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 precipitates which 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 archaea working in syntrophic co-operation with sulfate-reducing bacteria. Diagnostic lipid biomarkers of archaeal origin include 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 the biomarkers are strongly depleted in13C with values ofd13C 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 ofÿ45.4 andÿ48.7%. This suggests that methane-oxidizing
archaea have accumulated within the microbial community, which is active at the cold seep sites. The dominance of crocetane 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 ¯uid venting and active archaea. Comparison with other biomarker studies suggests that various archaeal assemblages might 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 inhabiting sediments 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 supports benthic 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). Growth and metabolism of the associated vent macrofauna are based on a chemoautotrophic food chain which starts with the microbially mediated oxidation of reduced compounds, 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. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 1 1 - X
* 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: melvert@mpi- bremen.de.
oxidation to carbon dioxide occurs either by aerobic (Childress et al., 1986) or anaerobic processes (Suess and Whiticar, 1989), with the latter still not completely understood. However, subsequent incorporation of car-bon dioxide by organisms in tissues or precipitation of carbonates from oversaturated microenvironments causes a strong carbon isotope shift towards13
C-deple-ted values often ciC-deple-ted as evidence for methane oxidation; e.g. mytilid mussels, vestimentiferan and pogonophoran tube worms, and carbonates are depleted in13C 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 evidence supporting 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 isotope values as low asÿ123.8%PDB from various anaerobic settings. 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 methanogenic archaea, with such extremely low carbon isotope values prompted these authors to suggest that either certain methanogens themselves are involved in the consumption of methane, operating in reverse in syntrophic co-operation with sulfate reducers (Elvert et al., 1999; Thiel et al., 1999; Pancost, 2000), or that until now unknown methanogens within archaeal lineages evolved to being capable 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 the eastern Aleutian subduction zone, adjacent to the Aleutian deep-sea trench. These cold seeps are among the deepest observed (4800 m) and therefore, being far removed from the photic zone, are well suited to study chemoauto-trophic processes because very little metabolizable particulate organic matter reaches this depth. We especially examined the abundance, carbon isotope values, and signi®cance of biomarkers diagnostic of anaerobic methane oxidation. Moreover, we evaluated the variability of the speci®c biomarkers found in this study compared to those observed at other cold seep environments.
2. Materials and methods
2.1. Study area
The study area at the eastern Aleutian subduction zone, referred to as SHUMAGIN sector, was surveyed and 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, and the general sampling strategy have been described earlier by Suess et al. (1998). Widespread methane venting was observed along the entire margin and speci®cally o SHUMAGIN at the intersection of accretionary ridges with tensional faults. These faults occur in canyons landward of the deformation front at water depths around 4800 m and are the result of oblique subduction of the Paci®c plate underneath the Aleutian arc. Colonies of typical seep macrofauna and authigenic carbonate crusts were found. The seep biota consists of bacterial mats, pogonophorans, vestimentiferans, and large colonies of bivalves. The carbon isotope composition of tissues from the seep fauna ranged fromÿ57.1 toÿ64.3%and
thus identi®es methanotrophy as the dominant carbon metabolizing pathway (Suess et al., 1998). Similarly, for authigenic carbonates, d13C values between
ÿ42.7 and
ÿ50.8%were reported (Greinert, 1998), suggesting that a
mixture of biogenic methane, via anaerobic oxidation, and carbon dioxide supplied by sulfate-reducing bacteria was the ultimate carbon source of the authigenic mineralogies.
2.2. Sediment, pore water, and carbonate analysis
Contents of Corgwere determined from the carbonate
free, dried, and homogenized sediment material using a Carlo Erba Nitrogen Analyzer 1500. For carbonate removal, 3 g of wet sediment were treated over night with 15 ml of 10% HCl. After freeze-drying, samples were homogenized by using an agate ball mill. Standard deviations of this method were 0.02%. Sulfate measure-ments were carried out by ion chromatography and detection by conductivity. Sulfate values are reported in mM and were calibrated with IAPSO-standard seawater. Using duplicate measurements, standard deviations were within 1.5%. The authigenic carbonates were identi®ed by standard X-ray diraction analysis. The speci®c calcite sample selected for extraction of biomarkers was a high Mg-calcite (Greinert, 1998).
2.3. Extraction, chromatographic separation, hydrogenation and derivatization
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 were extracted 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). Chromatographic separation 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) was removed by passing the fractions over separate short columns ®lled with 1 g of activated copper powder using
n-hexane as eluent.
