Environmental evidence for net methane production and oxidation in putative ANaerobic ...

17 Pages·2011·1.15 MB·English

EnvironmentalMicrobiology(2011)13(9),2548–2564 doi:10.1111/j.1462-2920.2011.02526.x
Environmental evidence for net methane production
and oxidation in putative ANaerobic MEthanotrophic
(ANME) archaea
emi_25262548..2564
KarenG.Lloyd,1,2*MarcJ.Alperin2and Introduction
AndreasTeske2
Anoxic coastal sediments produce large amounts of the
1CenterforGeomicrobiology,NyMunkegade114,Bldg
greenhouse gas methane, but biological anaerobic
1540,AarhusUniversity,8000-AarhusC,Denmark.
methane oxidation consumes most of the upward diffus-
2DepartmentofMarineSciences,CB#3300,University
ing methane before it reaches the overlying water (Ree-
ofNorthCarolinaatChapelHill,ChapelHill,NC27599,
burgh, 2007). This process is generally attributed to
USA.
groupsofmethanogen-likearchaeathathaveneverbeen
obtained in pure culture, but have been identified by
Summary molecular phylogeny and collectively named ‘ANME’(for
ANaerobic MEthanotroph) with suffixes 1, 2 and 3 (Hin-
Uncultured ANaerobic MEthanotrophic (ANME)
richs etal., 1999; Knittel and Boetius, 2009). It is widely
archaea are often assumed to be obligate methan-
assumed thatANME archaea are incapable of obtaining
otrophs that are incapable of net methanogenesis,
energyfromthenetproductionofmethane(Parkesetal.,
and are therefore used as proxies for anaerobic
2007;KnittelandBoetius,2009).Hence,thepresenceof
methane oxidation in many environments in spite of
ANME DNA sequences has been assumed to indicate
uncertainty regarding their metabolic capabilities.
anaerobicmethaneoxidationinawidevarietyofenviron-
Anaerobic methane oxidation regulates methane
ments including the oxycline of seawater, freshwater
emissionsinmarinesedimentsandappearstooccur
lakes,brinelakes,terrestrialaquifers,terrestrialmudvol-
through a reversal of a methane-producing metabo-
canoes, microbial mats, hydrothermal vents, ancient
lism. We tested the assumption that ANME are obli-
marine sediments, petroleum-contaminated sediments,
gate methanotrophs by detecting and quantifying
methane-producingmarinesedimentsandoceanichydro-
gene transcription of ANME-1 across zones of
carbonseeps(KnittelandBoetius,2009).
methane oxidation versus methane production in
The possibility that ANME archaea may be able
sediments from the White Oak River estuary, North
to function as normal methanogens has not been
Carolina. ANME-1 consistently transcribe 16S rRNA
adequately addressed. When Hinrichs and colleagues
and mRNAof methyl coenzyme M reductase (mcrA),
(1999) first coined the term ‘ANME’ to describe the
the key gene for methanogenesis, up to 45cm into
archaea in methane seep sediments from Eel River
methanogenic sediments. CARD-FISH shows that
Basin, they recognized that these organisms could be
ANME-1 exist as single rod-shaped cells or pairs of
obligate or ‘dominant’ methanotrophs, normally function-
cells. Integrating normalized depth distributions of
ing methanogens or methanogens operating in reverse.
16S rDNA and rRNA (measured with qPCR and
ThepossibilitythatANMEarchaeacouldbemethanogens
RT-qPCR respectively) shows that 26–77% of the
operatinginreversewasdismissedonthreegrounds:(i)
rDNA(aproxyforANME-1cellnumbers),and18–76%
ANME archaea had never been detected in methano-
of the rRNA (a proxy for ANME-1 activity) occurs
genic sediments, (ii) ANME-1 would represent a new
within methane-producing sediments. These results,
order of methanogens and (iii) a metabolic pathway for
along with a re-assessment of the published Iitera-
reversemethanogenesiswasunknown.Severalplausible
ture,changetheperspectivetoANME-1asmethano-
pathways for reverse methanogenesis have been pro-
gensthatarealsocapableofmethaneoxidation.
posed (Hoehler and Alperin, 1996), one of which was
recentlydemonstratedinvitro(Schelleretal.,2010).
Here,weaddressthefirstargument(i)ofHinrichsand
colleagues that ANME-1 archaea are not methanogens
Received12February,2011;accepted5April,2011.*Forcorrespon-
operating in reverse, by presenting data on the
[email protected];Tel.[phone];Fax
([phone]. occurrence and activity of ANME archaea in both
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd
MethanogenesisandmethanotrophyinANME-1archaea 2549
methane-oxidizing and methane-producing sediments. oxidation zone is less constrained as there is minimal
We applied RNA- and DNA-based analyses to brackish curvature in the concentration profile in near-surface
sedimentsoftheWhiteOakRiverestuary,NorthCarolina, sediments.
