Archaeal lipid biomarkers and isotopic evidence of anaerobic

Organic Geochemistry 34 (2003) 827–836
www.elsevier.com/locate/orggeochem
Archaeal lipid biomarkers and isotopic evidence of
anaerobic methane oxidation associated with gas
hydrates in the Gulf of Mexico
Chuanlun L. Zhanga,*, Richard D. Pancostb, Roger Sassenc,
Yaorong Qianc, Stephen A. Mackod
a
Department of Geological Sciences, University of Missouri, Columbia, MO 65211, USA
Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK
c
Geochemical and Environmental Research Group, Texas A&M University, College Station, TX 77845, USA
d
Department of Environmental Sciences, The University of Virginia, Charlottesville, VA 22903, USA
b
Received 6 May 2002; accepted 16 December 2002
(returned to author for revision 27 August 2002)
Abstract
Anaerobic oxidation of methane (AOM) occurs in the Gulf of Mexico gas hydrate systems. Here we show lipid
biomarker and isotopic evidence that archaea are involved in AOM. The estimated abundance of total archaeal lipids
ranges from 44.8 to 60.4 mg/g (dry sediment) in hydrate-bearing samples but is below detection limit in the hydrate-free
sample. The 13C values of archaeal lipids range from 69 to 99 % in hydrate-bearing samples. These results suggest
that biomass of archaea is significantly enhanced through AOM at the gas hydrate deposits. These data also support a
currently acknowledged mechanism of AOM mediated by a consortium of sulfate-reducing bacteria and archaea
observed in a variety of methane-rich marine settings. Anaerobic oxidation of oil hydrocarbons also occurs in the Gulf
of Mexico gas hydrate systems as shown by degradation of n-alkanes ( >C15) in the anoxic sediments. These processes
convert hydrocarbons to carbon dioxide and increase pore water alkalinity, which promote the precipitation of enormous volumes of authigenic carbonate rock depleted in 13C. This long-term geologic sequestration of carbon may
affect models of global climate change.
# 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
Gas hydrate occurs widely along continental margins
in the world’s oceans (Henriet and Mienert, 1998;
Kastner, 2001; Kvenvolden and Lorenson, 2001). Estimated methane carbon in the world’s gas hydrates is on
the order of 1019 g (Kvenvolden and Lorenson, 2001),
which is a vast potential energy resource. On the other
* Corresponding author at current address: Savannah River
Ecology Laboratory, The University of Georgia, Drawer E,
Aiken, SC 29802, USA Tel.: +1-803-725-5299; fax: +1-803725-3309.
E-mail address: zhang@srel.edu (C. L. Zhang).
hand, methane in gas hydrates may be released from the
subsurface into the water column and the atmosphere,
and may cause dramatic climate changes (Dickens et al.,
1995; Kennett et al., 2000; Dickens, 2001) because of the
potency of methane as a greenhouse gas (DeLong, 2000;
Kastner, 2001).
Anaerobic oxidation of methane (AOM) is estimated
to be equivalent to 5–20% of the net modern atmospheric methane flux (Valentine and Reeburgh, 2000).
Phylogenetic analyses in combination with lipid biomarkers and stable isotopes suggest that consortia of
sulfate-reducing bacteria and archaea work in syntrophy to mediate AOM in methane-rich sediments
(Hinrichs et al., 1999; Boetius et al., 2000; Orphan et al.,
0146-6380/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0146-6380(03)00003-2
828
C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
2001). Abundant gas hydrates have been found in the
Gulf of Mexico (Brooks et al., 1986; Roberts and Carney, 1997; Sassen et al., 1998). Geochemical evidence
indicates that AOM plays an important role in carbon
cycling and the development of biological communities
in the Gulf of Mexico (Sassen et al., 1993; Aharon and
Fu, 2000). By increasing alkalinity, AOM and concomitant bacterial sulfate reduction enhance carbonate
precipitation, which increases the seafloor stability
(Roberts and Aharon, 1994) and provides favorable
ground for the development of invertebrate communities in the deep ocean (Nelson & Fisher, 1995; Sassen
et al., 1994, 1998).
