROV(Roalkvametal.,2011),whichisconsid-
erable higher than the maximum value of 5.7% at 270cmbsf in
15GC.
DIVERSITYINDICESANDOTUDISTRIBUTION
Shannon–Weaver index and Rao’s quadrate entropy index were
used to evaluate and compare the microbial diversity of the
communities in CN03 with those in the G11 pockmark, based
onallreadsintheampliconlibrariesfrom15GCand29ROV.The
main difference between the indices used is that Rao’s quadrate
entropyindexincludesthedistancebetweenOTUsinadditionto
theabundanceofsequences.Atotalof3322OTUswereobtained
from 29ROV and 15GC combined,based on 193363 16S rRNA
genesequencesfromampliconlibraries.Thisapproachrevealeda
verticalvariationinmicrobialdiversityinbothcores,whereatrend
ofdecreasingdiversitywithincreasingdepthwasobservedregard-
lessoftheindexused(Figure4).Forcore15GC,thediversitywas
decreasing gradually with depth, except at 120cmbsf where the
trend was interrupted by the particularly low diversity estimate.
Thecore29ROVhadadifferentdiversityprofile,withagradual
decrease in the upper part of the core, followed by a consider-
able decline in the deeper part (Figure 4). The lowest diversity
was found at 270cmbsf in 15GC and 14–16cmbsf in 29ROV,
which corresponds to the horizon in each core with the highest
abundanceofANME-1.
Sequencesfrom29ROVwereclusteredinto2370OTUs(1908
uniqueOTUs)and15GCwereassignedto1414OTUs(952unique
OTUssamplingsite)(Table2),whereonly462OTUswereshared
between the sampling sites. The majority of the taxa from both
cores were clustered into several OTUs,where at least one OTU FIGURE4|DiversityestimationsusingShannon–Weaverindex((cid:7))and
wascommon.Predominatingtaxawithineachcorewerepresentin Rao’squadrateentropyindex(♦)for15GC(A)and29ROV(B).The
commonOTUs,howeversomelow-abundanttaxawithinArchaea standarddeviationforsubsampleswithin15GCand29ROVwerecalculated
[suchasMarineBenthicGroupA,Archaeoglobaceae,Thermococ- tobe0.041–0.068and0–0.078fortheShannon–Weaverindex,
respectively,andbetween3.04–8.67×10−3and0–9.05×10−3fortheRao’s
cales,SouthAfricanGoldmineEuryarchaeotalGroup(SAGMEG),
quadrateentropyindex,respectively.Allstandarddeviationbarsaresmaller
andMarineGroupIIwithinThermoplasmata]andBacteria(such
thanthesizeofsymbolsdisplayedinthefigure.Thegrayareaindicatesthe
asCandidatedivisionOP11,Chlorobiale,Thermotogales,andtaxa sulfate-methanetransitionzonein15GC(A).
withinBacteroidetes,Chloroflexi,Firmicutes,andProteobacteria)
wereonlypresentinOTUsthatwereuniqueforoneofthecores.
TheunculturedANMEclade,withsequencesaffiliatedwiththe the sequences assigned to ANME-1 were present in one OTU
ANME-1,ANME-2a/b,andANME-2csubgroups,werethemost (OTU_542; Figure 5), comprising 80.0–98.5% of the reads in
dominating taxa in both 15GC and 29ROV. To study the distri- 29ROV and 72.8–100% in 15GC (Table S1 in Supplementary
bution of ANME at the two sampling sites in more detail, the Material). The remaining ANME-1 sequences were present in
OTUsassignedtoallANMEsubgroupswereextractedfromthe two additional OTUs, which also included sequences from the
dataset.Atotalof65180readsfrom15GCand30273readsfrom shallow horizons above the ANME-dominated zone in both
29ROVgroupedinto34OTUs.Ofthese,19OTUswereexcluded cores. The ANME-2a/b affiliated sequences were mainly dis-
astheywerebasedonsinglesequences.Hence,14and7sequences tributed in two dominating OTUs (Figure 5), one specific
were removed from the 15GC and 29ROV dataset, respectively. OTU for each core. OTU_442 comprised between 78.6 and
Theremaining15OTUshadthefollowingdistributionamongthe 99.9% of ANME-2a/b sequences in 15GC, while OTU_50 con-
ANMEclades:ANME-1(4),ANME-2a/b(6),andANME-2c(5). stituted 54.5–95.2% of the sequences in 29ROV (0–10cmbsf).
