Archaea mediating anaerobic methane oxidation in deep-sea ...directory.umm.ac.id/Data Elmu/jurnal/O/Organic Geochemistry/Vol31...Archaea mediating anaerobic methane oxidation in deep-sea

Archaea mediating anaerobic methane oxidation indeep-sea sediments at cold seeps of the eastern Aleutian

subduction zone

Marcus Elvert a,*,1, Erwin Suess a, Jens Greinert a, Michael J. Whiticar b

aGEOMAR, Research Center for Marine Geosciences, Wischhofstr. 1-3, D-24148 Kiel, GermanybSchool of Earth and Ocean Sciences, University of Victoria, PO Box 3050, British Columbia, V8W 2Y2, Canada

Received 12 October 1999; accepted 1 August 2000

(returned to author for revision 7 January 2000)

Abstract

Cold seeps in the Aleutian deep-sea trench support proli®c benthic communities and generate carbonate precipitateswhich are dependent on carbon dioxide delivered from anaerobic methane oxidation. This process is active in the

anaerobic sediments at the sulfate reduction-methane production boundary and is probably performed by archaeaworking in syntrophic co-operation with sulfate-reducing bacteria. Diagnostic lipid biomarkers of archaeal origininclude irregular isoprenoids such as 2,6,11,15-tetramethylhexadecane (crocetane) and 2,6,10,15,19-pentamethylicosane(PMI) as well as the glycerol ether lipid archaeol (2,3-di-O-phytanyl-sn-glycerol). These biomarkers are prominent lipid

constituents in the anaerobic sediments as well as in the carbonate precipitates. Carbon isotopic compositions of thebiomarkers are strongly depleted in 13C with values of d13C as low as ÿ130.3% PDB. The process of anaerobic meth-ane oxidation is also re¯ected in the carbon isotope composition of organic matter with d13C-values of ÿ39.2 and

ÿ41.8% and of the carbonate precipitates with values of ÿ45.4 and ÿ48.7%. This suggests that methane-oxidizingarchaea have accumulated within the microbial community, which is active at the cold seep sites. The dominance ofcrocetane in sediments at one station indicates that, probably due to decreased methane venting, archaea might no

longer be growing, whereas high amounts of crocetenes found at other more active stations may indicate recent ¯uidventing and active archaea. Comparison with other biomarker studies suggests that various archaeal assemblagesmight be involved in the anaerobic consumption of methane. The assemblages are apparently dependent on speci®c

conditions found at each cold seep environment. Selective conditions probably include water depth, temperature,degree of anoxia, and supply of free methane. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Aleutian subduction zone; Cold seeps; Authigenic carbonates; Biomarkers; Irregular isoprenoids; Carbon isotopic com-

position; Crocetane; Crocetenes; PMI; Archaeol

1. Introduction

Chemoautotrophic microbial communities inhabitingsediments at cold seeps or living as symbionts in vent

macrofauna are important for carbon cycling in deep-sea

environments, preferentially along convergent continental

margins. At the cold seeps, ¯uid venting supportsbenthic communities and generates authigenic carbo-nates from the biogeochemical turnover and interaction

between ¯uids and ambient bottom water (Suess et al.,1985; Kulm et al., 1986; Wallmann et al., 1997). Growthand metabolism of the associated vent macrofauna arebased on a chemoautotrophic food chain which starts

with the microbially mediated oxidation of reducedcompounds, such as methane or hydrogen sul®de,delivered by active ¯uid venting. For methane, the

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PI I : S0146-6380(00 )00111-X

Organic Geochemistry 31 (2000) 1175±1187

www.elsevier.nl/locate/orggeochem

* Corresponding author. Fax: +1-49-431-600-2928.1 Present address: Max-Planck-Institute for Marine Micro-

biology, Celsiussor. 1, 28359 Bremen, Germany. Fax: +1-49-

421-2028-690; e-mail: [email protected].

E-mail address: [email protected] (M. Elvert).

oxidation to carbon dioxide occurs either by aerobic(Childress et al., 1986) or anaerobic processes (Suessand Whiticar, 1989), with the latter still not completelyunderstood. However, subsequent incorporation of car-

bon dioxide by organisms in tissues or precipitation ofcarbonates from oversaturated microenvironmentscauses a strong carbon isotope shift towards 13C-deple-

ted values often cited as evidence for methane oxidation;e.g. mytilid mussels, vestimentiferan and pogonophorantube worms, and carbonates are depleted in 13C to values

as low asÿ77% PDB (e.g. Paull et al., 1985; Brooks et al.,1987; Ritger et al., 1987; Suess et al., 1998).Biomarkers found at ancient and recent methane

seeps have provided another important piece of evidencesupporting methane oxidation under anaerobic condi-tions (Elvert et al., 1999; Hinrichs et al., 1999; Thiel et al.,1999; Pancost et al., 2000). These authors predominantly

identi®ed irregular tail-to-tail isoprenoids and isopranyl-glycerol diethers such as 2,6,11,15-tetramethylhexadecane(crocetane), 2,6,10,15,19-pentamethylicosane (PMI), 2,3-di-

O-phytanyl-sn-glycerol (archaeol), 2-O-3-hydroxyphytanyl-3-O-phytanyl-sn-glycerol (sn-2-hydroxyarchaeol), and 3-O-3-hydroxyphytanyl-3-O-phytanyl-sn-glycerol (sn-3-

hydroxyarchaeol) with highly depleted carbon isotopevalues as low as ÿ123.8% PDB from various anaerobicsettings. Characteristic settings include methane seeps

associated with marine gas hydrates (Elvert et al., 1999;Hinrichs et al., 1999), ancient methane vent systems(Thiel et al., 1999), and methane-rich mud volcanoes(Pancost et al., 2000). The detection of irregular iso-

prenoids and/or isopranylglycerol diethers, both tradi-tionally believed to be biosynthesized by methanogenicarchaea, with such extremely low carbon isotope values

prompted these authors to suggest that either certainmethanogens themselves are involved in the consumptionof methane, operating in reverse in syntrophic co-

operation with sulfate reducers (Elvert et al., 1999; Thielet al., 1999; Pancost, 2000), or that until now unknownmethanogens within archaeal lineages evolved to beingcapable of using methane as their predominant or even

exclusive carbon source (Hinrichs et al., 1999).Following these ideas, we analyzed speci®c biomarkers

related to anaerobic methane-oxidizing processes from

sediments and carbonates at cold seep settings of theeastern Aleutian subduction zone, adjacent to the Aleutiandeep-sea trench. These cold seeps are among the deepest

observed (�4800 m) and therefore, being far removedfrom the photic zone, are well suited to study chemoauto-trophic processes because very little metabolizable

particulate organic matter reaches this depth. Weespecially examined the abundance, carbon isotopevalues, and signi®cance of biomarkers diagnostic ofanaerobic methane oxidation. Moreover, we evaluated

the variability of the speci®c biomarkers found in thisstudy compared to those observed at other cold seepenvironments.