Recently, Lanoil et al. (2001) reported molecular
DNA evidence of diverse bacterial species but limited
archaeal species associated with gas hydrate, and Zhang
et al. (2002) reported the lipid biomarker and isotopic
evidence of AOM associated with sulfate-reducing bacteria in the Gulf of Mexico. Results of the present study
reveal extremely 13C-depleted archaeal lipid biomarkers,
thus supporting the hypothesis that AOM in the Gulf of
Mexico may be mediated by archaea in consortia with
sulfate-reducing bacteria (Zhang et al., 2002). Geochemical evidence also indicates that microbial oxidation of oil hydrocarbons occurs in the hydrate systems.
These microorganisms may impact global climate
change by oxidizing hydrocarbons and sequestering
enormous volumes of carbon dioxide as authigenic carbonate rock.
2. Material and methods
2.1. Sample collection
The Johnson Sea-Link (JSL) research submersible
was used to recover hydrate-associated sediment from
the Green Canyon (GC) 234 site (27 44.80 N and
91 13.30 W) at 543 m water depth. The site was initially identified as a fault related seismic amplitude zone
over shallow salt. A new outcrop of vein-filling gas
hydrate was discovered and sampled near a chemosynthetic community consisting of tube worms, mussels,
and clams (Fisher et al., 2000). Bacterial mats (Beggiatoa) covered the sediment surface over the exposed gas
hydrate. A sample of hemipelagic mud was collected
using the robot arm of the submersible by means of a
30-cm push core within 0.5 m of the outcropping gas
hydrate. The sediment contained decomposing nodules
of gas hydrate, was stained with crude oil, and smelled
of hydrogen sulfide. The invertebrate community was
avoided during push-core collection.
The JSL was also used to sample the newly discovered
GC 286 site (27 40.40 N and 90 49.70 W) at 839 m
water depth. Significant venting was not observed at the
site until research submersible operations disturbed a
fragile seal related to an isolated patch of living and
dead chemosynthetic clams. The minor disturbance of
the biologic seal instantly released free gas bubbles and
buoyant oil droplets that vented copiously to the water
column. Sediment at this site consisted of under-consolidated gassy oil-stained hemipelagic mud that smelled
of hydrogen sulfide, small nodules of gas hydrate, and
nodules and crusts of oil-stained authigenic carbonate
rock up to 10 cm across. The hydrate-bearing mud
sample was collected beneath a thick white bacterial mat
near the living chemosynthetic clams. Sample GC-cntrl
was a hydrate-free sample collected 15 m from sample
GC 286 during the same dive. These samples were not
intended for fine scale analysis, thus their precise depths
were unknown. All samples were frozen at 20 C
immediately upon recovery at the sea surface and kept
frozen until analysis.
2.2. Lipid extraction and biomarker identification
Lipid extraction followed the procedure of Pancost et
al. (2000). About 25 g of freeze-dried samples were
extracted via sonication in a sequence of solvent mixtures with increasing dichloromethane/methanol ratios:
0:1 three times, 1:1 three times, and 1:0 three times.
Total solvent was about three times the sediment
volume. Elemental sulfur was removed from the total
extracts by reaction with ca. 20 g of activated copper (24
h).
Total extracts were separated into acetone-soluble
and insoluble fractions (Pancost et al., 2000). The soluble component was further separated into apolar and
polar fractions on an alumina column (40 g activated
alumina). The apolar fraction was collected using pentane/dichloromethane (9:1, vol:vol) as the eluent, and
the polar fraction was collected using methanol as the
eluent. Phospholipid fatty acids in the polar fraction
have been determined in Zhang et al. (2002). Here we
report the abundances and 13C values of archaeal lipids
and hopanoids in the polar and apolar fractions.
Hydrocarbons (C15–C33) in the apolar fractions were
also determined.
Quantification of gas chromatography (GC)-amenable archaeal lipids and hopanoids was performed using
a combination of GC and GC-mass spectrometry (MS).
GC was conducted on a Carlo Erba HRGC 5300 Mega
Series instrument, equipped with a flame ionization
detector and a CP Sil-5CB (dimethylpolysiloxane, 0.12
mm df) fused silica capillary column (25 m, 0.32 mm id).