In horizons dominated by ANME, meaning horizons below Furthermore, some ANME-2a/b sequences from the ANME-1
4–5cm for 29ROV and 120cm for 15GC, the majority of dominated horizons in 29ROV were also assigned OTU_442.
www.frontiersin.org June2012|Volume3|Article216|7
Roalkvametal. ANME-stratificationinfluencedbymethaneflux
Table2|DistributionofOTUsin15GCand29ROV. whereas only 5.7% of all reads were assigned to this taxonomic
group in 15GC. It is possible that the observed difference in
OTUs 15GC 29ROV ANME-2c abundances is an effect of the much wider methane
gradientin15GCandthatANME-2catthissamplingsitearefond
Totalnumber 1414 2370
inhighernumbersinhorizonsdeeperthan270cmbsf,andthus
Numberofunique 952 1908
notdetectedinourstudy.
Numberofshared 462 462
Themethanefluxseemstohavelittleeffectonthestratifica-
Singletons 541 959
tion of the ANME groups 2a/b, 1, and 2c in the Nyegga field,
Archaeal 160 267
as a similar stratification of these clades was found in both the
Bacterial 1242 2085
29ROV(Roalkvametal.,2011)and15GClocations(Figure3B).
Unassigned 12 18
This suggests that shifts in ANME clades through the cores are
determinedbyotherfactorsthanmethaneavailability.Moreover,
Although the abundance of ANME-2c was considerably higher both cores are dominated by the same OTUs of ANME-1 and
in 29ROV than 15GC, the majority of the reads were clus- ANME-2c,indicating that organisms potentially adapted to dif-
tered into two common OTUs (OTU_168 and OTU_1280; ferentmethanefluxescannotbedistinguishedontheOTUlevel
Figure 5). In addition, a substantial number of reads from within these clades. On the other hand, differences in methane
29ROV were assigned to OTU_1800, which was unique for fluxmaypartlyexplainwhydifferentOTUsofANME-2a/bdom-
thiscore. inateinthecores.Themethanefluxseemstolargelyinfluencethe
specificdensityofANMEinthecoresasthenumberofarchaeal
DISCUSSION 16SrRNAgenespergramofsedimentintheANME-dominated
METHANEFLUXANDSTRATIFICATIONOFMETHANOTROPHS horizons was observed to be two orders of magnitude lower in
The Nyegga area is characterized by numerous pockmarks and 15GC(Figure2)thanin29ROV(Roalkvametal.,2011).Thisis
methaneseepagestructuresindifferentdevelopmentalstagesindi- congruentwithpreviousstudiesof sedimentswithvariationsin
cating a dynamic, temporal, and spatial system where different methanefluxanddepthoftheSMTZwhereANMEcompriseup
geochemical settings may influence the microbial community to 3×109cells/cm3 sediment in marine sediments with shallow
structure. In this study, 454-pyrosequencing of 16S rRNA gene gashydrates,suchasGulfofMexico(Orcuttetal.,2008),Hydrate
taggedampliconswereusedtocomparethemicrobialstratifica- Ridge (Knittel et al.,2005),and Eckernförde Bay (Treude et al.,
tioninagravitycore(15GC)fromthelessactiveCN03areawith 2005a), whereas in low methane seepage areas with deeper gas
that in a push core (29ROV) from the active seepage structure hydrates,suchasSantaBarbaraandPeruMargin,ANMEarerare
G11pockmark.TheCN03areaischaracterizedbymicro-seepage orabsent(Biddleetal.,2006;Inagakietal.,2006;Harrisonetal.,
ofmethane,fewpockmarks,aSMTZlocatedat∼200–250cmbsf 2009).