where the gene transcription patterns ofANME archaea The inverse model also predicts that rates of net
can be observed in clearly separated zones of net methaneproductionarehighestjustbelowthebaseofthe
methane production or oxidation. Since DNA can be methane oxidation zone and in most cases, decrease
stableincoldanoxicsedimentsontheorderof105years rather abruptly with depth (Fig.1, column B). Our cores,
(Willerslev etal., 2004), its presence is not a sufficient however, may not have penetrated through the entire
indicator for active microorganisms. In contrast, RNA methane production zone. This limitation, combined with
is much more readily degraded (Srivastava and increased‘noise’inthemethaneconcentrationdataatthe
Schlessinger, 1990), and is therefore a more specific bottom of the cores, makes it impossible to accurately
biomarkerforinsitumetabolicactivity. constrain the shape of the methane production rate
profile. The inverse model provides reliable estimates of
Results thedepthmarkingthetopofthemethaneproductionzone
as well as the depth-integrated methane production rate
Geochemicaldepthdistributions
(which by mass-balance must equal the flux of methane
Sulfate concentrations in brackish White Oak River intothemethaneoxidationzoneplusthefluxofmethane
estuary sediments (Fig.1, columnA) generally decrease thatisburieddeepbelowthemethaneproductionzone).
from~10mMatthesediment–waterinterfaceto~0.1mM Ourmethaneconcentrationdata,however,areinsufficient
atdepth.Sulfate:chlorideratiosalsodecreasewithdepth todeterminewhethermethaneproductionisfocusedina
(notshown),indicatingthatsulfateisconsumed,presum- relatively narrow (~20cm) zone just below the methane
ablybythesulfate-reducingbacteria.Wewillrefertothe oxidationzone,orwhethermethaneproductionoccursat
sediment region where sulfate concentrations exceed slower rates over a broader depth interval. A multi-
0.1mMasthesulfatereductionzone. component reaction-transport model applied to this site
Methaneconcentrationprofiles(Fig.1,columnA)have (Martens etal., 1998) is consistent with the latter case.
the characteristic sigmoidal shape that is common in This model – which uses the benthic flux of dissolved
marinesedimentswheremoleculardiffusionisthedomi- inorganic carbon and the sulfate depletion depth to con-
nant transport process (Reeburgh, 2007). Methane con- strain the quantity and reactivity of sedimentary organic
centrations are low ((cid:2)0.01mM) throughout most of the matter – predicts that more than half of the depth-
sulfate reduction zone where sulfate concentrations are integrated methane production occurs at depths greater
greaterthan1mM,increaserapidlywithdepthassulfate than80cm.Resolvingwhichmodelbestreflectsthetrue
becomes depleted, and remain relatively high (~1mM) methaneproductionrateprofilewillrequiremoredata.
but variable towards the bottom of the core. Based on Depthdistributionsofd13C-methaneweremeasuredfor
Fick’s Second Law of Diffusion applied to porous sedi- two cores in July 2008 (Fig.1, column E) and provide a
ments, the concave-up portion of the methane profile check on the location of the lower boundary of the
denotesthezoneofnetmethaneoxidation,theconcave- methane oxidation zone predicted by the inverse model.
down portion denotes the zone of net methane produc- Methane oxidation causes the d13C-methane profile to
tion,andtheinflectionpointmarkstheboundarybetween shifttowards‘heavier’(lessnegative)valuesatshallower
methane oxidation and methane production zones depths(Fig.1,columnE)since12C-methaneispreferen-
(Berner,1980). tiallyoxidizedasitdiffusesupthecore(Reeburgh,2007).
An inverse model (Alperin etal., 1988) provides esti- d13C-methane profiles predicted by an isotope reaction-
mates of net methane oxidation and production rates by transport model using the methane reaction rate profiles
quantifying curvature (i.e., the second derivative) in the in Fig.1 (column B) are consistent with the measured
methaneconcentrationprofile.Themodelpredictsasub- values.Thisconfirmsthatnetmethaneoxidationinthese
surfacemaximuminnetmethaneoxidationratenearthe two cores does not occur at depths below 40cm. The
base of the sulfate reduction zone (Fig.1, column B) as predictedkineticisotopeeffect(a)foranaerobicmethane
has been measured with 14C-labelled methane in a wide oxidation (1.015 for Jul 08-1 and 1.019 for Jul 08-2) is
variety of marine sediments (Reeburgh, 2007). The within the range measured for enrichments of ANME-2
boundary between methane oxidation and methane pro- fromthreedifferentmethaneseeps(1.012–1.039)(Holler
duction zones for each core (marked by the dashed etal.,2009).