Samples were injected at 70 C using an on-column
injector and the oven was heated to 130 C at 20 C/min
then at 4 C/min to 300 C, at which the temperature
was held for 20 min. The carrier gas was H2. GC–MS
was performed using a Thermoquest Finnigan TRACE
GC interfaced to a Thermoquest Finnigan TRACE MS
operated with electron ionization at 70 eV and scanning
C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
a m/z range of 50–800. GC column and temperature
program were the same as those used in GC analyses,
but the carrier gas was He. Identification of archaeol,
sn-2-hydroxyarchaeol, tetrahymanol, bishomohopanol,
and C20 and C25 isoprenoids (e.g., crocetane: 2, 6, 11,
15-tetramethylhexadecane and PMI: 2, 6, 10, 15,
19-pentamethylicosane) were based on Pancost et al.
(2000) and references therein.
Identification of tetraethers was performed using high
performance liquid chromatograph (HPLC)-atmospheric pressure chemical ionization (APCI)-MS (Hopmans et al., 2000; Pancost et al., 2001) on a Waters 600
MS liquid chromatograph interfaced to a Finnigan
MAT TSQ 700 triple quadrupole mass spectrometer.
Separation was achieved using a Spherisorb NH2 column (4.6250 mm, 5 mm df) maintained at ambient
temperature and with tetraethers eluted isocratically
with a 1 ml/min flow rate of 99% hexane/1% iso-propanol for 25 min. The APCI-MS detection condition
includes 60 psi Nebulizer pressure, 400 C vaporizer
temperature, 200 C capillary temperature, 14 V, and a
7 ma corona.
2.3. Carbon isotopes of biomarkers
GC–C-IRMS was conducted using a Hewlett Packard
gas chromatograph interfaced via a Thermoquest Finnigan GC III combustion interface to a Thermoquest
Finnigan Delta S mass spectrometer. The GC was
equipped and operated as for the GC–MS analyses.
Measurements were performed in duplicate and values
are reported as parts per thousand (%) relative to the
V-PDB standard. Errors were typically less than 1%
based on duplicate measurements and internal or coinjected standards. These errors are somewhat larger
than those normally observed (ca. 0.3%) and reflect the
relatively high baseline and/or co-elution with small
background peaks.
3. Results
3.1. Lipid biomarkers
The two hydrate-bearing samples (GC 234 and GC
286) contain diagnostic biomarkers of the Archaea,
which include archaeol, sn-2-hydroxyarchaeol (Fig. 1),
and isoprenoidal hydrocarbons (e.g., crocetane and
PMI) (Table 1). These biomarkers, however, are below
the detection limits in the hydrate-free sample
(GC-cntrl). In GC 234 and GC 286, archaeol and sn-2hydroxyarchaeol are the most abundant of the quantified archaeal biomarkers (7.5–41.6 mg/g dry sediment).
Furthermore, sn-2-hydroxyarchaeol is about twice as
abundant as archaeol in GC 234 and about four times
more abundant than archaeol in GC 286 (Table 1). The
829
unsaturated crocetene and PMI are present at low (< 1
mg/g dry sediment) concentrations (Table 1).
Intact isoprenoid glycerol dialkyl glycerol tetraethers
(GDGTs), although not quantified, are also present in
GC 234 and GC 286, as indicated by the five major
peaks in the LC-MS chromatogram (Fig. 2). These are
all caldarchaeol skeletons with varying numbers of cyclic moieties (Fig. 2): an acyclic-acyclic GDGT (0–0); a
monocyclic-acyclic GDGT (0–1); a mixture of acyclicbicyclic and monocyclic-monocyclic (0–2/1–1) GDGTs;
a monocyclic-bicyclic GDGT (1–2); and crenarchaeol, a
GDGT comprised of a bicyclic tetraether and a tricyclic
tetraether in which one cyclic group is a cyclohexane
rather than cyclopentane moiety (Schouten et al., 2000;
see Pancost et al., 2001 for a detailed interpretation of
cold seep GDGT mass spectra). The acyclic caldarchaeol and the crenarchaeol are also present in GC-cntrl,
and thus appear to reflect pelagic archaeal input independent of the hydrate community. This is consistent
with the observation that acyclic caldarchaeol and crenarchaeol are commonly observed in pelagic sediments
(Schouten et al., 2000). Furthermore, the isotopic composition of the pelagic crenarchaeal biomarkers in
Mediterranean carbonate crusts has been shown to be
consistent with a pelagic source (Bouloubassi and others, personal communication).