2. Materials and methods

2.1. Study area

The study area at the eastern Aleutian subductionzone, referred to as SHUMAGIN sector, was surveyedand sampled during R/V SONNE cruises 97 (SO 97)

and 110 (SO 110-lb and SO 110-2), and is shown in Fig.1a. The tectonic setting, manifestations of venting, andthe general sampling strategy have been described earlier

by Suess et al. (1998). Widespread methane venting wasobserved along the entire margin and speci®cally o�SHUMAGIN at the intersection of accretionary ridges

with tensional faults. These faults occur in canyonslandward of the deformation front at water depthsaround 4800 m and are the result of oblique subductionof the Paci®c plate underneath the Aleutian arc. Colonies

of typical seep macrofauna and authigenic carbonatecrusts were found. The seep biota consists of bacterialmats, pogonophorans, vestimentiferans, and large colonies

of bivalves. The carbon isotope composition of tissuesfrom the seep fauna ranged from ÿ57.1 to ÿ64.3% andthus identi®es methanotrophy as the dominant carbon

metabolizing pathway (Suess et al., 1998). Similarly, forauthigenic carbonates, d13C values between ÿ42.7 andÿ50.8% were reported (Greinert, 1998), suggesting that a

mixture of biogenic methane, via anaerobic oxidation, andcarbon dioxide supplied by sulfate-reducing bacteria wasthe ultimate carbon source of the authigenic mineralogies.

2.2. Sediment, pore water, and carbonate analysis

Contents of Corg were determined from the carbonate

free, dried, and homogenized sediment material using aCarlo Erba Nitrogen Analyzer 1500. For carbonateremoval, 3 g of wet sediment were treated over night

with 15 ml of 10% HCl. After freeze-drying, sampleswere homogenized by using an agate ball mill. Standarddeviations of this method were 0.02%. Sulfate measure-ments were carried out by ion chromatography and

detection by conductivity. Sulfate values are reported inmM and were calibrated with IAPSO-standard seawater.Using duplicate measurements, standard deviations were

within 1.5%. The authigenic carbonates were identi®ed bystandard X-ray di�raction analysis. The speci®c calcitesample selected for extraction of biomarkers was a high

Mg-calcite (Greinert, 1998).

2.3. Extraction, chromatographic separation,

hydrogenation and derivatization

Lipids were extracted ultrasonically from the samples(20±25 g of wet sediment) with 50 ml of methanol/

dichloromethane (2:1, v/v), 50 ml of methanol/dichloro-methane (1:2, v/v), and twice with 50 ml of dichloro-methane. For the carbonate, 25 g were washed with

1176 M. Elvert et al. / Organic Geochemistry 31 (2000) 1175±1187

acetone and dissolved in a 1 l round bottom ¯ask by

adding stepwise 500 ml of 1 N HCl and stirring for 6 h.After centrifugation for 5 min at 4000 rpm and decan-tation of the supernatant, the residue was washed two

times with pre-extracted water and the lipids wereextracted as described above for wet sediment material.Fractions were separated from the lipid extracts by

medium pressure liquid chromatography on 1.3 g silica

gel (70±230 mesh, 5% deactivated). Chromatographicseparation was by elution with (I) 13 ml of n-hexane(hydrocarbons), (II) 10 ml of dichloromethane/n-hexane

(20:80, v/v; esters and ketones), (III) 10 ml of dichloro-methane (alcohols), and (IV) 10 ml of methanol/di-chloromethane (50:50, v/v; glyco- and phospholipids).

Elemental sulfur in the hydrocarbon fractions (I) wasremoved by passing the fractions over separate shortcolumns ®lled with 1 g of activated copper powder using

n-hexane as eluent.Hydrogenation of hydrocarbon fractions was carried

out by saturation of 50 ml n-hexane withH2 in fusible glassampoules pre-®lled with 10 mg of PtO2 and subsequent

adding of 50 ml of sample (12 aliquot in n-hexane). After¯ushing with H2, the ampoules were closed and storedat room temperature for 1 h. Finally, the samples were

directly analyzed by gas chromatography±mass spec-

trometry (GC±MS).To facilitate gas chromatographic analysis of alcohols,

trimethylsilyl (TMS) derivatives were produced. Alcohol

fractions were evaporated under a stream of pure nitrogento near dryness, mixed with 100 ml BSTFA (bis(tri-methylsilyl)tri¯uoroacetamide; Supelco), and heated inclosed glass ampoules for 2 h at 80�C. Following eva-

poration to near dryness under nitrogen, the residue wastaken up in n-hexane and subsequently analyzed bymass spectrometry.

2.4. Gas chromatography (GC)

Gas chromatographic analyses of hydrocarbons wereperformed using a 30 m apolar DB-5 fused silica capillarycolumn (0.25 mm internal diameter (ID), ®lm thickness

0.25 mm; J&W Scienti®c) in a Carlo Erba 5160 gas chro-matograph equipped with an on-column injector and a¯ame ionization detector. The samples were injected at60�C. After a 1 min hold time, the oven temperature was

raised to 140�C at 10�C/min, then to 310�C at 5�C/minand ®nally kept at 310�C for 25 min. The carrier gas wasH2 at a ¯ow rate of 2.5 ml/min. Concentrations for each

Fig. 1. Aleutian subduction zone with (a) area of investigation (SHUMAGIN sector) and (b) detailed map of coring stations TV-G 97

(= TV-guided grab sampler), TV-GKG 40 (= TV-guided box corer), TV-G 43, and TV-G 48 inside a canyon landward of the

deformation front.

M. Elvert et al. / Organic Geochemistry 31 (2000) 1175±1187 1177

compound were determined by adding internal stan-dards (3-methylnonadecane, 2-methylicosane, 5b(H)-cholane) with known concentrations prior to GC analysisand are reported in mg/g Corg. Standard deviations for

single compounds are below s=2 mg/g Corg except forcompounds with more than 50 mg/g Corg (below �=10mg/g Corg). Loss of material during analysis was mon-

itored by adding a recovery standard (n-C40) prior to theoverall analytical procedure. In general, typical recov-eries were 70±80% relative to n-C40.

2.5. Gas chromatography±mass spectrometry (GC±MS)

Hydrocarbons and alcohols (as TMS-derivatives)were identi®ed by GC±MS using a Carlo Erba 8000 gaschromatograph interfaced to a Fisons MD 800 massspectrometer operated in electron impact (EI-) mode at

70 eV (cycle time 0.9 s, resolution 1000) with a massrange of m/z 40±600 for hydrocarbons and m/z 40±800for alcohols. The gas chromatograph was equipped with

a DB-1 fused silica capillary column (30 m, 0.25 mm ID)coated with cross-linked methyl silicone (®lm thickness0.25 mm; J&W Scienti®c) using He as carrier gas. The

samples were injected in splitless mode (hot needletechnique; injector temperature: 285�C) and subjected tothe same temperature program given for GC measure-

ments (see Section 2.4.).