andaBSRat250–300mbsf.TheG11pockmarkincomparison,is BothinthesedimentsfromtheG11pockmarkandin15GC,
characterizedbyshallowgashydratesandSMTZ,authigeniccar- ANME-2a/bwasfoundtodominateinhorizonswithlowercon-
bonates,and pingo structures within the pockmark. The higher centrationsofsulfidecomparedtotheANME-1dominatedhori-
methanefluxsustainsmacro-faunaandbacterialmats(Hovland zons.Theseresultsareinaccordancewithpreviousstudiessuggest-
and Svensen, 2006; Chen et al., 2010; Ivanov et al., 2010). The ingthatANME-2issensitivetoH SproducedduringAOMwith
2
methane fluid flux was ∼80mmol/m2year for 15GC and 300– sulfate (Meulepas et al., 2009a,b). Furthermore, the abundance
540mmol/m2year for 29ROV (Chen et al.,2010). The methane of ANME-2a/b was found to be negatively correlated with high
fluxintheCN03areaiswithinthesamerangeasotherseepareas methaneandsulfideconcentrationsinGuaymasBasinsediments,
whereANMEhavebeenfoundtobeabundant,suchastheSanta whereasanoppositecorrelationwasfoundforANME-1(Biddle
BarbaraBasin(164–200mmol/m2year;Harrisonetal.,2009)and et al.,2011).Also,azonation of ANME-2 communitiesin areas
GulfofMexico(20–200mmol/m2year;Coffinetal.,2008;Lloyd withefficientH SremovalandANME-1inzoneswithhighercon-
2
etal.,2010).In29ROV,thefluxisapparentlysohighthatmethane centrationsofH Swasobservedinmultilayeredmicrobialmatsin
2
reachesthesedimentsurfacewhereitstimulatesadominanceof theBlackSea(Krügeretal.,2008).Duetothehighermethaneflux
aerobicmethanotrophicGammaproteobacteria(Roalkvametal., in G11 the ANME-2a/b population is exposed to methane-rich
2011). In 15GC,aerobic methanotrophic Gammaproteobacteria fluidswhereasinCN03,thedominatingANME-2a/bisfoundin
werenotidentified,indicatingthatthemethaneseepingthrough horizonswithlowmethaneconcentrationsof0.035–0.13mmol/L.
the sediments were consumed by ANME which were increas- However,thepipingstructuresandshellfragmentsinthesehori-
ingly dominating the amplicon libraries from 120cmbsf and zons shows that the methane concentration has probably been
below (Figure 3). A dominance of ANME in deeper horizons higherinearliertimes,indicatingthattheANME-2a/bpopulation
wasalsoobservedin29ROV,however,theANME-dominatedsed- couldhavebeenestablishedinamethane-enrichedenvironment
iment horizons extended over a wider depth interval in 15GC. inthepastandthattheANME-2a/bpopulationpresentin15GC
Furthermore,similar relative abundances and equivalent transi- now is sustained by lower methane concentrations. Given the
tions between ANME-2a/b and ANME-1 with increasing depth ability to survive in methane depleted environments, ANME-
(Figure3B)wereobservedinboth29ROVand15GCcores.How- 2a/b could be the seed population in new methane-enriched
ever,the proportion of ANME-2c seemed to differ between the systems, as suggested by Knittel and Boetius (2009). Thus, in
cores as 60% of all reads were assigned toANME-2c in 29ROV, ordertofullyunderstandthestratificationanddynamicsofANME
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Roalkvametal. ANME-stratificationinfluencedbymethaneflux
FIGURE5|Anaerobicmethanotrophs(ANME)-affiliatedsequencesfrom indicatesthetotalnumberofreadsassignedtoeachofthethreeANME
the15GCand29ROVampliconlibrarieswereclusteredintoOTUs(97% subgroups.Thisnumberissummedto100%anddisplayedonthex-axis.All
cut-off).TherelativedistributionofdifferentOTUsaffiliatedtoeither OTUsrepresentedbyonlyonesequencearepooledandpresentedinthe
ANME-2a/b,ANME-2c,orANME-1withineachsedimenthorizonisshownin category“singletonOTUs.”Thesubsample270cmbsffrom15GCwas
separategraphsfor15GCand29ROV.Thenumberslistedbesideeachbar analyzedintriplicates,hencethemarkingA,B,andConthey-axis.