horizontal lines in Fig.1) is well constrained as the con- The methane reaction rate profiles derived from
centration profile is highly curved and the methane data the geochemical data are consistent with our current
are relatively smooth in this portion of the sediment understanding of the vertical zonation of organic matter
column. In contrast, the upper boundary of the methane remineralization in most anoxic marine sediments. In
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
2550 K.G.Lloyd,M.J.AlperinandA.Teske
Fig.1. Geochemistryandmicrobialdepthdistributions.ColumnA:Concentrationsofmethane((cid:2))andsulfate((cid:3)),withmethane
concentrationspredictedbyareaction-transportmodel(solidline)drivenbyratesshowninColumnB.ColumnB:Methaneoxidationrates
(filledblackarea)andmethaneproductionrates(dottedline)estimatedfromaninversemodelappliedtothemethaneconcentrationsdata,
exceptforthecaseofcoresJul08-1andJul08-2,whered13C-CH4dataprovideanadditionalconstraintontheupperboundaryofthe
methaneoxidationzone.Thesmallblackarrowspointtothelowerboundaryofthemethaneoxidationrateprofilethatisinferredfromthe
d13C-CH4data.Thedottedlinesformethaneproductionratesdenotethattheshapeoftheseprofilesisnotwell-constrainedbecausethedeep
methanedataare‘noisy’andmaynotextendthroughtheentiremethaneproductionzone.ColumnsCandD:ANME-116SrRNAindex
measuredwithRT-qPCR,andANME-116SrDNAindexmeasuredwithqPCRincopiespergramofsedimentateachdepthdividedbythe
highestvalueofcopiespergramofsedimentforthatcore,witherrorbarsrepresentingthestandarddeviationsoftwotofourmeasurements.
ColumnE:d13Cofmethane,withprofilespredictedbyanisotope-reaction-transportmodel(solidline)forakineticisotopeeffect(a)for
anaerobicmethaneoxidationof1.015(Jul08-1)and1.019(Jul08-2).Dashedhorizontallinesindicatethedepthoftransitionbetween
methaneoxidationandproductionzones.Cores,toptobottom,areJul05-1(qPCRandd13Cnotmeasured),Jul05-2(RT-qPCR,qPCRand
d13Cnotmeasured,butanalysedforcDNAsequencesandFISH),Dec06(d13Cnotmeasured),Jul08-1andJul08-2.Thepercentageoftotal
depth-integratedcopiesofrRNAorrDNAthatarepresentinthemethaneproductionzonearelistedforeachcore.(continuedonnextpage)
sulfate-containing sediments, sulfate-reducing bacteria allows methanogenic archaea to assimilate the fermen-
out-compete methanogens for common substrates that tation products and convert them to methane (Hoehler
are produced by organic matter fermentation, and main- etal., 1994).The methane that diffuses upward from the
tainH atverylowconcentrations(<1nM)(Lovleyetal., methane production zone is nearly quantitatively oxi-
2
1982; Hoehler etal., 1998). In sulfate-depleted sedi- dized near the base of the sulfate reduction zone where
ments,lackofcompetitionfromsulfate-reducingbacteria low H concentrations make methane oxidation to CO
2 2
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
MethanogenesisandmethanotrophyinANME-1archaea 2551
Fig.1. cont.
and H energetically favourable (Hoehler etal., 1994; numberofribosomesperrDNAcopyisbiasedbydifferent
2
1998). extractionefficienciesforrRNAandrDNAandtheaddition
of a reverse transcription step for rRNA(and possibly by
the persistence of rDNAin dead cells), down-core varia-
Microbialdepthdistributions
tionsintheratioofANME-1rRNA:rDNAindicesprovidea
ExtractionofDNAandespeciallyRNAfromnaturalsedi- proxy of relative changes in the growth rate of ANME-1
mentsmakesabsolutequantificationdifficultsinceextrac- archaea with depth (lower values of rRNA:rDNAindicate
tion efficiencies can be variable and are difficult to relativelylowergrowthrates).
measure accurately (Lloyd etal., 2010). Therefore, 16S Profiles of ANME-1 16S rRNA and rDNA indices are
rRNAand 16S rDNAcopy numbers are not reported as showninFig.1(columnsC,D).Bothindicesaregenerally
absolutequantitiesbutareindexedtothehighestaverage lowthroughoutmuchofthesulfatereductionzonewhere
copynumberpermeasurementtypepercore.Thatis,an methaneproductionsuffersfromcompetitionfromsulfate-
index value of 1.0 marks the depth with the highest reducingbacteria,andmethaneoxidationislimitedbylow
average rRNA or rDNA copy number per gram of dry methaneconcentrations((cid:2)0.01mM).Asexpected,both
sediment; an index of 0.5 marks the depth with half as indices have a peak that coincides with the subsurface
manycopynumbers. peakinmethaneoxidationrate,consistentwiththewidely
The 16S rDNAand rRNAindices provide complemen- heldviewthatANME-1archaeaarecapableofanaerobic
tary information regarding the depth distribution of abun- methane oxidation.The abrupt decline in both indices at
dance and activity of ANME-1 archaea. The 16S rDNA the boundary between methane oxidation and methane
index reflects the relative abundance of ANME-1 cells, production suggests that the abundance of ANME-1
sincepublishedgenomesofenvironmentalANME-1cells archaea declines in this horizon, and that the persistent
haveone16SrDNAcopypergenome(Meyerdierksetal., cellsbecomedormant(therRNAindex–whichindicates
2010),andthisvalueisnotexpectedtochangemuchwith activity–approacheszerointhreeofthefourcores).The
growthrate.The16SrRNAindexreflectsboththerelative sharp decline in the rDNA index suggests that in these
abundance and the activity of ANME-1 cells, since the sediments,16SrDNAfromANME-1archaeadegradeson
numberofribosomespercellisproportionaltothecellular timescalesofyears.Thus,weconsidertherDNAindexto
growth rate (Kemp etal., 1993). Although the absolute closelyreflecttheabundanceofANME-1archaealivingin
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
2552 K.G.Lloyd,M.J.AlperinandA.Teske
each depth interval rather than the cumulative mass of trations has been shown to halt anaerobic methane
deadcells. oxidation, presumably since reverse methanogenesis no
For the three cores that were analysed for gene tran- longer provides the organism with a free energy gain
scription and that penetrated well into the methane pro- (Hoehler etal., 1994). As sulfate continues to decline,
ductionzone(Jul05-1,Dec06andJul08-1),bothrRNA sulfate reducers decrease in activity, as shown by the
andrDNAindiceshavenon-zerovaluesinmethanogenic inconsistent amplification of dsrAB mRNA (see below),
sediments (Fig.1, column C, D). In fact, a majority of and H continues to rise until it eventually becomes high
2
theseprofiles(Jul05-1/rRNA,Dec06/rDNAandJul08-1/ enough to support the minimum energy requirements of
rRNA)showaclearsecondarypeakinthemethanepro- methanogens.Thedepthintervalwhereneithermethane
ductionzone.TherRNAandrDNAindicesdonottrackthe oxidation nor production can support microbial mainte-
methane production rate profiles nearly as well as they nance energies has been called the ‘no reaction’ zone
trackthemethaneoxidationrateprofiles.Thisisnotsur- (Hoehleretal.,1994).