2.6. Stable carbon isotope analysis

Carbon isotope compositions of hydrocarbons andalcohols were determined using a coupled gas chromato-graph±combustion±isotope ratio mass spectrometer

(GC±C±IRMS). The mass spectrometer (Finnigan MAT252) was connected to a Varian 3300 GC equipped witha 50 m CP-Sil 5 CB-MS (0.25 mm ID, 0.4 mm stationary

phase; Chrompack). The carrier gas was He at a ¯owrate of 1.5 ml/min. The samples were on-column injectedat 60�C and after 1 min the oven temperature was raisedto 140�C at 10�C/min, then to 230�C at 3�C/min, and

®nally to 310�C at 2�C/min at which it was held for 35 min.Carbon isotope ratios are reported in the d notation as permil (%) deviation from the Pee Dee Belemnite standard

(PDB). Internal standards (5b(H)-cholane and n-C36 forhydrocarbons; n-C20 and n-C36 for alcohols) of knownisotopic composition were co-injected with each sample for

monitoring reproducibility and precision during the pro-ject. Analytical reproducibility was on average within�0.2±0.3% for an n-alkane standard (n-C13 to n-C38)

with no background, but was much more variable forthe complex mixtures analyzed here (up to �1.6%) dueto factors such as co-elution or signal to backgroundratio. Isotopic compositions of alcohols were measured in

the form of TMS-derivatives and corrected for the iso-topic shift associated with the addition of carbon atomsduring derivatization according to Huang et al. (1995).

d13Corg was measured by elemental analysis±isotoperatio mass spectrometry (EA±IRMS) using a CarloErba Elemental Analyzer connected via a ConFloTM

interface to the Finnigan MAT 252. Analytical repro-

ducibility for duplicate runs was below �0.1%.

3. Results

Three sediments and one carbonate sample from four

di�erent stations at cold seeps were analyzed for bio-markers indicative of anaerobic methane oxidation. Theactive seep sites along with extensive carbonate crusts

and methane anomalies of the bottom water columnwere observed at a location from the SHUMAGIN sec-tor inside a canyon which crosscuts the third accre-tionary ridge (Suess et al., 1998) (Fig. 1b). The canyon

itself is cut by two faults along N±S and NNW±SSEdirection, which probably provide ¯uid pathways andfocus di�usive ¯uid venting. Pore water analysis showed

that sediments analyzed were well within the sulfatereduction zone which starts right below the sedimentsurface (Fig. 2). Sulfate concentrations reach 10 mM at

station TV-G 43 (35 cmbsf) and concentrations <5 mMat station TV-G 48 (12 cmbsf). Sample TV-GKG 40,from which no sulfate pro®le was available due to very

low sample recovery, was probably derived from nearthe suboxic±anoxic interface.

3.1. Hydrocarbons: structures, abundance, and isotopic

compositions

Two sediment samples (stations TV-GKG 40 and TV-

G 48) and the carbonate sample (station TV-G 97) werefound to contain signi®cant amounts of crocetane(Robson and Rowland, 1993; Elvert et al., 1999) and to

a minor degree PMI and pentamethylicosenes (Brassellet al., 1981; Rowland et al., 1982; Schouten et al., 1997).These compounds were absent from sediments at stationTV-G 43. Three representative partial gas chromato-

grams of the hydrocarbon fractions obtained from theanoxic sediments are shown in Fig. 3a±c. Irregular C25

isoprenoids such as PMI have especially been identi®ed

in methanogenic archaea (Holzer et al., 1979; Torna-bene et al., 1979; Risatti et al., 1984) although fre-quently a source from photoautotrophic organisms has

been inferred (Kohnen et al., 1992; Freeman et al.,1994). Nevertheless, Elvert et al. (1999) and Thiel et al.(1999) described that this compound may also be

derived from archaea involved in the anaerobic oxida-tion of methane. In the same study, Elvert et al. (1999)detected a four times unsaturated pentamethylicosene(PMI:4) which was previously identi®ed by Sinninghe

DamsteÂ et al. (1997) in the methanogenic archaeonMethanosarcina mazei. Moreover, pentamethylicosenespossessing three to ®ve double bonds were isolated from


the marine methanogenic archaeon Methanolobus bom-bayensis (Schouten et al., 1997). Similar pentamethyli-cosenes were encountered in hydrocarbon fractions

from stations TV-GKG 40, TV-G 48, and TV-G 97.Crocetane is the most prominent hydrocarbon in the

samples at stations TV-GKG 40, TV-G 48, and TV-G

97. This biomarker has recently been found in highamounts in anaerobic sediments and in carbonatesassociated with recent and ancient methane seeps and

methane-rich mud volcanoes (Elvert et al., 1999; Thiel etal., 1999; Pancost et al., 2000). Based on structural andisotope evidence, crocetane was linked to archaeainvolved in anaerobic methane oxidation. In contrast to

these samples containing crocetane or PMI, such bio-markers are absent from the sediment at station TV-G43 (Fig. 3c). Sulfate reduction at this location is less

intensive compared to station TV-G 48 (Fig. 2) andtherefore does probably not favour near-surface anae-robic methane oxidation. Furthermore, the shape of the

sulfate pro®le appears di�usion-controlled, suggestingthat the actual sulfate reduction site is deeper in the corethan the location of the sample. The samples obtainedfrom stations TV-GKG 40, TV-G 48, and TV-G 97 also

show the presence of unsaturated hydrocarbons elutingslightly ahead and after crocetane. On the basis of theirmass spectra and retention times, these compounds were

tentatively identi®ed as 2,6,11,15-tetramethylhexadecenes(crocetenes) containing one or two double bonds (Fig.4). This was further con®rmed by a hydrogenation

experiment which yielded crocetane as the only irregularC20 compound (see Section 2.3). The exact positions ofthe double bond(s) within each compound were not

determined.The concentrations of hydrocarbons present in the

anaerobic sediments ranged from 3 up to 830 mg/g Corg,with the main compounds being crocetane and its unsa-

turated counterparts (Table 1). The highest amounts ofcrocetane (2400 mg/g Corg) and other irregular isoprenoidswere obtained from the carbonate extract, which in turn

showed only trace amounts of non-isoprenoid com-pounds (Fig. 3a). Apparently, the carbonate initiallyprecipitated at a site of maximum activity by methane-

oxidizing organisms.Other microbial hydrocarbons obtained from the seep

sediments are the irregular C30 isoprenoid squalene

(2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene), an universal precursor molecule in lipid bio-synthesis of many living organisms, and the C30 hopanoid

diploptene (hop-22(29)-ene) (Fig. 3b). Sources knownfor the latter biomarker are various bacteria such ascyanobacteria, ammonia-oxidizing bacteria, and methylo-trophic and methanotrophic bacteria (Rohmer et al.,

1984; Ourisson et al., 1987; Summons et al., 1994) but ithas yet not been observed in anaerobic bacteria. Squalenewas only present at station TV-GKG 40 and absent at

all other stations. In contrast, diploptene was found inall sediments which also contained irregular C20 and C25

isoprenoids. Its relative abundance was moderate at

station TV-GKG 40 and low at the stations TV-G 48and TV-G 97 (Table 1).Carbon isotope analyses revealed two distinct groups

of microbial biomarkers both highly depleted in 13C

(Table 2). First, irregular saturated and unsaturated C20

and C25 isoprenoids, indicative of archaea, with isotopevalues betweenÿ93.5 andÿ130.3% (group I) and second,

non-speci®c bacterial biomarkers such as squalene anddiploptene with isotope values of ÿ60.5 to ÿ74.4%(group II). The extremely low isotope values of group I

in subsurface samples at the seep sites strongly indicatean origin from organisms which utilize methane anae-robically. In contrast, the higher d13C values obtained

for compounds of group II might indicate an originfrom as yet undetected anaerobic bacteria growing noton methane but still on a 13C-depleted carbon source orre¯ect oxidation of methane to some degree by aerobic

methanotrophs. Oxygen may be transported periodicallyinto the shallow anoxic habitat by the pumping activityof clams such as Calyptogena sp. known to be living at

Fig. 2. Pore-water sulfate pro®les at stations TV-G 43 and TV-G 48; sample intervals are indicated. Station TV-G 48 shows much

stronger sulfate reduction than station TV-G 43. Sample TV-GKG 40 probably comes from near the suboxic±anoxic interface; no

sulfate data are available.


the sediment±water interface of cold seep environments(Wallmann et al., 1997).