communitiesatNyegga,moreknowledgeontemporalvariations associatedwithsulfate-reducerswithinDesulfosarcinaandDesul-
inthemicrobialcommunitystructuresmightbeneeded. fococcus (DSS; Knittel et al., 2005; Schreiber et al., 2010), while
ANME-3 are associated with sulfate-reducers within Desulfob-
SYNTROPHICPARTNERSOFANME ulbus (DBB; Niemann et al., 2006; Lösekann et al., 2007). The
The AOM with sulfate has in previous studies been shown share of Deltaproteobacteria in horizons with the highest rel-
to be performed by ANME in syntrophy with sulfate-reducing ative abundance of ANME-2a/b was ranging between 9.0 and
Deltaproteobacteria, where ANME-1 and ANME-2 are mainly 9.1%in29ROV(Roalkvametal.,2011),butwasbelow0.7%in
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Roalkvametal. ANME-stratificationinfluencedbymethaneflux
15GC (Figure 3A) of which most sequences were assigned to a ANME-1populationinG11andCN03,independentofaclosely
clade that is not assumed to be a syntrophic partner of ANME associatedsulfate-reducingpartner.
(TableS1inSupplementaryMaterial).Fromthelow-abundance
ofdetectedDeltaproteobacteria,itwasnotobvioustouswhichof COMMUNITYSTRUCTURESINSHALLOWSEDIMENTHORIZONS
thedetectedorganismsthatactedasasulfate-reducingsyntrophic The total organic carbon (TOC) content in Nyegga sediments
partnerforANME-2a/b,atleastnotin15GC.Onepossibilityis is 0.55–0.74% in G11 pockmark and 0.40–0.54% at CN03 area
thattheabundanceofDeltaproteobacteriaisunderestimateddue (Ivanov et al., 2010), which corresponds well with the average
tobiasinthePCRamplificationof16SrRNAgenes.Anotherpos- TOCvalues(∼0.5–1%)fortheregion(HölemannandHenrich,
sibilityisthatotherorganismsthanDeltaproteobacteriaactasthe 1994). In sedimentary environments,the organic matter buried
sulfate-reducingsyntrophicpartnerfortheANME-2a/bdetected overgeologicaltimescalesisutilizedasanenergysourcebyorgan-
in the Nyegga field. JS-1 was present in allANME-2 dominated otrophs(Kujawinski,2011;Orcuttetal.,2011).Throughmicrobial
horizonsin15GC,butwasevenmoredominatinginothersed- remineralization,degradationproductsareformedwhichcanbe
imenthorizons,indicatingnoobligaterelationshipwithANME. utilized by diverse heterotrophic taxa. The microbial distribu-
TheJS-1groupisubiquitousinmarinesediments,atdepthsrang- tion in 15GC showed a high abundance of taxa that generally
ingfrom<10to>200mbsf (Rochelleetal.,1994;Inagakietal., occurs in high numbers in marine sediments, such as Plancto-
2006;Parkesetal.,2007;Websteretal.,2007).Inagakietal.(2006) mycetes, Chloroflexi, Bacteroidetes, JS-1, MBG-B/DSAG, MG-1,
hypothesizedthatJS-1couldbeadaptedtoanaerobicconditionsin MCG,and MBG-D (Figure3A;Reed et al.,2002;Inagaki et al.,
organic-richsedimentsassociatedwithmethanehydrates,which 2006;Harrisonetal.,2009;BlazejakandSchippers,2010).In15GC,
is similar to the environment at Nyegga. Further indications of ArchaeaoutnumberedBacteriainallhorizons(Figure2)witha
JS-1 being a heterotrophic sulfate-reducing bacterium are based clearstratificationof thearchaealphyla(Figure3A).TheMG-1
onenrichmentcultureswheresulfatewasdepletedinwellswith predominated in horizons probably depleted in oxygen at 10–
acetate (Webster et al.,2011). In 29ROV,the abundance of JS-1 120cmbsf.Cultivatedrepresentativesof MG-1havebeenshown
throughoutthecorewascorrelatedtotheabundanceofANME-2 to perform aerobic ammonium oxidation (Hallam et al., 2006;
(Roalkvametal.,2011),andANME-2maybenefitfromtheactivity NicolandSchleper,2006;Walkeretal.,2010).Ammonium,poten-
ofJS-1bytransferofreducingequivalentsderivedthoughAOM tiallyderivedfromthedegradationofnitrogen-containingorganic