prising given uncertainty in the shape of our methane Below the no reaction zone, rRNA and rDNA indices
productionrateprofilesstemmingfrominsufficientdatato generally increase to a secondary peak within the
define the base of the methane production zone and methane production zone. Of the total depth-integrated
scatter in the methane concentration data towards the rDNAand rRNAindices for each core, 26–77% of rDNA
bottomofthecore.Depth-integratingtherDNAindexsug- and 18–76% of rRNA are located in the methane pro-
gests that a sizable fraction (26–77%) of the whole-core duction zone, indicating that substantial fractions of
ANME-1populationispresentinthemethaneproduction ANME-1 cell numbers and activity occur here. All these
zone. Depth-integrating the rRNA index suggests that a dataareconsistentwiththenotionthatANME-1function
similar fraction (18–76%) of whole-core ANME-1 activity as methanotrophs in the methane oxidation zone, and
occurs below the methane oxidation zone.These values asmethanogensinthemethaneproductionzone.Thisis
represent lower limits as the cores may not have pen- consistent with the ‘reverse methanogenesis’ mecha-
etratedthroughtheentiremethaneproductionzone. nism in which methanogens are able to switch their
ThreecoreswereanalysedforbothANME-116SrRNA metabolism from methane production to methane oxida-
and rDNA (Dec 06, Jul 08-1, Jul 08-2). For all three of tion depending on which process is thermodynamically
thesecores,therRNAindexattenuatesmorequicklywith favourable (Zehnder and Brock, 1979; Hoehler and
depth than the rDNA index, suggesting that ANME-1 Alperin, 1996). This hypothesis is based on the obser-
archaeagrowfasterinthemethaneoxidationzonethanin vation that cultured methanogens are able to oxidize
the methane production zone. This implies that there is methane to CO while engaged in net methane produc-
2
more metabolic power available to ANME-1 in the tion (Zehnder and Brock, 1979), and that net methane
methane oxidation zone than in the methane production oxidation can be induced in methanogenic sediments by
zone.Thepoweravailablefromametabolicprocessisthe experimentally manipulating the H concentration to
2
product of the Gibbs free energy of reaction and the make methane oxidation thermodynamically feasible
reaction rate. Studies of the energy requirements of (Hoehler etal., 1994).
methanogenesis(Hoehleretal.,2001)andmethaneoxi-
dation(Hoehleretal.,1994)suggestthatbothprocesses
Clonelibraries
are supported by similar energy yields (c. -10 to
-15kJmol-1 CH ). If methane production and consump- General archaeal 16S rRNA clone libraries yielded
4
tion have comparable energy yields in these sediments, ANME-1 archaea within the methane oxidation zone as
fasterANME-1growthratesintheoxidationzonesuggest well as at least 45cm into the methane production zone
thatmethaneoxidationratesarehigher,butfocusedina (Table1A), providing additional evidence that ANME-1
relatively narrow depth interval, compared with methane remain active in methanogenic sediments. These
productionrates. sequences grouped with other ANME-1 16S rRNA
OurrRNAandrDNAdataareconsistentwiththestan- sequences derived from both methane seeps and diffu-
dard model of stratification of sulfate reduction linked to sive sediments (Fig.S1). They did not form a separate
methane oxidation and methanogenesis (Hoehler etal., WhiteOakRiver-specificclade,anddidnothavedistinct
1994).AsharpdecreaseinrRNAandrDNAindicesatthe clades between the methane oxidation and production
base of the methane oxidation zone is consistent with a zones. ANME-1 comprised a small (<12%) part of
regionwhereneithermethaneoxidationnormethanepro- archaeal16SrRNAclonelibraries,whichweredominated
duction can support cellular energy requirements. Here byuncultured,benthicCrenarchaeota(datanotshown).
sulfate-reducingbacteriabecomesulfate-limitedandtheir Astringent test to detect metabolically active methane
rateofH consumptioncannotkeeppacewithH genera- producers is the presence of mcrA mRNA, which has a
2 2
tion via fermentation.Aresulting increase in H concen- lifetime of minutes once metabolic activity stops (Henni-
2
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MethanogenesisandmethanotrophyinANME-1archaea 2553
Table1. cDNAclonelibraryresults,witharchaeal16SrRNA,mcrAmRNAanddsrABmRNA.