3.2. Alcohols: structures, abundance, and isotopic

compositions

Archaeol was the dominant alcohol in the sediment at

station TV-G 48 and the carbonate at station TV-G 97,but was absent at the stations TV-GKG 40 and TV-G43. Archaeol is one of the most common core ether

lipids in archaea and especially prominent in methano-

gens and halophiles (De Rosa and Gambacorta, 1986).A representative GC±MS chromatogram of the alcoholfraction obtained from the carbonate sample at stationTV-G 97 is shown in Fig. 5. Ether lipids other than

archaeol were detected only in trace amounts and wereassigned to be monounsaturated archaeols as identi®edearlier in the Antarctic methanogen Methanococcoides

burtonii (Nichols and Franzmann, 1992). These com-pounds are probably breakdown products of hydroxy-archaeols (e.g. both sn-2 and sn-3 isomers) which were

found to be acid-labile even under mild conditions(Sprott et al., 1990). Intact hydroxyarchaeols as detectedin cultures of members of the families Methanococcales

and Methanosarcinales (Koga et al., 1993; Sprott et al.,1993) were completely absent. In addition to etherlipids, signi®cant amounts of phytanol and n-alcoholsfrom n-C14:0 to n-C18:0 with a maximum at n-C16:1 were

found in the carbonate sample at station TV-G 97, butwere absent in the sediment sample at station TV-G 48.Carbon isotope analyses of archaeol obtained from

the samples at stations TV-G 48 and TV-G 97 revealhighly depleted 13C values as low as ÿ123.5% (Table 3).They are in the same range as those detected for croce-

tane and the crocetenes. This coincidence suggests acommon source for these compounds from organismswhich use methane as their carbon source for lipid bio-

synthesis. The ®nding of 13C-depleted archaeol at deep-sea cold seeps is in agreement with surveys of archaeal16S rRNA genes and biomarkers from seep-related sedi-ments of the Eel River Basin (Hinrichs et al., 1999). This

study revealed the predominance of a new group ofmethanogens which co-exist with isotopically 13C-depletedbiomarkers such as archaeol and sn-2-hydroxyarchaeol.

Furthermore, highly 13C-depleted archaeol has also beenidenti®ed in sediments from mud volcanoes on theMediterranean Ridge, another important convergent

margin setting (Pancost et al., 2000). The carbon isotopevalue of phytanol (3,7,11,15-tetramethlhexadecanol) atstation TV-G 97 is similar to the irregular isoprenoidsand archaeol (�ÿ120%) and thus, links it to anaerobic

methane-oxidizing archaea at this station. However, atstation TV-G 48 the d13C value of phytanol is distinctlydi�erent (ÿ82.2%). This lowered isotope signal may be

caused by a small but unknown portion of phytanolderived from organisms di�erent than anaerobic meth-ane-oxidizing archaea. A plausible source appears to be

phytanol via hydrogenation of chloro-phyll-derivedphytol, which would certainly show an isotope valuemuch more enriched in 13C relative to archaeal-derived

phytanol.Intracrystalline n-alcohols released from the Mg-calcite

at station TV-G 97 in the range of n-C14:0 to n-C17:1

show d13C values betweenÿ96.5 andÿ111.2% (Table 3).

These values document that these biomarkers are derivedfrom an organism which plays a signi®cant role in thenet oxidation of methane at this location. However, care

Fig. 3. Gas chromatograms of hydrocarbon fractions obtained

from samples at stations TV-G 97 (a), TV-GKG 40 (b), and

TV-G 43 (c). n-Alkanes are indicated by solid circles. Numbers

represent total number of carbon atoms. (Pr: 2,6,10,14-tetra-

methylpentadecane; Ph: 2,6,10,14-tetramethylhexadecane; Cr:

crocetane, Cr: 1, Cr:2: crocetenes containing one or two double

bonds, respectively; PMI: 2,6,10,15,19-pentamethylicosane;

PMI:3, PMI:4, and PMI:5: pentamethylicosenes containing

three, four, or ®ve double bonds, respectively; IS: internal

standards; ?: unknown cyclic compounds).


should be taken for the assignment of an archaeal originbecause these lipids have never been reported as mem-brane components in methanogenic archaea (see Kogaet al., 1993 for a review). In general, short-chain n-alcohols

are thought to be of marine origin. They may derive fromthe degradation of wax esters biosynthesized by zoo-plankton andmarine invertebrates (Grimalt andAlbaigeÂ s,

1990). Nevertheless, given their extremely low d13Cvalues in the samples analyzed here, an in situ produc-tion of these compounds seems very probable. Onepossible source of such 13C-depleted alcohols may be

sulfate reducers using not methane but an intermediatesubstrate with still isotopically depleted organic carbon.This was previously suggested from the presence of

Fig. 4. Electron impact mass spectra and corresponding retention indices on an apolar DB-1 column used for GC±MS analysis of

crocetane and crocetenes obtained at station TV-GKG 40.


13C-depleted C15:0 iso- and anteiso-, and C16:0 mid-chainbranched alcohols from a Miocene limestone (Thiel et

al., 1999). These compounds showed distinctly higherd13C values (by 20±30%) than phytanol and a new ten-tatively identi®ed ether lipid, containing one phytanyl

and one hexadecyl moiety from the same sample. In oursamples, alcohols with branched carbon chains wereabsent. Nevertheless, the clear distinction of isotope

values obtained for phytanol and archaeol compared tothe n-alcohols likewise indicates di�erent source organ-isms for these compounds. Therefore, an origin of thesen-alcohols from sulfate reducers living in syntrophy with

archaea and using an isotopically depleted type oforganic carbon (e.g. 13C-depleted pore water carbondioxide or acetate) is a plausible interpretation.

3.3. Bulk organic carbon

The high concentrations of irregular C20 and C25 iso-prenoids at station TV-G 48 represent roughly 0.1% of

the total organic carbon. This extraordinary amount ofbiomarkers diagnostic of archaea growing on methaneis also re¯ected in the carbon isotopic composition ofthe total organic matter (ÿ41.8%; Table 4). Considering

that lipids only comprise a very small part of the sedi-mentary Corg (Sinninghe DamsteÂ and Schouten, 1997),this value is extraordinarily low compared to normal

marine organic matter. Because substantial contributorsto sedimentary Corg such as proteins and carbohydratesare enriched in 13C compared to lipids (Meyers, 1997),

the d13C value observed either indicates contributionsfrom considerable quantities of 13C-depleted lipids otherthan irregular isoprenoids or that proteins and carbo-

hydrates also contain methane-derived carbon at thisstation. In contrast, the less sulfate-depleted sediment atstation TV-G 43 shows an isotope value of ÿ25.5% fortotal Corg. This most likely indicates a nutritional path-

way di�erent from that of anaerobic methane oxidationof the microbial assemblage at this station or that mostof the organic matter was derived from pelagic inputs.