Nonisoprenoidal dialkyl glycerol diethers, which are
inferred to derive from Bacteria rather than Archaea
(Pancost et al., 2001), are present in GC 234 and GC
286 but not in GC-cntrl. In GC 234, at least six such
compounds exist and apparently comprise two pseudohomologous series. However, the abundances of these
diethers are too low to permit precise identification. In
GC 286, two diethers (diether 1 and diether 2) are predominant (Table 1). Based on previously reported
occurrences of diethers (Pancost et al., 2001) and the
available mass spectrometric data, diether 1 probably
has a cyclic group (perhaps a cyclopropane) in one of
the chains and diether 2 has a cyclic group in both
chains.
Pentacyclic triterpenoids, including bishomohopanol
and tetrahymanol, are present in GC 234 and GC 286
but not in GC-cntrl (Fig. 1, Table 1). Bishomohopanol
is diagnostic of aerobic bacteria (Rohmer et al., 1984).
In hydrocarbon seeps, it may be derived from a H2Soxidizing chemoautotroph, such as Beggiatoa (Pancost
and Sinninghe Damste´, in press). The particularly high
abundance of bishomohopanol at GC 234 (8.7 mg/g dry
sediment) is consistent with the wide occurrence of
Beggiatoa mats at this site. The source of tetrahymanol
is less clear (see Sinninghe Damste´ et al., 1995), but this
compound is common in other cold seep sediments
where it has been tentatively ascribed to ciliates grazing
exclusively on prokaryotes (Pancost and Sinninghe
Damste´, in press). Another possible source of these pentacyclic triterpenoids is methylotrophic bacteria, which
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C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
Fig. 1. Partial gas chromatogram of TMS derivatives of polar lipids, including bacterial and archaeal biomarkers, in GC 286. Stars
denote steroids, solid circles denote unidentified non-isoprenoidal diethers, and C24–C32 denote n-alkanols. Note that both the monoand di-TMS ethers of sn-2-hydroxyarchaeol are shown on the chromatogram using the derivatization method in this study.
may be present in water column above hydrocarbon
seeps (LaRock et al., 1994).
Petroleum hydrocarbons were analysed to assess the
nature of seeping hydrocarbons and the extent of biodegradation. At all sites, the hydrocarbons are represented by n-alkanes, pristane, phytane, steranes, and
hopanes with thermally mature stereochemical distributions typical for petroleum. In all three samples (Fig. 3),
a strongly elevated baseline is associated with an unresolved complex mixture (UCM), which suggests that the
oil hydrocarbons are heavily biodegraded (Sassen et al.,
1994), perhaps under anaerobic conditions suggested by
the strong smell of H2S. Based on the abundance of
n-alkanes relative to the UCM, hydrocarbons at GC 234
and GC cntrl are most severely biodegraded (Fig. 3). In
GC 234, the inferred degradation was so pronounced
that individual n-alkanes could not be distinguished
from the complex mixture even in the m/z 85 mass
chromatogram. This is consistent with early observations of oil hydrocarbon degradation at this site (Sassen
et al., 1994). Sassen et al. (1994, 2001) attribute this differential degradation to the rate of hydrocarbon flux at
these sites. At a high fluxes (such as GC 286), degradation of hydrocarbons is less significant because oil
bypasses the sediment and enters the water column (Sassen et al., 1994). At a slow flux (such as GC cntrl and GC
234), the hydrocarbons reside in sediment and are less
likely to escape the effect of microbial degradation.
In all cases, microbial degradation of hydrocarbons
5C15 appears to be an important process in the Gulf of
Mexico gas hydrate systems. However, this process may
not involve archaea as indicated by the absence of their
lipid biomarkers in GC-cntrl. Other organisms, such as
sulfate-reducing bacteria, may be responsible for degradation of these hydrocarbons. This is supported by
bacterial fatty acids and carbon isotopic data (Zhang et
al., 2002). In addition, degradation of n-alkanes by sulfate-reducing bacteria has been reported in several culture studies (Rueter et al., 1994; Zengler et al., 1999; So
and Young, 1999; Kroop et al., 2000).