A. 16SrRNA
Coreanddepth CH oxidizedorproduced NumberofANME-1clones Othermethanogens(n) Numberoftotalclones
4
Dec0624–27cm Oxidized 3 ND 52
Jul05-254–57cm Oxidized 7 ND 91
Jul08-248–51cm Produced 4 ND 33
Jul05-160–63cm Produced 3 ND 68
Dec0669–72cm Produced 2 ND 90
Jul05-287–90cm Produced 5 ND 98
B. mcrAmRNA
Coreanddepth CH oxidizedorproduced NumberofANME-1clones Othermethanogens(n) Numberoftotalclones
4
Jul08-23–6cm Oxidized 12 ND 12
Jul08-29–12cm Oxidized 26 ND 26
Jul08-218–21cm Oxidized 30 ND 30
Jul05-254–57cm Oxidized 18 ND 18
Jul08-248–51cm Produced 36 ND 36
Jul05-160–63cm Produced 24 Methanomicrobiales(19) 43
Jul05-187–90cm Produced 2 Methanosaetasp.(37) 39
Jul05-287–90cm Produced 3 ND 3
C. dsrABmRNA
Coreanddepth CH oxidizedorproduced Sulfatereducers(n) Numberoftotalclones
4
Dec0624–27cm Oxidized ClusterB(32) 32
Jul05-251–54cm Oxidized ClusterB(72),Desulfobacteriumanilinii(7) 79
Jul05-160–63cm Produced ClusterB(7) 7
Jul05-184–87cm Produced Desulfobaccaacetoxidans(29) 29
NDstandsforNoneDetected,andimpliesthataparticulargroupwasnotrepresentedinourclonelibrary,notthatitwasnecessarilyabsentin
thesample.
gan and Reeve, 1994) and encodes the enzyme that transcribed in sulfate-reducing bacteria while they are
catalysestheenergy-conservingstepinmethanogenesis, reducingsulfateaswellaswhentheyuseanon-sulfate-
methylcoenzymeMreductase.Thisenzymeispresumed reducing metabolism (Neretin etal., 2003). dsrAB phylo-
to also mediate anaerobic methane oxidation since it is types in the methane oxidation zone grouped within the
reversible(Schelleretal.,2010).Withinboththemethane Desulfobacteraceae(Fig.S2);othermembersoftheDes-
oxidationandproductionzones,mcrAmRNAspecificfor ulfobacteraceaephysicallyassociatewithANMEarchaea
ANME-1dominatedmcrAclones(Table1B).Aswith16S in methane seep sites (Knittel and Boetius, 2009). In
rRNA, the ANME-1 mcrA grouped with sequences from methanogenic sediments, dsrAB mRNA was amplified
methaneseeps,withoutformingaseparateclade;andno onlysporadically(4outof29attempts),withasignificantly
phylogenetic distinctions were seen between sequences lower success rate than in the methane oxidation zone
from methane oxidation and production zones (Fig.2A). (c2=7.99, d.f.=1, P>98%, two-tailed Chi-squared test;
Intwooutoffourclonelibrarieswithinthemethanogenic Fig.S3). Sequences retrieved from two methanogenic
zone, mcrA mRNA from other methanogen-affiliated depths grouped either within the Desulfobacteraceae
archaea(Methanosaetasp.at87–90cmandmembersof or near Desulfobacca acetoxidans, an acetate-utilizing
the Methanomicrobiales at 60–63cm in core Jul 05-1) sulfate reducer. The low detection rate of dsrAB gene
were also identified (Table1B; Fig.2A). Non-ANME transcription was consistent with constitutive dsrAB tran-
methanogens were recovered with methanogen-specific scription in small amounts, as in fermentative sulfate
mcrA primers but not with three general archaeal 16S reducers(Neretinetal.,2003).
primers (Table1A, TablesS1 and S2), indicating low CAtalysed Reporter Deposition Fluorescent In Situ
numbersandactivitybelowdetectionlimitswitharchaeal Hybridization (CARD-FISH) with probes specific for
16SrRNAprimers. ANME-1, ANME-2 and the Desulfosarcinales/
In the methane oxidation zone, dsrAB mRNA, which Desulfosarcina group showed that ANME-1 archaea in
encodes dissimilatory sulfite reductase was reproducibly themethaneoxidationzoneexistedonlyassinglerodsor
amplified (Table1C). This enzyme is constitutively small conspecific clusters containing (cid:2)4 rod-shaped
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
2554 K.G.Lloyd,M.J.AlperinandA.Teske
Fig.2. PhylogenyofANME-1mcrAmRNAsequencesandCARD-FISHphotomicrographofANME-1.