Hydrogenation of hydrocarbon fractions was carried out by saturation of 50mln-hexane with H2in fusible glass
ampoules pre-®lled with 10 mg of PtO2and subsequent
adding of 50ml of sample (1
2aliquot inn-hexane). After
¯ushing with H2, the ampoules were closed and stored
at 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 nitrogen to near dryness, mixed with 100 ml BSTFA (bis(tri-methylsilyl)tri¯uoroacetamide; Supelco), and heated in closed glass ampoules for 2 h at 80C. Following
eva-poration to near dryness under nitrogen, the residue was taken up in n-hexane and subsequently analyzed by mass spectrometry.
2.4. Gas chromatography (GC)
Gas chromatographic analyses of hydrocarbons were performed using a 30 m apolar DB-5 fused silica capillary column (0.25 mm internal diameter (ID), ®lm thickness 0.25mm; 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 at 60C. After a 1 min hold time, the oven temperature was
raised to 140C at 10C/min, then to 310C at 5C/min
and ®nally kept at 310C for 25 min. The carrier gas was
compound were determined by adding internal stan-dards (3-methylnonadecane, 2-methylicosane, 5b (H)-cholane) with known concentrations prior to GC analysis and are reported in mg/g Corg. Standard deviations for
single compounds are belows=2mg/g Corg except for
compounds with more than 50mg/g Corg(below=10
mg/g Corg). Loss of material during analysis was
mon-itored by adding a recovery standard (n-C40) prior to the
overall analytical procedure. In general, typical recov-eries were 70±80% relative ton-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 gas chromatograph interfaced to a Fisons MD 800 mass spectrometer operated in electron impact (EI-) mode at 70 eV (cycle time 0.9 s, resolution 1000) with a mass range ofm/z40±600 for hydrocarbons andm/z40±800 for 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 thickness 0.25 mm; J&W Scienti®c) using He as carrier gas. The samples were injected in splitless mode (hot needle technique; injector temperature: 285C) and subjected to
the same temperature program given for GC measure-ments (see Section 2.4.).
2.6. Stable carbon isotope analysis
Carbon isotope compositions of hydrocarbons and alcohols were determined using a coupled gas chromato-graph±combustion±isotope ratio mass spectrometer (GC±C±IRMS). The mass spectrometer (Finnigan MAT 252) was connected to a Varian 3300 GC equipped with a 50 m CP-Sil 5 CB-MS (0.25 mm ID, 0.4mm stationary phase; Chrompack). The carrier gas was He at a ¯ow rate of 1.5 ml/min. The samples were on-column injected at 60C and after 1 min the oven temperature was raised
to 140C at 10C/min, then to 230C at 3C/min, and
®nally to 310C at 2C/min at which it was held for 35 min.
Carbon isotope ratios are reported in thednotation as per mil (%) deviation from the Pee Dee Belemnite standard
(PDB). Internal standards (5b(H)-cholane and n-C36for
hydrocarbons; n-C20 and n-C36 for alcohols) of known
isotopic 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 for the complex mixtures analyzed here (up to1.6%) due
to factors such as co-elution or signal to background ratio. 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 atoms during derivatization according to Huang et al. (1995).
d13C
org was measured by elemental analysis±isotope
ratio mass spectrometry (EA±IRMS) using a Carlo Erba Elemental Analyzer connected via a ConFloTM
interface to the Finnigan MAT 252. Analytical repro-ducibility for duplicate runs was below0.1%.
3. Results
Three sediments and one carbonate sample from four dierent stations at cold seeps were analyzed for bio-markers indicative of anaerobic methane oxidation. The active seep sites along with extensive carbonate crusts and methane anomalies of the bottom water column were 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±SSE direction, which probably provide ¯uid pathways and focus diusive ¯uid venting. Pore water analysis showed that sediments analyzed were well within the sulfate reduction zone which starts right below the sediment surface (Fig. 2). Sulfate concentrations reach 10 mM at station TV-G 43 (35 cmbsf) and concentrations <5 mM at 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 near the suboxic±anoxic interface.