Table 1

Concentrations of hydrocarbons (in mg/g Corg) obtained from

anaerobic cold seep sediments and a methane-derived carbon-

ate of the SHUMAGIN sector

TV-G 97

(Mg-calcite)

TV-GKG

40 (3±5

cmbsf)

TV-G

43 (20±22

cmbsf)

TV-G

48 (13±16

cmsbf)

Hydrocarbon [mg/g Cog]

n-C17 110 13 24 21

Pr 65 26 42 52

Cr:1 230 46 n.d. 15

n-C18 81 7 19 16

Cr 2400 280 n.d. 830

Ph n.d.a n.d. 10 n.d.

Cr:10 230 51 n.d. 10

Cr:100 150 35 n.d. 10

Cr:1000 390 130 n.d. 23

Cr:2 340 97 n.d. 11

n-C19 96 15 25 21

n-C20 72 10 21 19

n-C21 36 14 33 26

n-C22 25 12 28 26

PMI 85 41 n.d. 44

n-C23 17 12 47 29

PMI:3 52 12 n.d. 16

PMI:4 77 21 n.d. 17

PMI:5 15 13 n.d. n.d.

n-C24 12 10 43 24

n-C25 13 14 64 34

n-C26 4 10 32 22

n-C27 15 18 73 41

n-C28 7 12 25 21

Squalene n.d. 110 n.d. n.d.

n-C29 10 76 110 68

n-C30 4 15 11 10

n-C31 7 24 96 38

n-C32 3 9 13 8

n-C33 5 11 32 18

Diploptene 30 87 n.d. 37

a n.d., not detected.

Table 2

Carbon isotopic compositions of hydrocarbons related to

methane oxidation obtained from anaerobic cold seep sedi-

ments and a methane-derived carbonate of the SHUMAGIN

sector; also shown are hydrocarbons derived from photo-

autotrophic sources (n-C17, Pr, and n-C19)

TV-G 97

(Mg-calcite)

TV-GKG 40

(3±5 cmbsf)

TV-G 48

(13±16 cmbsf)

Hydrocarbon d13C (%)

n-C17 ÿ32.0�0.7a ÿ34.6�0.1 ÿ31.4Pr ÿ34.3�0.0 ÿ31.0�0.1 ÿ29.4Cr:1+n-C18 ÿ100.0�0.4 ÿ114.0�0.3 ÿ71.7Cr ÿ129.9�0.6 ÿ130.3�0.3 ÿ124.6Cr:10 ÿ123.1�1.1 ÿ123.6�1.4 n.m.

Cr:100 ÿ128.9�1.2 ÿ129.9�0.1 n.m.

Cr:100 0 ÿ124.7�1.4 ÿ128.2�0.3 ÿ93.5Cr:2 ÿ123.7�0.0 ÿ122.7�0.0 n.m.

n-C19 ÿ31.8�0.5 ÿ31.6�0.4 ÿ27.0PMI ÿ107.0�0.4 ÿ71.1�1.1 ÿ104.4PMI:3 ÿ116.8�0.7 ÿ98.0�0.7 n.m.

PMI:4 ÿ110.6�0.8 ÿ111.8�1.4 n.m.

PMI:5 n.d.c ÿ98.0�1.1 n.d.

Squalene n.d. ÿ60.5�1.1 n.d.

Diploptene ÿ72.7�1.2b ÿ72.5�0.3 ÿ74.4b

a Indicated uncertanties are standard errors of means of 2

(TV-G 97) or 3 (TV-GKG 40) measurements. Values for SO

110-2 TV-G 48 represent single measurement.b Values given for diploptene at coring sites TV-G 97 and

TV-G 48 represent small signal to noise ratio.c n.d., not detected; n.m. not measured, insu�cient amount

for carbon isotope analysis.


4. Discussion

4.1. Evidence for anaerobic methane oxidation at coldseeps

Anaerobic methane oxidation in marine sediments isunambiguously identi®able at the sulfate reduction±methane production boundary (Iversen and Jùrgensen,

1985; Blair and Aller, 1995; Burns, 1998; NiewoÈ hner et al.,1998). Radiotracer experiments, stable carbon isotopes,and methane and sulfate mass balances point to methaneoxidation at the expense of sulfate (e.g. Reeburgh, 1980;

Devol and Ahmed, 1981; Iversen and Jùrgensen, 1985;Blair and Aller, 1995; Burns, 1998; NiewoÈ hner et al.,1998). Indeed, laboratory and ®eld investigations indicate

that a syntrophic consortium of methanogenic archaea,operating in reverse, and sulfate reducers is responsiblefor the net methane oxidation under anaerobic condi-tions using water as the formal oxidant (Hoehler et al.,

1994; Hansen et al., 1998). Moreover, indications frombiomarker studies tracing anaerobic methane oxidationhave now been found for ancient (Thiel et al., 1999) and

recent methane seeps (Elvert et al., 1999; Hinrichs et al.,1999), and methane-rich mud volcanoes (Pancost et al.,2000). However, if the overall reaction is actually a syn-

trophic co-operation involving a methanogen, runningmethane formation reversely, and a sulfate-reducingbacterium, it is obvious considering the thermodynamic

controls that only one of the partners could gain metabolicuseful energy from the reaction and that the other partnerhas to run this process only as a co-metabolic activity(Schink, 1997). Therefore, it is conceivable that one

bacterium might exist which is able to oxidize methaneand reduce sulfate at the same time and thus obtains theentire energy from this transformation. Recently, Hinrichs

et al. (1999) suggested, based on ribosomal RNA andbiomarker analysis from the same sample, that newly,up to now unidenti®ed methanogens may have evolved

within archaeal lineages for which methane oxidation isthe predominant or even exclusive metabolic pathway(methanotrophic archaea). Nevertheless, the identity of

the terminal electron acceptor of the overall process hasnot been documented.Our biomarker results clearly indicate that anaerobic

methane oxidation is a major process in sulfate-depleted

sediments (TV-G 48) at cold seeps of the eastern Aleutiansubduction zone whereas less sulfate-depleted sediments(TV-G 43) showed no such evidence. Moreover, con-

sidering the above discussion and the geochemical con-ditions at the Aleutian subduction zone (bottom watermethane anomalies, pore water sulfate concentrations,

authigenic carbonates), biomarkers (irregular isoprenoids,ether lipids, n-alcohols), and carbon isotopes reportedhere, it seems very likely that anaerobic methane oxida-tion is mediated by methanogenic archaea living in syn-

trophy with sulfate reducers. Thermodynamically, sucha process would be favorable for methanogenic archaeaat very low hydrogen concentrations caused by the sul-

fate reducers (Hoehler et al., 1994). Nevertheless, theexact identity of the archaea, oxidizing methane throughan up to now unrecognized, enzymatic or non-enzymatic

pathway, still remains to be documented. Presently, it isnot resolvable whether this process is actually accom-plished by methanogenic archaea operating in reverse

(Elvert et al., 1999; Thiel et al., 1999; Pancost, 2000), bynovel obligate methanotrophic archaea (Hinrichs et al.,1999), or even by both types of organisms.It should be noted that the 13C-depleted isotope

values of the biomarkers may also be derived frommethanogenic archaea growing on highly 13C-depletedpore water carbon dioxide (Suess and Whiticar, 1989).