A.Neighbour-joiningtreeofaminoacidtranslationsofmcrAmRNAcDNA,withdistance-andthenparsimony-basedbootstrapsupport
(>60%,1000repetitions)listedatthenodes.Numbersofclonesrepresentedbyeach97%OTUgroup(determinedfromnucleicacids)are
showninparentheses,usingAforcoreJul05-1,BforcoreJul05-2,EforcoreDec06andGforcoreJul08-2.Scalebarshows10%
distance.Redclonesarefromthemethaneoxidationzoneandblueclonesarefromthemethaneproductionzone.
B.CARD-FISHlabelledwithprobeANME-1-350ingreenfromcoreJul05-251–54cm.Scalebaris5mm.
C.BlueDAPIsignalfromthesameimage.
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
MethanogenesisandmethanotrophyinANME-1archaea 2555
cells, and did not physically associate with other phylo- reductase) was not assigned to ANME-1 (Meyerdierks
types (Fig.2B and C); the lack of associated bacteria is etal.,2010).ANME-1archaeaalsocontaingenesencod-
common for ANME-1, ANME-2 and ANME-3 archaea ingthesamecarbonfixationpathwayasculturedmetha-
(Table2). ANME-2 archaea were never detected with nogens (Meyerdierks etal., 2010). ANME-2 archaea
CARD-FISH. ANME-1 signals were only found in the have been shown to fix nitrogen (Dekas etal., 2009), as
methane oxidation zone (Jul 05-2 51–54cm) using do cultured methanogens in the Methanosarcinales
CARD-FISH.Wenotethatcelldensitiesweretoolowfor (MurrayandZinder,1984),andANMElipidstructuresare
accurate quantification using this method, since many verysimilartothoseofculturedmethanogens(Elvertand
fieldsofviewhadtobeexaminedtofindasingleANME-1 Suess, 1999; Hinrichs etal., 1999). Microbial mats con-
signal.Therefore,thelackofANME-1orANME-2signals taining ANME archaea have been shown to express
in other depths (Jul 05-2, 0–3cm, 3–6cm, 9–12cm, methyl coenzyme M reductase (Krüger etal., 2003), an
18–21cm, 42–45cm, 72–75 cm and 87–90cm) should enzyme found in all orders of cultivated methanogens
not be taken as evidence for absence of ANME-1 or (Luton etal., 2002), and its evolutionary path in ANME
ANME-2. archaea mirrors that of cultured methanogens (Hallam
etal., 2003).ANME archaea fluoresce blue-green under
ultravioletlight(Michaelisetal.,2002;Knitteletal.,2005;
Discussion Lösekannetal.,2007),acharacteristicofculturedmetha-
nogens due to the presence of an F flavin-derived
420
We have shown that ANME-1 archaea are present and coenzyme.ANMEarchaeaareoftenfoundalongsidecul-
active in sediments from the White Oak River estuary at turedmethanogensatdeep-seabiogenicmethaneseeps
depthswherethereisnetmethaneoxidationandalsoat (Lanoil etal., 2001) and in methane-producing marine
depths where there is net methane production. We sediments (Table3, and references therein).At methane
found ANME-1-specific 16S rRNA and mcrA mRNA, seepswithhighorganicmatter,ANMEarchaeaareoften
moleculesthatarerapidlydegradedinnon-metabolically found in clusters with sulfate reducers (Boetius etal.,
activecells,inbothofthesetwosedimentaryzones.The 2000), a trait shared with methanogens and sulfate
rDNA index (a proxy for ANME-1 cell number) and the reducers in high organic matter wastewater bioreactors
rRNA index (a proxy for ANME-1 metabolic activity) (Santegoeds etal., 1999). ANME-2 and ANME-3 often
increasewithinthemethaneoxidationzone,decreaseat appear as coccoid cells and form clusters (Orphan
the base of the methane oxidation zone, and then gen- etal., 2002), as do cultured methanogens of the Metha-
erally increase to a secondary peak within the methane nosarcinales (Boone etal., 1993); and ANME-1 are
production zone. Of the total depth-integrated rDNAand often rod-shaped (Orphan etal., 2002), as are cultured
rRNA indices for each core, 26–77% of rDNA and methanogensoftheMethanobacterialesandMethanomi-
18–76% of rRNAare located in the methane production crobiales (Boone etal., 1993). Methane production has
zone, indicating that substantial fractions ofANME-1 cell been demonstrated in ANME-rich methane seep sedi-
numbers and activity occur here.All these data are con- ments (Orcutt etal., 2008), hydrocarbon-contaminated
sistent with the notion that ANME-1 in these sediments sediments (Siegert etal., 2010) and microbial mats
function as methanotrophs in the methane oxidation (Pimenov etal., 1997; Seifert etal., 2006; Treude etal.,
zone, and as methanogens in the methane production 2007).
zone. Despite the remarkable genetic, metabolic, structural
EvidencethatANMEarchaeaarecapableofmethano- and ecological similarities between uncultured ANME
genesis comes from the substantial similarities they and cultured methanogenic archaea, it is widely
share with cultured methanogens such as phylogenetic assumed that ANME archaea are obligate methanotro-
affiliations,predictedgenefunctions,habitatranges,mor- phs that cannot gain energy from methane production.