3.1. Hydrocarbons: structures, abundance, and isotopic compositions
Two sediment samples (stations GKG 40 and TV-G 48) and the carbonate sample (station TV-TV-G 97) were found to contain signi®cant amounts of crocetane (Robson and Rowland, 1993; Elvert et al., 1999) and to a minor degree PMI and pentamethylicosenes (Brassell et al., 1981; Rowland et al., 1982; Schouten et al., 1997). These compounds were absent from sediments at station TV-G 43. Three representative partial gas chromato-grams of the hydrocarbon fractions obtained from the anoxic 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 Damste et al. (1997) in the methanogenic archaeon
the marine methanogenic archaeonMethanolobus 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 high amounts in anaerobic sediments and in carbonates associated with recent and ancient methane seeps and methane-rich mud volcanoes (Elvert et al., 1999; Thiel et al., 1999; Pancost et al., 2000). Based on structural and isotope evidence, crocetane was linked to archaea involved in anaerobic methane oxidation. In contrast to these samples containing crocetane or PMI, such bio-markers are absent from the sediment at station TV-G 43 (Fig. 3c). Sulfate reduction at this location is less intensive compared to station TV-G 48 (Fig. 2) and therefore does probably not favour near-surface anae-robic methane oxidation. Furthermore, the shape of the sulfate pro®le appears diusion-controlled, suggesting that the actual sulfate reduction site is deeper in the core than the location of the sample. The samples obtained from stations TV-GKG 40, TV-G 48, and TV-G 97 also show the presence of unsaturated hydrocarbons eluting slightly ahead and after crocetane. On the basis of their mass 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 irregular C20compound (see Section 2.3). The exact positions of
the double bond(s) within each compound were not determined.
The concentrations of hydrocarbons present in the anaerobic sediments ranged from 3 up to 830mg/g Corg,
with the main compounds being crocetane and its unsa-turated counterparts (Table 1). The highest amounts of crocetane (2400mg/g Corg) and other irregular isoprenoids
were obtained from the carbonate extract, which in turn
showed only trace amounts of non-isoprenoid com-pounds (Fig. 3a). Apparently, the carbonate initially precipitated 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 C30hopanoid
diploptene (hop-22(29)-ene) (Fig. 3b). Sources known for the latter biomarker are various bacteria such as cyanobacteria, ammonia-oxidizing bacteria, and methylo-trophic and methanomethylo-trophic bacteria (Rohmer et al., 1984; Ourisson et al., 1987; Summons et al., 1994) but it has yet not been observed in anaerobic bacteria. Squalene was only present at station TV-GKG 40 and absent at all other stations. In contrast, diploptene was found in all sediments which also contained irregular C20and C25
isoprenoids. Its relative abundance was moderate at station TV-GKG 40 and low at the stations TV-G 48 and 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 C25isoprenoids, indicative of archaea, with isotope
values betweenÿ93.5 andÿ130.3%(group I) and second,
non-speci®c bacterial biomarkers such as squalene and diploptene 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 indicate an origin from organisms which utilize methane anae-robically. In contrast, the higher d13C values obtained
for compounds of group II might indicate an origin from as yet undetected anaerobic bacteria growing not on methane but still on a13C-depleted carbon source or
re¯ect oxidation of methane to some degree by aerobic methanotrophs. Oxygen may be transported periodically into the shallow anoxic habitat by the pumping activity of clams such asCalyptogenasp. known to be living at
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-G 43. 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 alcohol fraction obtained from the carbonate sample at station TV-G 97 is shown in Fig. 5. Ether lipids other than archaeol were detected only in trace amounts and were assigned to be monounsaturated archaeols as identi®ed earlier in the Antarctic methanogen Methanococcoides burtonii (Nichols and Franzmann, 1992). These com-pounds are probably breakdown products of hydroxy-archaeols (e.g. bothsn-2 andsn-3 isomers) which were found to be acid-labile even under mild conditions (Sprott et al., 1990). Intact hydroxyarchaeols as detected in cultures of members of the familiesMethanococcales
andMethanosarcinales(Koga et al., 1993; Sprott et al., 1993) were completely absent. In addition to ether lipids, signi®cant amounts of phytanol and n-alcohols fromn-C14:0ton-C18:0with a maximum atn-C16:1were
found in the carbonate sample at station TV-G 97, but were 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 reveal highly depleted13C 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 a common source for these compounds from organisms which use methane as their carbon source for lipid bio-synthesis. The ®nding of13C-depleted archaeol at
deep-sea cold seeps is in agreement with surveys of archaeal 16S 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 of methanogens which co-exist with isotopically13C-depleted
biomarkers such as archaeol and sn-2-hydroxyarchaeol. Furthermore, highly13C-depleted archaeol has also been
identi®ed in sediments from mud volcanoes on the Mediterranean Ridge, another important convergent margin setting (Pancost et al., 2000). The carbon isotope value of phytanol (3,7,11,15-tetramethlhexadecanol) at station TV-G 97 is similar to the irregular isoprenoids and archaeol (ÿ120%) and thus, links it to anaerobic
methane-oxidizing archaea at this station. However, at station TV-G 48 thed13C value of phytanol is distinctly
dierent (ÿ82.2%). This lowered isotope signal may be
caused by a small but unknown portion of phytanol derived from organisms dierent than anaerobic meth-ane-oxidizing archaea. A plausible source appears to be phytanol via hydrogenation of chloro-phyll-derived phytol, which would certainly show an isotope value much more enriched in13C relative to archaeal-derived
phytanol.
