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Organic Geochemistry 31 (2000) 1175±1187
www.elsevier.nl/locate/orggeochem

Archaea mediating anaerobic methane oxidation in
deep-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
a
GEOMAR, Research Center for Marine Geosciences, Wischhofstr. 1-3, D-24148 Kiel, Germany
School of Earth and Ocean Sciences, University of Victoria, PO Box 3050, British Columbia, V8W 2Y2, Canada

b

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 in 13C with values of d13C as low as ÿ130.3% PDB. The process of anaerobic methane 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-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 composition; 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
* Corresponding author. Fax: +1-49-431-600-2928.
1

Present address: Max-Planck-Institute for Marine Microbiology, Celsiussor. 1, 28359 Bremen, Germany. Fax: +1-49421-2028-690; e-mail: melvert@mpi- bremen.de.
E-mail address: melvert@geomar.de (M. Elvert).

environments, preferentially along convergent continental
margins. At the cold seeps, ¯uid venting supports
benthic communities and generates authigenic carbonates 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.
PII: S0146-6380(00)00111-X

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

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 carbon dioxide by organisms in tissues or precipitation of
carbonates from oversaturated microenvironments
causes a strong carbon isotope shift towards 13C-depleted values often cited as evidence for methane oxidation;
e.g. mytilid mussels, vestimentiferan and pogonophoran
tube worms, and carbonates are depleted in 13C to values
as low as ÿ77% 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 conditions (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 isopranylglycerol diethers such as 2,6,11,15-tetramethylhexadecane
(crocetane), 2,6,10,15,19-pentamethylicosane (PMI), 2,3-diO-phytanyl-sn-glycerol (archaeol), 2-O-3-hydroxyphytanyl3-O-phytanyl-sn-glycerol (sn-2-hydroxyarchaeol), and 3O-3-hydroxyphytanyl-3-O-phytanyl-sn-glycerol (sn-3hydroxyarchaeol) with highly depleted carbon isotope
values as low as ÿ123.8% 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 isoprenoids and/or isopranylglycerol diethers, both traditionally 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 cooperation 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 chemoautotrophic 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 from ÿ57.1 to ÿ64.3% 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 Corg were 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 measurements 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 di€raction 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
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/dichloromethane (1:2, v/v), and twice with 50 ml of dichloromethane. For the carbonate, 25 g were washed with

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

1177

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.

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 decantation 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 dichloromethane (alcohols), and (IV) 10 ml of methanol/dichloromethane (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 50 ml n-hexane with H2 in fusible glass
ampoules 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 stored
at room temperature for 1 h. Finally, the samples were

directly analyzed by gas chromatography±mass spectrometry (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(trimethylsilyl)tri¯uoroacetamide; Supelco), and heated in
closed glass ampoules for 2 h at 80 C. Following evaporation 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
0.25 mm; J&W Scienti®c) in a Carlo Erba 5160 gas chromatograph equipped with an on-column injector and a
¯ame ionization detector. The samples were injected at
60 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/min

and ®nally kept at 310 C for 25 min. The carrier gas was
H2 at a ¯ow rate of 2.5 ml/min. Concentrations for each

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

compound were determined by adding internal standards (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 below s=2 mg/g Corg except for
compounds with more than 50 mg/g Corg (below =10
mg/g Corg). Loss of material during analysis was monitored by adding a recovery standard (n-C40) prior to the
overall analytical procedure. In general, typical recoveries 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 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 of m/z 40±600 for hydrocarbons and m/z 40±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: 285 C) and subjected to
the same temperature program given for GC measurements (see Section 2.4.).

d13Corg 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 reproducibility 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 biomarkers 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 sector inside a canyon which crosscuts the third accretionary 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 di€usive ¯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