3.2. Stable carbon isotopes
Carbon isotopic compositions were determined for
selected biomarkers (Table 1). Others were not determined because of either co-elution of peaks, the presence of the UCM, or low abundance of the biomarker.
In GC 234, the 13C value of archaeol is 85% and that
C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
Table 1
Abundance and carbon isotopic composition of ether lipid
biomarkers and hopanoids in the gas hydrate samples from the
Gulf of Mexico
Lipid
biomarkers
GC 286
GC 234
mg/g 13C (%)
mg/g 13C (%)
Ether lipid biomarkers
Archaeol
sn-2 Hydroxyarchaeol
Crocetane+phytane
Cr:2
PMI:3
Diether 1 (Nonisoprenoid)
Diether 2 (Nonisoprenoid)
7.5
30.8
n.a.a
0.5
0.7
2.6
1.4
18.8
41.6
n.a.
n.a.
n.a.
n.a.
n.a.
Hopanoids
Tetrahymanol
Bishomohopanol
1.7 63
n.a. n.a.
a
98
89
38
13
Cdepletedb
99
83
74
85
82
n.a.
n.a.
n.a.
n.a.
n.a.
5.6 n.d.
8.7 n.d.
n.a.=These compounds were present but not available for
isotopic analysis due to co-elution or other problems (see
below).
b
Small and co-eluting peaks. Co-elution and the presence of
unresolved complex mixture made it impossible to obtain an
accurate measurement, but the ratio trace clearly indicated a
profound depletion in 13C relative to normal marine organic
matter. Other co-eluting peaks include crocetane (Cr), Cr:1,
and PMI (pentamethylicosane):4. The number following the
colon sign indicates the numbers of double bonds.
831
of sn-2-hydroxyarchaeol is 82% (Table 1). In GC 286,
the 13C values of archaeol and sn-2-hydroxyarchaeol
are even more depleted in 13C, 98 and 89%, respectively (Table 1). In the same sample, the 13C value of
triunsaturated PMI (PMI:3) is 99%, whereas that of
crocetane plus phytane is 38% (Table 1). Crocetane
likely derives from a methane oxidizer with 13C-depleted
isotopic compositions, whereas phytane largely derives
from the phytol moiety of chlorophyll with 13C-enriched
isotopic compositions (Pancost and Sinnighe Damste´, in
press). The 13C value for the composite phytane+crocetane peak will be intermediate between the two endmember values such that the crocetane 13C value is
likely to be significantly lower than 38%. This suggestion is consistent with the 13C depletion (13C values
are <45%) in diunsaturated crocetene (Cr:2)
(Table 1), which may be derived from the same source
as crocetane. The two unidentified diethers (diether 1
and diether 2) have 13C values of 83 and 74%,
respectively (Table 1). The 13C value for tetrahymanol
is 63% (Table 1), which supports its origin from
aerobic methanotrophs or sulfide-oxidizers living on
13
C-depleted CO2 from methane oxidation (Pancost &
Sinninghe Damste´, in press).
The 13C values of the oil n-alkanes (C15–C33) range
from 27 to 31% (Fig. 4). These values are consistent
with bulk analyses of oil hydrocarbons (Sassen et al.,
1994). Moreover, they are similar to those of C2–C4
Fig. 2. HPLC/APCI/MS partial total ion current traces of glycerol dialkyl glycerol tetraethers (GDGTs) in cold seep sediments from
(a) the Gulf of Mexico (GC234) and (b) Amsterdam mud volcano in the Eastern Mediterranean Sea (Pancost et al., 2001), which
serves as a reference for the Gulf of Mexico sample. Numbers in the chromatograph refer to the number of cyclopentane moieties in
the two biphytane components of the GDGTs (c) and ‘P’ refers to a pelagic crenarchaeal GDGT (Schouten et al., 2000) as discussed in
the text.
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C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
Fig. 3. Total ion current (TIC) chromatograms of apolar (hydrocarbon) fractions from GC 286 (a), GC cntrl (b), and GC 234 (c).