Intracrystallinen-alcohols released from the Mg-calcite at station TV-G 97 in the range of n-C14:0 to n-C17:1
showd13C values between
ÿ96.5 andÿ111.2%(Table 3).
These values document that these biomarkers are derived from an organism which plays a signi®cant role in the net oxidation of methane at this location. However, care
should be taken for the assignment of an archaeal origin because these lipids have never been reported as mem-brane components in methanogenic archaea (see Koga et al., 1993 for a review). In general, short-chainn-alcohols are thought to be of marine origin. They may derive from the degradation of wax esters biosynthesized by zoo-plankton and marine invertebrates (Grimalt and AlbaigeÂs,
1990). Nevertheless, given their extremely low d13C
values in the samples analyzed here, an in situ produc-tion of these compounds seems very probable. One possible source of such 13C-depleted alcohols may be
sulfate reducers using not methane but an intermediate substrate with still isotopically depleted organic carbon. This was previously suggested from the presence of
13C-depleted C
15:0iso- andanteiso-, and C16:0mid-chain
branched alcohols from a Miocene limestone (Thiel et al., 1999). These compounds showed distinctly higher
d13C 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 our samples, alcohols with branched carbon chains were absent. Nevertheless, the clear distinction of isotope values obtained for phytanol and archaeol compared to then-alcohols likewise indicates dierent source organ-isms for these compounds. Therefore, an origin of these
n-alcohols from sulfate reducers living in syntrophy with archaea and using an isotopically depleted type of organic carbon (e.g. 13C-depleted pore water carbon
dioxide or acetate) is a plausible interpretation.
3.3. Bulk organic carbon
The high concentrations of irregular C20and C25
iso-prenoids at station TV-G 48 represent roughly 0.1% of the total organic carbon. This extraordinary amount of biomarkers diagnostic of archaea growing on methane is also re¯ected in the carbon isotopic composition of the total organic matter (ÿ41.8%; Table 4). Considering
that lipids only comprise a very small part of the sedi-mentary Corg (Sinninghe Damste and Schouten, 1997),
this value is extraordinarily low compared to normal marine organic matter. Because substantial contributors to sedimentary Corgsuch as proteins and carbohydrates
are enriched in13C compared to lipids (Meyers, 1997),
the d13C value observed either indicates contributions
from considerable quantities of13C-depleted lipids other
than irregular isoprenoids or that proteins and carbo-hydrates also contain methane-derived carbon at this station. In contrast, the less sulfate-depleted sediment at station TV-G 43 shows an isotope value ofÿ25.5%for
total Corg. This most likely indicates a nutritional
path-way dierent from that of anaerobic methane oxidation of the microbial assemblage at this station or that most of the organic matter was derived from pelagic inputs.
Table 1
Concentrations of hydrocarbons (inmg/g Corg) obtained from anaerobic cold seep sediments and a methane-derived carbon-ate of the SHUMAGIN sector
TV-G 97
Hydrocarbon [mg/g Cog]
n-C17 110 13 24 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, andn-C19)
TV-G 97
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.