phologies and aggregation habits. 16S rRNA phylogeny ThisassumptioncanbetracedbacktoHinrichsandcol-
places ANME-1 among the methanogens, and ANME-2 leagues who preferred obligate or dominant methanotro-
andANME-3withinthemethanogenicOrderMethanosa- phy for ANME-1 largely on the grounds that ANME-1
rcinales, withANME-3 grouping within the genus Metha- archaea had never been detected in sediments where
nococcoides (Fig.S1). All cultured members of all five methane production was known to be active (Hinrichs
Orders of methanogens have been shown to produce etal., 1999). Our results from the White Oak River
methane (Boone etal., 1993). The genomes ofANME-1 estuary show the presence and activity of ANME-1
and ANME-2 have been sequenced from environmental archaea in methanogenic sediments. At least five other
samples and contain homologous genes for enzymes studies (Table3) found DNA from ANME archaea in
involved in all seven steps of methanogenesis, although methanogenic sediments, suggesting that our findings
one enzyme (N5,N10-methenyltetrahydromethanopterin are not unique to one environment.
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
2556 K.G.Lloyd,M.J.AlperinandA.Teske
Table2.ObservationsofphysicalassociationsofANME-1,ANME-2andANME-3withsulfate-reducingbacteria(WithSRB),otherbacteria(WithBac)ornootherorganisms(Alone)madeusingaFluorescenceInSituHybridization(FISH). ANME-1ANME-2a/bandANME-2cANME-3 bcdbcebceWithBacAloneWithSRBWithBacAloneWithSRBWithBacAloneTypeofenvironmentLocationWithSRB MethaneseepsBlackSeamats1,5,153,153,1533EelRiverBasin111111,1610–12,16,1811,1211,1618GulfofMexico9999,13139HydrateRidge333EasternMediterranean87,88,137,8fNorthSea171313HaakonMosbyMudVolcano444,64,134SeasonalmethaneseepEckernfordeBay1414FreshwaterLakePlußseefreshwater22Marinenon-seepWhiteOakRiverestuaryThisstudy a.Listedarereferencenumbersforthestudieswheretheseaggregations(orlackthereof)aredocumented.Thistableisnotexhaustive;theremaybeotherreportsthatarenotincluded.EmptycellsshouldnotnecessarilybetakenasevidencefortheabsenceofacertainaggregationbehaviourforacertaintypeofANMEatacertainlocationsincesomestudiesonlyfocusedonasubsetofcelltypes.b.ThiscategoryincludessphericalclustersofANMEcellsontheinside,surroundedbysulfate-reducingbacterialcells,matsofANMEcellspock-markedwithsulfate-reducingbacterialcells,andmixedclusterswhereANMEcellsandsulfate-reducingbacterialcellsappearevenlydistributed.c.ExceptforPernthalerandcolleagues(2008),wherethebacteriawereidentifiedasAlphaproteobacteriaandBetaproteobacteria,theidentitiesofthesebacteriaareunknown.d.Thiscategoryincludeslongfilamentsofrod-shapedANME-1cellsorsingleANME-1rods.e.ThiscategoryincludessphericalclustersofmonospecificANME-2orANME-3cellsorsingleANME-2orANME-3cocci.f.TheauthorsconcludedthatsingleANME-2cellsweretheresultofsonicationbeforehybridization.12345678(Blumenbergetal.,2004),(Elleretal.,2005),(Knitteletal.,2005),(Lösekannetal.,2007),(Michaelisetal.,2002),(Niemannetal.,2006),(Omoregieetal.,2008),(OmoregieDatasources:910111213141516(Orcuttetal.,2005),(Orphanetal.,2001),(Orphanetal.,2002),(Pernthaleretal.,2008),(Schreiberetal.,2010),(Treudeetal.,2005),(Treudeetal.,2007),(Houseetal.,etal.,2009),1718(Niemannetal.,2005),(Orphanetal.,2001).2009),
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564
MethanogenesisandmethanotrophyinANME-1archaea 2557
Reference Senioretal.(1982);Munsonetal.(1997) Thomsenetal.(2001) Kendalletal.(2007) Parkesetal.(2007) Harrisonetal.(2009)Currentstudy performedforthisdepth (a(tTwhd1rhi1ac9tA3ehht9Cnya9aoae(cid:2))ctdsaho1se-tu3h(uC1rlHdam0itkon0eefr‰damiyc-ct)h3echas0tooahr‰butgeasnmutn,etmaeebrlvt.aeh,feosnoa1dertxn9diido9bntago9yhtlen)ieiponHatinihdclisneesoarxisiadrtccmrthevthretsamaihbrleeiueutaasetnepordmd1ern3o-Celtecydoa-ovudsaAlcelpuelNeuprraoealMlegcidtptuieEoiifoedso-nn1sssr.