Insets show the m/z 57 mass chromatogram of each sample, illustrating specifically the n-alkane distributions. No n-alkanes could be
identified in GC 234 due to extensive biodegradation. Solid circles denote n-alkanes, letter ‘‘o’’ crocetenes, and letter ‘‘H’’ hopanes.
hydrocarbons previously observed in the Gulf of Mexico (Brooks et al., 1986; Sassen et al., 1998), possibly
indicating a common source. The 13C values of nalkanes decrease with increasing carbon numbers
(Fig. 4), as commonly observed in subsurface reservoirs
of the Gulf of Mexico (e.g., Sassen et al., 2001). The
13C values could not be determined for hopanes and
steranes due to their low abundances and co-elution
with UCM.
4. Discussion
Pancost et al. (2001) compared the ratios of crocetane, PMI, and hydroxyarchaeol to archaeol among
different mud volcanoes. The results suggest that even
at a single site, multiple archaeal species may be present (Pancost et al., 2001). A summary is provided on
the diversity and isotopic compositions of archaea in
several sedimentary environments where detailed
C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
833
Fig. 4. Carbon isotopic compositions of n-alkanes in GC 286.
organic geochemical analyses have been published
(Table 2).
The distribution of archaeal lipids in the Gulf of
Mexico hydrate sites is similar to that observed at the
California Margin (Hinrichs et al., 1999, 2000; Orphan
et al., 2001) and the Hydrate Ridge (Elvert et al., 1999;
2001), but different from that observed in the Mediterranean mud volcanos (Pancost et al., 2000, 2001)
(Table 2). In the Gulf of Mexico and Hydrate Ridge, for
example, archaeol and sn-2-hydroxyarchaeol are most
abundant and concentrations of PMI and crocetane are
low (Table 2). In the Mediterranean, however, crocetane
is as abundant as or even more abundant than archaeol
in some samples (Table 2). In addition, in the Mediterranean samples, hydroxyarchaeol is almost always
less abundant than archaeol, while the opposite is
observed for the Gulf of Mexico and other seep environments. Furthermore, sn-3-hydroxyarchaeol is present
at some Mediterranean sites but has not been reported
for any other cold seep setting. Sprott et al. (1993)
observed that sn-2-hydroxyarchaeol is mainly produced
by Methanosarcina spp. whereas sn-3-hydroxyarchaeol
is found in Methanosaeta concilii. This suggests that
archaeal lipids in methane-rich environments may be
derived from a variety of archaea closely related to the
methanogens. Finally, the presence of tetraether lipids in
the Gulf of Mexico confirms the wide occurrence of these
compounds as they have been found in the Mediterranean (Pancost et al., 2001), the Black Sea (Schouten et
al., 2001; Thiel et al., 2001; Michaelis et al., 2002), and
the hydrothermal sediments in the Guaymas Basin
(Teske et al., 2002). These biomarkers, however, can
derive from archaea that may or may not participate in
AOM (Pancost et al., 2001; Teske et al., 2002). For
example, In the Mediterranean, a sample from the
Amsterdam seep site showed elevated contribution of
GDGTs with 1 or 2 cyclopentane rings (GDGT #=0–1,
0–2/1–1, or 1–2; Fig. 2), which are depleted in 13C (54
to 77%), suggesting a source of methane-consuming
archaea. On the other hand, the GDGT with three
cyclopentane rings in the same sample was enriched in
13
C (19%) and indicate the contribution from nonthermophilic crenarchaeota (peak ‘‘P’’ in Fig. 2; Pancost
et al., 2001). The strong similarity in GDGT profile of
the Gulf of Mexico sample with the Amsterdam sample
of the Mediterranean (Fig. 2) suggest a similar contribution of archaeal lipids from both methane-consuming archaea and crenarchaeotal sources. This has to
wait for a definitive answer, however, because of the
unavailability of the carbon isotope data in this study
(see below).