4. Discussion
4.1. Evidence for anaerobic methane oxidation at cold seeps
Anaerobic methane oxidation in marine sediments is unambiguously identi®able at the sulfate reduction± methane production boundary (Iversen and Jùrgensen, 1985; Blair and Aller, 1995; Burns, 1998; NiewoÈhner et al., 1998). Radiotracer experiments, stable carbon isotopes, and methane and sulfate mass balances point to methane oxidation at the expense of sulfate (e.g. Reeburgh, 1980; Devol and Ahmed, 1981; Iversen and Jùrgensen, 1985; Blair and Aller, 1995; Burns, 1998; NiewoÈhner et al., 1998). Indeed, laboratory and ®eld investigations indicate
bacterium, it is obvious considering the thermodynamic controls that only one of the partners could gain metabolic useful energy from the reaction and that the other partner has 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 methane and reduce sulfate at the same time and thus obtains the entire energy from this transformation. Recently, Hinrichs et al. (1999) suggested, based on ribosomal RNA and biomarker analysis from the same sample, that newly, up to now unidenti®ed methanogens may have evolved within archaeal lineages for which methane oxidation is the predominant or even exclusive metabolic pathway (methanotrophic archaea). Nevertheless, the identity of the terminal electron acceptor of the overall process has not 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 Aleutian subduction zone whereas less sulfate-depleted sediments (TV-G 43) showed no such evidence. Moreover, sidering the above discussion and the geochemical con-ditions at the Aleutian subduction zone (bottom water methane anomalies, pore water sulfate concentrations, authigenic carbonates), biomarkers (irregular isoprenoids, ether lipids, n-alcohols), and carbon isotopes reported here, it seems very likely that anaerobic methane oxida-tion is mediated by methanogenic archaea living in syn-trophy with sulfate reducers. Thermodynamically, such a process would be favorable for methanogenic archaea at very low hydrogen concentrations caused by the sul-fate reducers (Hoehler et al., 1994). Nevertheless, the exact identity of the archaea, oxidizing methane through an up to now unrecognized, enzymatic or non-enzymatic pathway, still remains to be documented. Presently, it is not resolvable whether this process is actually accom-plished by methanogenic archaea operating in reverse (Elvert et al., 1999; Thiel et al., 1999; Pancost, 2000), by novel 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 from methanogenic archaea growing on highly13C-depleted
pore water carbon dioxide (Suess and Whiticar, 1989).
n-C16:100 ÿ111.21.5 n.m.
n-C16:0 ÿ102.91.0 n.m.
n-C17:1 ÿ102.31.3 n.m.
Phytanol ÿ121.30.1 ÿ82.2
n-C18:0 ÿ48.8 n.m.
Archaeol ÿ123.50.6 ÿ120.2
a Indicated uncertainties are standard errors of means of two measurements. Where no uncertainty is given, value represents single measurement.
b n.m., not measured, insucient amount for carbon isotope analysis.
Table 4
%Corg and carbon isotopic compositions of sediments and carbonates at cold seeps of the SHUMAGIN sector; d13C values of carbonates are from Greinert (1998)
Sample Corg(%) d13C (%)
TV-G 97 (Mg-calcite) 0.61 ÿ48.72.7 (n=24) TV-GKG 40 (3±5 cmbsf) 1.67 ÿ39.2 TV-GKG 40 (Mg-calcite/aragonite) n.a.a
ÿ45.43.7 (n=9) TV-G 43 (20±22 cmbsf) 0.72 ÿ25.5 TV-G 48 (13±16 cmbsf) 0.58 ÿ41.8
Extensive oxidation of methane performed by any hypothetical organism would clearly generate very low
d13C values of carbon dioxide and therefore, considering
the large carbon isotopic fractionation associated with carbonate reduction (Whiticar, 1996), methanogens grow-ing on this carbon source would also have quite low
d13C values. Due to the fact that we did not observe any 13C-depleted biomarker diagnostic of methanotrophic
bacteria (e.g. methylhopanoids; Summons and Jahnke, 1990; Summons et al., 1994) and that other bacterial biomarkers such as diploptene or squalene, which may be assigned to aerobic methanotrophs, are much less abundant than archaeal biomarkers, such an organism is 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 very lowd13C values of carbon dioxide at the sulfate
reduc-tion±methane production boundary, which then could be 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 eectively methane to carbon dioxide and back, would most likely result in highly13C-depleted carbon isotope values and
various biomarker patterns as observed in our study. Isotope dierences of over 50%between the methane
substrate and the lipids observed in other studies (Elvert et al., 1999; Hinrichs et al., 1999) indicate that both carbon atoms of acetate used for the biosynthesis of irregular isoprenoids and archaeol within the archaea are derived from methane. Nevertheless, large isotope shifts have also been found in investigations on methylo-trophic methanogenesis under non-limiting substrate conditions (Summons et al., 1998). This study revealed that these organisms, anaerobically capable of dis-proportionating methylated C1-substrates such as
methanol, methyl amines, or methyl sul®des into
meth-ane and carbon dioxide, also biosynthesize ether lipids in which the phytanyl moieties are depleted in13C
rela-tive to the substrate by up to 71.6%. Such isotope shifts
might explain the highly13C-depleted values found for
archaeal biomarkers here. However, considering the fact that methylated substrates are probably much less abundant than methane, which is venting into the bot-tom water column at this setting (Suess et al., 1998), and that methylotrophic methanogenesis cannot account for the high amounts of 13C-depleted pore water carbon
dioxide manifested in authigenic carbonates, methylo-trophic methanogens may be present in either low abundance or be absent.