toJS-1.Although,detailedknowledgeontheenergymetabolism matter,couldsupportthepopulationofMG-1in15GCandmay
of JS-1 is needed to assess any syntrophic relationship between possibly be oxidized anaerobically. The MG-1 is a diverse clade
ANME-2andJS-1.However,ourresultsmightimplythatANME- withseveralsubgroupsthatarenotwellcharacterized(Durbinand
2a/b can be adapted to performAOM with Deltaproteobacteria Teske,2010),andtheMG-1couldpossibilityhaveawiderrange
asasulfate-reducingpartnerinsystemswithhighmethanecon- of useful metabolisms in this environment.Within theArchaea,
centrations(Boetiusetal.,2000;Orphanetal.,2001;Knitteletal., theDSAGispredominantinseveralmarineenvironments,such
2005), such as 29ROV. Finally, it should be kept in mind that asdeepseasediments(Vetrianietal.,1999;Fryetal.,2008;Wang
ANME-2a/b possibly live in syntrophy with organisms reducing et al.,2010),sediments overlaying shallow gas hydrates (Inagaki
otherelectronacceptorsthansulfate.Previouswork,hasdemon- etal.,2006)andwithintheSMTZatSantaBarbaraBasin(Harri-
stratedthatalsoFe,Mn,orNO2−maybeusedaselectronacceptors sonetal.,2009)andPeruMargin(SørensenandTeske,2006).In
3
inAOM(Raghoebarsingetal.,2006;Ettwigetal.,2008;Bealetal., 15GC,DSAGwasuniformlydistributedinhorizonsbetween10
2009) which will provide a higher energy yield than the use of and240cmbsfwheretheconcentrationofmethaneislow,butwas
sulfate(Boetiusetal.,2000;Nauhausetal.,2002;Caldwelletal., outcompetedbyANME-1at270cmbsf wheretheconcentration
2008).LinkingAOMeitherinsyntrophyorbyafree-livinglifestyle of methane is higher. Hence,DSAG may rather perform organ-
tosuchelectronacceptormaythussustainalifeinlowmethane otrophicsulfatereductionassuggestedbyBiddleetal.(2006)and
concentrations.However,ithasbeenarguedthatthekeyenzyme Inagakietal.(2006)thanconsumptionofmethanein15GC.This
Methyl-CoM reductase in the reverse methanogenesis pathway isalsosupportedbythelow-abundanceofDSAGin29ROV,where
willnotcatalyzethereductionofFeandMnduetotheinactiva- thehorizonsareexposedtomethane-richfluids.
tionoftheenzymecausedbythehighlypositiveredox-potential ThehighermethanefluxintheG11pockmarkapparentlyalso
fortheseelectronacceptors(ShimaandThauer,2005;Thauerand influenced the absolute numbers of microorganisms, with two
Shima,2008). to three orders of magnitude higher number of 16S rRNA gene
Recently, the in situ metabolism of the free-living ANME- copiespergramofsedimentcomparedtotheCN03area.Hence,
1 enriched horizon in 29ROV was studied by using a coupled eventhoughtaxasuchasPlanctomycetes,ChloroflexiJS-1,MG-1,
metagenomicandmetaproteomicapproach(Stokkeetal.,2012). andDSAGwerepresentwithabundancesbetween<1and10%in
Allenzymesinthereversemethanogenesispathway(exceptN5, 29ROV,theirabsolutenumbersareinthesameorderof magni-
N10-methylene tetrahydromethanopterin reductase) and corre- tudeasinthe15GCcoreasthetotalcellnumberinthesehorizons
spondingelectronacceptingcomplexeswerefoundexpressedby weretwotothreeorderofmagnitudeshigherthaninthe15GC.
ANME-1. Furthermore, the key enzymes for dissimilatory sul- Thisindicatesthatbothsamplingsiteshaveequivalentamounts
fate reduction were found to be expressed in the environment ofmicroorganismspotentiallyinvolvedindegradationoforganic
by Deltaproteobacteria,and in addition,anAPS-reductase affil- matter, which is consistent with the relatively even distribution
iated with previously unknown ANME-partners was identified. oforganicmatterinNyeggasediments(HölemannandHenrich,
FromthiswededucethehypothesisthatAOMisperformedbythe 1994;Ivanovetal.,2010).
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