as methanogenic biomarkers in an Eocene lake shale with
3 w
E- s estimated porewater d13C-CO =+1‰; temperature=
e M si 2
eazon er mer AN naly 25°C (Hayes etal., 1987). Recent experimental (Londry
Methanogen-likearchainmethaneproduction MethanomicrobialesaANME-2a/bandothMethanosarcinalesAmplificationwithANME-1-specificpriANME-2 ANME-2aandotherMethanosarcinalesMethanomicrobiales,ANME-1ANME-1cMethanosarcinalescMethanomicrobiales Boetiusetal.,2000).d0.8m.No16SrDNAa tfeesseutextumntaardcpel.tie,imemosr2neea0itltsnyh0uhga8rdoen)ewme<apenl1etstdh0tehea°eatdtChpnesoui(noAgnrwed1lep3neiCtetisrhcr.iancclpoaao(nAnnrdedlpiwtpHeioarroonitneedsrhualccendeor1d,3mC2lHmi0p-oC0iode9Onsh)2,letn~torho,-ard24tme00aea0‰lpr9ley-);
Table3.Methanogen-likearchaeafoundinsedimentswherezonesofmethaneoxidationandproductionhavebeenclearlydefined. EvidenceformethaneEvidenceforMethanogen-likearchaeaLocationoxidation/productionmicroorganismsinmethaneoxidationzone 1414COconvertedtoCHandCHColnePointsaltmarsh,Essex,UK16SrDNANotexamined244effluxintooverlyingwatercolumn AarhusBay,DenmarkModelledratesandcurveofCH16SrDNAANME-1,ANME-2and4profileotherMethanosarcinalesSkanBay,Alaska,USARadiotracerratesandcurveofCH16SrDNAANME-1,ANME-2and413concentrationandCprofilesotherMethanosarcinalesdMethanomicrobialesbSkagerrakBay,DenmarkRadiotracerratesandcurveofCH16SrDNANotexamined4concentrationprofile SantaBarbaraBasin,California,USACurveofCHconcentrationprofile16SrDNAANME-14WhiteOakRiverestuary,Modelledrates,curveofCH16SrRNA,mcrAANME-1413NorthCarolina,USAconcentrationandCprofilesmRNA,16SrDNAd a.Theseclones(2MT7and2C83forANME-2a)wererecoveredbeforeBoetiusandcolleaguesnamedtheANME-2groupin2000(b.Methaneconcentrationprofilesandradiotracerratesareingoodagreementthatnetmethaneoxidationtakesplacebetween0.5an~interval.c.FoundonlywithmcrAmRNA. EoTnseob1adcbrtsgtGAtocitT(sm(stHshohhheS-4uouoapbovfenaaechhvNC2mueaeaa8qTOesinbcnmaltldytnieeddioldMtrittt-1u(nrsgtfui-coscch/troslef9aeeeweo1mamiecaeaetrleanErea(cid:3)pr;tttu.oinrurnrbuethhesnre4tn.uaseneleh-dftaecdocePtsdaeresr1ttFtdettr1tseee(cid:3)mmnreeheeocuhlhutedMamlu‰atdt>deamosfmaaalchsarseAiwtrttnnba2ae.edtreiAhsnenturuestt8oio)ssNphdieeyhl.ohnxmtFaeletl.gN30hnvbwhlttfhe,ipanaadMssirtutcccgi%oawioaMp2wrponhrst,nc(,[ooaAremEewiaannO+br0rumgthcaeBneo,ElpcoshoNsnloHah0oreornserlostatd-edemtcsbatpdtccoh6iMlatrth2ra-pernulbiuoruhunheocu]ikpuetwnao)riacgrdgeldlEscnleccke-c,ntebdaseoctpvwfhteutrisuetatehsrsamtSrlgueedtAhiiSviueneahtoovhroditeacbmt)ubrasmuseeieoNeeedseatrefnannsaeeteacccirftinnayisareaufetgMtc,a((wtthtttstewerimhiet2TilohniAesstorsavo.osmairhEotoec,ast0ratmdtnetNhte,fnnaemeia7oeborms2wa0mtsasetc.wnaraaMun.nsiwhu(0t8oeoet0aeanneesrwuKtdeuiflmans0)WEcftnathlwlrdotmelhelhdi(cid:3)nhadbndha8hcecrimmoptedecaoieeiaimcooeat)aoataetcebrstehe.rntui1aoerthnnotnesssheettlhencdiedSweti.tiltndaedda.glml5sahhaorcemdpsieiinoueuiastisalbatatpottanaaetrou.fdhhet[cxcoeitc,ornynernchclmgrfieltitopetotaut9mevhdceo2eaoiteenouhoameadeutsi-open0xndrhidpeeaenmoanditosuBlllprtnt0tieaeeidslnnoabvrroiacxctemcoltee7vtioeaheateiilnditneecbe(booenidtd)a3ildeegi2wc.oh]ut.danoinxynntat2enestsoa0ianaicprotahTmvuBnts-pdxubahlmt0tnti,tpneidtesamshehoigilhpealydg6oayifrofrar,ncpmeenreearcosdiosns)ncmtoaamyotoor.2errltdiithlkiphuedfeonmpdoigfiusrs0e,moTueielggerfin-ngcadptedyioS0vtoaurtcnhurdaahaetcbiiiihninie9rwnoenenenhHHetedcndaunnoelasil-r)egndgadddhecsyyy221-----tt,f.,l
©2011SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology,13,2548–2564