4.2. Variability of irregular isoprenoids associated with anaerobic methane oxidation
The results presented here predominantly include13
C-depleted irregular C20isoprenoids such as crocetane and
its unsaturated counterparts. However, previous bio-marker studies have revealed dierent biobio-marker pat-terns (Elvert et al., 1999; Hinrichs et al., 1999; Thiel et al., 1999; Pancost et al., 2000), which might indicate that more archaeal assemblages than one are part of syn-trophic consortia performing anaerobic methane oxida-tion (Table 5). This would explain why our samples show only a minor abundance of PMI compared to crocetane, both of which have been reported before to be of similar concentration in samples analyzed for the presence of anaerobic methane-oxidizing microbes (Elvert et al., 1999; Thiel et al., 1999; Pancost et al., 2000). It seems that the consortia strongly depend on speci®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 highly13C-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 a13C-depleted ether lipid containing a phytanyl moiety. b Archaeol not found at all stations studied (e.g. TV-GKG 40).
In our samples, the relative abundance of crocetenes compared to crocetane strongly decreases from TV-GKG 40 over TV-G 97 to TV-G 48 (Fig. 6). At station TV-G 48, crocetane comprises more than 90% of the overall amount of C20isoprenoids, inferring that this cold seep
environment may be dierent in terms of the speci®c conditions than the other two environments. Speci®cally TV-G 48 contains only a third as much organic carbon as TV-GKG 40 (Table 2). This may indicate that sub-sequent diagenetic processes could have decreased the
actual amount of Corgand that in the process crocetenes
could have undergone transformation reactions such as hydrogenation. Thus, archaea once living could have starved and biosynthesis via anaerobic methanotrophy would have ceased. Therefore, it is suggested that at station TV-G 48 methane venting might be very low or no longer active. Such conditions would result in the predominance of diagenetically stable crocetane which is known to be preserved in ancient carbonates as old as 20 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).
TV-GKG 40, high amounts of crocetenes may indicate an environment with recent methane venting. Thus, the accumulation of methane-consuming archaea within the sediments is an intense present day process at that sta-tion also favoring carbonate precipitasta-tion. This in turn would incrust living archaea and thereby would preserve evidence 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-operaoxida-tion with sulfate reducers. This interpretation is based on the co-occurrence of biomarkers speci®c for archaea such as crocetane, PMI, and archaeol withd13C values as low as
ÿ130.3%PDB
andn-alcohols, assigned to sulfate reducers, which reveal a positive oset in carbon isotope values of 10±25%.
Several archaeal assemblages are probably involved in the consumption of methane under anaerobic conditions near cold seep environments. Each of these assemblages may be dependant on speci®c conditions generated by the respective cold seep environment. Low methane venting, causing starvation of methane-oxidizing archaea, may be characterized by a predominance of diagenetically stable crocetane, whereas high amounts of more labile crocetenes 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. McQuoid for their help with the d13C
org measurements, and B.
Domeyer and A. Bleyer for laboratory support. We thank D. Schulz-Bull for providing laboratory space at the Son-derforschungsbereich 313 in Kiel and G. Petrick for hydrogenation and mass spectrometric analysis of selected hydrocarbon fractions. We also thank S. Grandel for comments on an earlier version of this manuscript. Cri-tical comments by R.D. Pancost, V. Thiel, and the associate editor were highly appreciated and greatly helped to improve the ®nal manuscript. Financial support was provided by the Bundesministerium fuÈr Bildung und Forschung (BMBF) through grant 03G0110A/B and the Deutsche Forschungsgemeinschaft (DFG) through the Graduiertenkolleg ``Dynamik globaler Kreislaufe im System Erde'' and grants SU 114/7-1 and 7-2.
Associate EditorÐJ.S. Sinninghe DamsteÂ
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