Table 3

Carbon isotopic compositions of selected alcohols obtained

from an anaerobic cold seep sediment and a methane-derived

carbonate of the SHUMAGIN sector

TV-G 97

(Mg-calcite)

TV-G 48

(13±16 cmbsf)

Alcohol d13C (%)

n-C14:0 ÿ96.5 n.m.

n-C15:0 ÿ97.4 n.m.

n-C16:1 ÿ109.4�0.3a n.m.b

n-C16:10 ÿ109.0�1.6 n.m.

n-C16:100 ÿ111.2�1.5 n.m.

n-C16:0 ÿ102.9�1.0 n.m.

n-C17:1 ÿ102.3�1.3 n.m.

Phytanol ÿ121.3�0.1 ÿ82.2n-C18:0 ÿ48.8 n.m.

Archaeol ÿ123.5�0.6 ÿ120.2a Indicated uncertainties are standard errors of means of two

measurements. Where no uncertainty is given, value represents

single measurement.b n.m., not measured, insu�cient amount for carbon isotope

analysis.

Table 4

%Corg and carbon isotopic compositions of sediments and

carbonates at cold seeps of the SHUMAGIN sector; d13Cvalues of carbonates are from Greinert (1998)

Sample Corg (%) d13C (%)

TV-G 97 (Mg-calcite) 0.61 ÿ48.7�2.7(n=24)

TV-GKG 40 (3±5 cmbsf) 1.67 ÿ39.2TV-GKG 40 (Mg-calcite/aragonite) n.a.a ÿ45.4�3.7

(n=9)

TV-G 43 (20±22 cmbsf) 0.72 ÿ25.5TV-G 48 (13±16 cmbsf) 0.58 ÿ41.8

a n.a., not analyzed.


Extensive oxidation of methane performed by any

hypothetical organism would clearly generate very lowd13C values of carbon dioxide and therefore, consideringthe large carbon isotopic fractionation associated with

carbonate reduction (Whiticar, 1996), methanogens grow-ing on this carbon source would also have quite lowd13C values. Due to the fact that we did not observe any13C-depleted biomarker diagnostic of methanotrophicbacteria (e.g. methylhopanoids; Summons and Jahnke,1990; Summons et al., 1994) and that other bacterialbiomarkers such as diploptene or squalene, which may

be assigned to aerobic methanotrophs, are much lessabundant than archaeal biomarkers, such an organismis not likely to be an aerobic methanotroph. On the

other hand, obligate methanotrophic archaea as pro-posed by Hinrichs et al. (1999) could account for verylow d13C values of carbon dioxide at the sulfate reduc-

tion±methane production boundary, which then couldbe used by methanogens for biosynthesis. Ultimately,such a combination of methanogenic and methano-trophic archaea, biosynthezising biomarkers indicative

of archaea and converting more or less e�ectivelymethane to carbon dioxide and back, would most likelyresult in highly 13C-depleted carbon isotope values and

various biomarker patterns as observed in our study.Isotope di�erences of over 50% between the methane

substrate and the lipids observed in other studies (Elvert

et al., 1999; Hinrichs et al., 1999) indicate that bothcarbon atoms of acetate used for the biosynthesis ofirregular isoprenoids and archaeol within the archaea

are derived from methane. Nevertheless, large isotopeshifts have also been found in investigations on methylo-trophic methanogenesis under non-limiting substrateconditions (Summons et al., 1998). This study revealed

that these organisms, anaerobically capable of dis-proportionating methylated C1-substrates such asmethanol, methyl amines, or methyl sul®des into meth-

ane and carbon dioxide, also biosynthesize ether lipids

in which the phytanyl moieties are depleted in 13C rela-tive to the substrate by up to 71.6%. Such isotope shiftsmight explain the highly 13C-depleted values found for

archaeal biomarkers here. However, considering the factthat methylated substrates are probably much lessabundant than methane, which is venting into the bot-

tom water column at this setting (Suess et al., 1998), andthat methylotrophic methanogenesis cannot account forthe high amounts of 13C-depleted pore water carbondioxide manifested in authigenic carbonates, methylo-

trophic methanogens may be present in either lowabundance or be absent.

4.2. Variability of irregular isoprenoids associated withanaerobic methane oxidation

The results presented here predominantly include 13C-depleted irregular C20 isoprenoids such as crocetane andits unsaturated counterparts. However, previous bio-marker studies have revealed di�erent biomarker pat-

terns (Elvert et al., 1999; Hinrichs et al., 1999; Thiel etal., 1999; Pancost et al., 2000), which might indicate thatmore archaeal assemblages than one are part of syn-

trophic consortia performing anaerobic methane oxida-tion (Table 5). This would explain why our samplesshow only a minor abundance of PMI compared to

crocetane, both of which have been reported before to beof similar concentration in samples analyzed for thepresence of anaerobic methane-oxidizing microbes

(Elvert et al., 1999; Thiel et al., 1999; Pancost et al.,2000). It seems that the consortia strongly depend onspeci®c conditions, characterizing the respective environ-ment. Critical conditioned may be for instance water

depth, temperature, degree of anoxia, and supply of su�-cient free methane, which are more suitable for one con-sortia and less so for another.

Table 5

Sequence of abundance of highly 13C-depleted biomarkers of archaeal origin from recent and ancient environments associated with

anaerobic methane oxidation (Cr: crocetane; PMI: 2,6,10,15,19-pentamethylicosane; Ar: archaeol; hyAr: hydroxyarchaeol(s); n.a.: not

analyzed)

Cold seep environment Cr PMI Ar hyAr Authors

Recent, gas hydrate vicinty (Eel river basin) None None Moderate Moderate Hinrichs et al. (1999)

Recent, gas hydrates, carbonates

(Hydrate ridge)

High High High Moderate Elvert et al. (1999);

Elvert (unpublished data);

Carsten Schubert (personal communication)

Fossil, carbonates (``Calcari a Lucina'') High High Nonea None Thiel et al. (1999)

Recent, mud volcanoes (Mediterranean ridge) High Moderate High High Pancost et al. (2000)

Recent, carbonates (Aleutian trench) High Minor Highb Nonec This study

a No archaeol, but detection of a 13C-depleted ether lipid containing a phytanyl moiety.b Archaeol not found at all stations studied (e.g. TV-GKG 40).c No free hydroxyarchaeol, but detection of monounsaturated archaeols.


In our samples, the relative abundance of crocetenescompared to crocetane strongly decreases from TV-GKG40 over TV-G 97 to TV-G 48 (Fig. 6). At station TV-G

48, crocetane comprises more than 90% of the overallamount of C20 isoprenoids, inferring that this cold seepenvironment may be di�erent in terms of the speci®cconditions than the other two environments. Speci®cally

TV-G 48 contains only a third as much organic carbonas TV-GKG 40 (Table 2). This may indicate that sub-sequent diagenetic processes could have decreased the

actual amount of Corg and that in the process crocetenescould have undergone transformation reactions such ashydrogenation. Thus, archaea once living could have

starved and biosynthesis via anaerobic methanotrophywould have ceased. Therefore, it is suggested that atstation TV-G 48 methane venting might be very low orno longer active. Such conditions would result in the

predominance of diagenetically stable crocetane whichis known to be preserved in ancient carbonates as old as20 million years (Thiel et al., 1999). In contrast, at station

Fig. 5. Reconstructed ion-current chromatogram of trimethylsilylated alcohol fraction obtained from a methane-derived carbonate

recovered at station TV-G 97 (archaeol: 2,3-di-O-phytanyl-sn-glycerol; *: contaminant).

Fig. 6. Relative abundance of crocetane and crocetenes at stations TV-GKG 40, TV-G 97, and TV-G 48.


TV-GKG 40, high amounts of crocetenes may indicatean environment with recent methane venting. Thus, theaccumulation of methane-consuming archaea within thesediments is an intense present day process at that sta-

tion also favoring carbonate precipitation. This in turnwould incrust living archaea and thereby would preserveevidence of methane emission from active ¯uid venting

such as found in the carbonate at station TV-G 97.

5. Conclusions

Sediments and authigenic carbonates from cold seeps

of the eastern Aleutian subduction zone provide evi-dence of archaea mediating anaerobic methane oxida-tion in syntrophic co-operation with sulfate reducers.This interpretation is based on the co-occurrence of

biomarkers speci®c for archaea such as crocetane, PMI,and archaeol with d13C values as low as ÿ130.3% PDBand n-alcohols, assigned to sulfate reducers, which reveal

a positive o�set in carbon isotope values of 10±25%.Several archaeal assemblages are probably involved inthe consumption of methane under anaerobic conditions

near cold seep environments. Each of these assemblagesmay be dependant on speci®c conditions generatedby the respective cold seep environment. Low methane

venting, causing starvation of methane-oxidizing archaea,may be characterized by a predominance of diageneticallystable crocetane, whereas high amounts of more labilecrocetenes seem to be indicative of recent ¯uid venting

and growing archaea. Therefore, crocetane is prob-ably the ultimate product which is preserved in sedi-ments and authigenic carbonates in ancient cold seep

environments.

Acknowledgements

We gratefully acknowledge M. Eek and M. McQuoidfor their help with the d13Corg measurements, and B.

Domeyer and A. Bleyer for laboratory support. We thankD. Schulz-Bull for providing laboratory space at the Son-derforschungsbereich 313 in Kiel and G. Petrick for

hydrogenation andmass spectrometric analysis of selectedhydrocarbon fractions. We also thank S. Grandel forcomments on an earlier version of this manuscript. Cri-

tical comments by R.D. Pancost, V. Thiel, and theassociate editor were highly appreciated and greatlyhelped to improve the ®nal manuscript. Financial support

was provided by the Bundesministerium fuÈ r Bildung undForschung (BMBF) through grant 03G0110A/B and theDeutsche Forschungsgemeinschaft (DFG) through theGraduiertenkolleg ``Dynamik globaler Kreislaufe im

System Erde'' and grants SU 114/7-1 and 7-2.

Associate EditorÐJ.S. Sinninghe DamsteÂ

References

Blair, N.E., Aller, R.C., 1995. Anaerobic methane oxidation on

the Amazon shelf. Geochimica et Cosmochimica Acta 59,

3707±3715.

Brassell, S.C., Wardroper, A.M.K., Thomson, I.D., Maxwell,

J.R., Eglington, G., 1981. Speci®c acyclic isoprenoids as

biological markers of methanogenic bacteria in marine sedi-

ments. Nature 290, 693±696.

Brooks, J.M., Kennicutt II, M.C., Fisher, C.R., Macko, S.A.,

Cole, K., Childress, J.J. et al., 1987. Deep-sea hydrocarbon

seep communities: evidence for energy and nutritional car-

bon sources. Science 238, 1138±1142.

Burns, S.J., 1998. Carbon isotopic evidence for coupled sulfate

reduction±methane oxidation in Amazon Fan sediments.

Geochimica et Cosmochimica Acta 62, 797±804.

Childress, J.J., Fisher, C.R., Brooks, M.C., Kennicutte II, R.,

Bidigare, A.E., Anderson, A.E., 1986. A methanotrophic

marine molluscan (Bivalva, Mytilidae) symbiosis: mussels

fueled by gas. Science 233, 1306±1308.

De Rosa, M., Gambacorta, A., 1986. Lipid biosynthesis in

archaebacteria. Systematic and Applied Microbiology 7, 278±

285.

Devol, A.H., Ahmed, S.I., 1981. Are high rates of sulphate

reduction associated with anaerobic oxidation of methane?

Nature 291, 407±408.

Elvert, M., Suess, E., Whiticar, M.J., 1999. Anaerobic methane

oxidation associated with marine gas hydrates: superlight C-

isotopes from saturated and unsaturated C20 and C25 irre-

gular isoprenoids. Naturwissenschaften 86, 295±300.

Freeman, K.H., Wakeham, S.G., Hayes, J.M., 1994. Predictive

isotopic biogeochemistry: hydrocarbons from anoxic marine

basins. Organic Geochemistry 21, 629±644.

Greinert, J., 1998. Rezente submarine Mineralbildungen:

Abbild geochemischer Prozesse an aktiven Fluidaustritts-

stellen im Aleuten-Cascadia Akkretionskomplex. PhD thesis,

Christian-Albrechts-University of Kiel, 190 pp.

Grimalt, J.O., AlbaigeÂ s, 1990. Characterzation of the deposional

environments of the Ebro Delta (western Mediterranean) by

the study of sedimentary lipids. Marine Geology 95, 207±224.

Hansen, L.B., Fisher, K., Fossing, H., Iversen, N., 1998.

Anaerobic methane oxidation in sulfate depleted sediments:

e�ects of sulfate and molydbate additions. Aquatic Micro-

bial Ecology 14, 195±204.

Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G.,

DeLong, E.F., 1999. Methane-consuming archaebacteria in

marine sediments. Nature 298, 802±805.

Hoehler, T.M., Alperin, M.J., Albert, D.B., Martens, C.S., 1994.

Field and laboratory studies of methane oxidation in an anoxic

marine sediments: evidence for a methanogenic±sulfate reducer

consortium. Global Biogeochemical Cycles 8, 451±463.

Holzer, G., OroÂ , J., Tornabene, T.G., 1979. Gas chromato-

graphic±mass spectrometric analysis of neutral lipids from

methanogenic and thermoacidophilic bacteria. Journal of

Chromatography 186, 795±809.

Huang, Y., Lockheart, M.J., Collister, J.W., Eglington, G.,

1995. Molecular and isotopic biochemistry of the Miocene

Clarkia Formation: hydrocarbons and alcohols. Organic

Geochemistry 23, 785±801.

Iversen, N., Jùrgensen, B.B., 1985. Anaerobic methane oxidation

rates at the sulfate±methane transition in marine sediments


from Kattegat and Skagerrak (Denmark). Limnology and

Oceanography 30, 944±955.

Koga, Y., Nishihara, M., Morii, H., Akagawa-Matsushita, M.,

1993. Ether polar lipids of methanogenic bacteria: structures,

comparative aspects, and biosyntheses. Microbiological

Reviews 57, 164±182.

Kohnen, M.E.L., Schouten, S., SinningheÂ Damste, J.S., de

Leeuw, J.W., Merritt, D.A., Hayes, J.M., 1992. Recognition

of paleobiochemicals by a combined molecular sulfur and

isotope geochemical approach. Science 256, 358±362.

Kulm, L.D., Suess, E., Moore, J.C., Carson, B., Lewis, B.T.,

Ritger, S.D. et al., 1986. Oregon subduction zone: Venting,

fauna, and carbonates. Science 231, 561±566.

Meyers, P.A., 1997. Organic geochemical proxies of paleocea-

nographic, paleolimnologic, and paleoclimatic processes.

Organic Geochemistry 27, 213±250.

Nichols, P.D., Franzmann, P.D., 1992. Unsaturated diether

phospholipids in the Antarctic methanogen Methano-

coccoides burtonii. FEMS Microbiology Letters 98, 205±208.

NiewoÈ hner, C., Hensen, C., Kasten, S., Zabel, M., Schulz, H.D.,

1998. Deep sulfate reduction completely mediated by anaerobic

methane oxidation in sediments of the upwelling area o�

Namibia. Geochimica et Cosmochimica Acta 62, 455±464.

Ourisson, G., Rohmer, M., Poralla, K., 1987. Prokaryotic

hopanoids and other polyterpenoid sterol surrogates. Annual

Reviews of Microbiology 41, 301±333.

Pancost, R.D., Sinninghe DamsteÂ , J.S., de Lint, S., van der

Maarel, M.J.E.C., Gottschal, J.C., The Medinaut Shipboard

Scienti®c Party, 2000. Biomarker evidence for widespread

anaerobic methane oxidation in Mediterranean sediments by

a consortium of methanogenic archea and bacteria. Applied

and Environmental Microbiology 66, 1126±1132.

Paull, C.K., Jull, A.J.T., Toolin, L.J., Linick, T., 1985. Stable

isotope evidence for chemosynthesis in an abyssal seep com-

munity. Nature 317, 709±711.

Reeburgh, W.S., 1980. Anaerobic methane oxidation: rate

depth distributions in Skan Bay sediments. Earth and Plane-

tary Science Letters 47, 345±352.

Risatti, J.B., Rowland, S.J., Yon, D.A., Maxwell, J.R., 1984.

Stereochemical studies of acyclic isoprenoidsÐXII. Lipids of

methanogenic bacteria and possible contributions to sedi-

ments. Organic Geochemistry 6, 93±104.

Ritger, S., Carson, B., Suess, E., 1987. Methane-derived authi-

genic carbonates formed by subduction-induced pore-water

expulsion along the Oregon/Washington margin. Geological

Society of American Bulletin 98, 147±156.

Robson, J.N., Rowland, S.J., 1993. Synthesis, chromatographic

and spectral chracterisation of 2,6,11,15-tetramethylhexa-

decane (crocetane) and 2,6,9,13-tetramethyltetradecane:

reference acyclic isoprenoids for geochemical studies.


Rohmer, M., Bouvier-Nave, P., Ourisson, G., 1984. Distribu-

tion of hopanoid triterpenes in prokaryotes. Journal of

General Microbiology 130, 1137±1150.

Rowland, S.J., Lamb, N.A., Wilkinson, C.F., Maxwell, J.R.,

1982. Con®rmation of 2,6,10,15,19-pentamethyleicosane in

methanogenic bacteria and sediments. Tetrahedron Letters

23, 101±104.

Schink, B., 1997. Energetics of syntrophic cooperation in

methanogenic degradation. Microbiology and Molecular

Biology Reviews 61, 262±280.

Schouten, S., van der Maarel, M.J.E.C., Huber, R., Sinninghe

DamsteÂ , J.S., 1997. 2,6,10,15,19-Pentamethylicosenes in Met-

hanolobus bombayensis, a marine methanogenic archaeon, and

in Methanosarcina mazei. Organic Geochemistry 26, 409±

414.

Sinninghe DamsteÂ , J.S., Schouten, S., 1997. Is there evidence

for a substantial contribution of prokaryotic biomass to

organic carbon in Phanerozoic carbonaceous sediments?


Sinninghe DamsteÂ , J.S., Schouten, S., van Vliet, N.H., Huber,

R., Geenevasen, T.A.J., 1997. A polyunsaturated irregular

acyclic C25 isoprenoid in a methanogenic archaeon. Tetra-

hedon Letters 38, 6881±6884.

Sprott, G.D., Ekiel, I., Dicaire, C., 1990. Novel, acid-labile,

hydroxydiether lipid cores in methanogenic bacteria. The

Journal of Biological Chemistry 265, 13735±13740.

Sprott, G.D., Dicaire, C.J., Choquet, C.G., Patel, G.B., Ekiel,

I., 1993. Hydroxydiether lipid structures in Methanosarcina

spp and Methanococcus voltae. Applied and Environmental

Microbiology 59, 912±914.

Suess, E., Carson, B., Ritger, S.D., Moore, J.C., Jones, M.L.,

Kulm, L.D. et al., 1985. Biological communities at vent sites

along the subduction zone o� Oregon. Biological Society of

the Washington Bulletin 6, 475±484.

Suess, E., Witicar, M.J., 1989. Methane-derived CO2 in pore

¯uids expelled from the Oregon subduction zone. Palaeo-

geography, Palaeoclimatology, Palaecology 71, 119±136.

Suess, E., Bohrmann, G., von Huene, R., Linke, P., Wallmann,

K.M., Lammers, S. et al., 1998. Fluid venting in the eastern

Aleutian subduction zone. Journal of Geophysical Reseaech

103, 2597±2614.

Summons, R.E., Jahnke, L.L., 1990. Identi®cation of the

methylhopanes in sediments and petroleum. Geochimica et

Cosmochimica Acta 54, 247±251.

Summons, R.E., Jahnke, L.L., Roksandic, Z., 1994. Carbon

isotopic fractionation in lipids from methanotrophic bac-

teria: relevance for interpretation of the geochemical record

of biomakers. Geochimica et Cosmochimca Acta 58, 2853±

2863.

Summons, R.E., Franzmann, P.D., Nicols, P.D., 1998. Carbon

isotopic fractionation associated with methylotrophic

methanogenesis. Organic Geochemistry 28, 465±475.

Thiel, V., Peckmann, J., Seifert, R., Wehrung, P., Reitner, J.,

Michaelis, W., 1999. Highly isotopically depleted iso-

prenoids: molecular markers for ancient methane venting.

Geochimica et Cosmochimica Acta 63, 3959±3966.

Tornabene, T.G., Langworthy, T.A., Holzer, G., Oro, J., 1979.

Squalenes, phytanes and other isoprenoids as major neutral

lipids of methanogenic and thermoacidophilic ``archae-

bacteria''. Journal of Molecular Evolution 13, 73±83.

Wallmann, K., Linke, P., Suess, E., Bohrmann, G., Sahling,

H., SchluÈ ter, M. et al., 1997. Quantifying ¯uid ¯ow, solute

mixing, and biogeochemical turnover at cold vents of the

eastern Aleutian subduction zone. Geochimica et Cosmochi-

mica Acta 61, 5209±5219.

Whiticar, M.J., 1996. Isotope tracking of microbial methane for-

mation and oxidation. In: Adams, D.D., Crill, P.M., Seitzinger,

S.P. (Eds.), Cycling of reduced gases in the hydroshpere, Mit-

teilungen. Internationale Vereinigung fuÈ r theoretische und

angewandte Limnologie, vol. 25. NaÈ gele u. Obermiller, E.

Schweizerbart'sche Verlagsbuchhandlung, pp. 39±54.


Documents

Archaea mediating anaerobic methane oxidation in deep-sea ...directory.umm.ac.id/Data Elmu/jurnal/O/Organic Geochemistry/Vol31...Archaea mediating anaerobic methane oxidation in deep-sea