Archaeal biomarkers often have extremely depleted
13C values in gas hydrate or cold seep enviornments. At
the California Margin or Hydrate Ridge sites, the 13C
values of sn-2-hydroxyarchaeol are as low as 128%
and the 13C values of PMI are as low as 129 %
(Table 2). In the Mediterranean and the Gulf of Mexico
cold seeps, low 13C values of 99 to 107% are
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C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
Table 2
Comparison of archaeal lipid biomarkers, carbon isotopes, and microbial species potentially involved in anaerobic methane oxidation
in methane-rich sedimentary basins
Archaeal biomarker
Archaeol (mg/g)
(13C,%)
sn-2-hydroxyarchaeol (mg/g)
(13C,%)
P
Crocetane (mg/g)
(13C,%)
P
PMI (mg/g)
(13C,%)
Potential Archaea involved
in methane oxidation
Potential Bacteria involved
in methane oxidation
Gulf of Mexico
Hydrate Ridge
California Marginj
Mediterranean
Black Sea
7.5–18.5
(85 to 98)
30.8–41.6
(82 to 89)
>0.5
(13C-depleted)
>0.7
(99)
ANME-1a
ANME-2a
Methanosaeta spp.a
Proteobacteriaa
Actinobacteriaa
Firmicutesa
8b
(114)
8b
(133)
0.02–1.06c*
(63 to 126)c
0.02–3.01c*
(37 to 128)c
ANME-2b
0.4–2.9d
(100 to 119)d
0.1–7.3d
(101 to 128)d
0.3d
(119)d
1.1d
(76 to 129)d
ANME-1e
ANME-2e
0.4–52.9f
(20 to 96)f
0.1–f10.2f
(57 to 105)f
2.0–55.4f
(45 to 89)f
0.5–2f
(34.2 to 107)f
ANME-1g
new Archaeal spp.g
not available
(88)h
not available
(90)h
not available
(95 to 107)h,i
not available
(71 to 106)i
ANME-1h
Desulfosarcinab
Desulfococcusb
Desulfosarcinae
Desulfococcuse
Proteobacteriag
Desulfosarcinah
Desulfococcush
a
Lanoil et al., 2001.
Boetius et al., 2000. Desulfosarcina and Desulfococcus both belong to the Proteobacteria.
c
Elvert et al., 1999, 2001. *Calculated based on% organic carbon (Corg) and mg biomarker/g Corg (Elvert et al., 2001).
d
Hinrichs et al., 1999, 2000.
e
Orphan et al. 2001.
f
Pancost et al., 2000, 2001.
g
Aloisi et al., in press. Please note that it is unknown whether ANME-2 is absent in Mediterranean sediments because the respective primers were not used in the analysis.
h
Michaelis et al., 2002. In addition, biphytanes in Back Sea (excluding biomarkers from nonthermophilic crenarchaeota) have 13C
values ranging from 58% in anoxic water (Schouten et al., 2001) to 92% in microbial mats (Michaelis et al., 2002) to 97% in
carbonate rocks (Thiel et al., 2001).
i
Thiel et al., 2001.
j
Include Eel River Basin and Santa Barbara Basin.
b
observed (Table 2). Extremely 13C-depleted archaeal
biomarkers have also been observed in water column,
sediments and carbonate crusts of the Back Sea
(Schouten et al., 2001; Thiel et al., 2001; Michaelis et al.,
2002) as well as ancient methane seep deposits (Thiel et
al., 1999).
The low 13C values of archaeal biomarkers indicate
that the sources of the archaeal lipids derive their carbon from methane, which is the most 13C-depleted
carbon source in the natural environment. At GC 234,
the 13C value of vent methane is about 49% (Sassen
et al., 1998). At GC 286, the 13C value of vent methane
is about 63% (Sassen, personal communication). The
difference in 13C of methane may explain the difference
in 13C of archaeal lipids between these two sites
(Table 1). The 13C values of methane at the Hydrate
Ridge site range from 62 to 72% (Elvert et al., 1999)
and those of methane at the California Margin site
range from 50 to 69% (Hinrichs et al., 1999). There
is not enough information to relate these values to isotopic variation of archaeal lipid biomarkers at these
sites. However, the maximum fractionation between
methane and archaeal lipids appears to be greater in the
California Margin or the Hydrate Ridge (> 59%) than
in the Gulf of Mexico (maximum 37%). These results
suggest that different fractionation kinetics or pathways
may exist for AOM in different geological settings.
Although our understanding of the mechanisms of
AOM is limited, the process has profound implications.
For example, in the Gulf of Mexico, this process is perhaps responsible for the accumulation over geologic
time of enormous volumes of carbonate on the ocean
floor, and development of chemosynthetic communities,
which are favored by the hard ground from carbonate
precipitation (Sassen et al., 1994). Future research
should quantify AOM process and develop models to
evaluate the relative volume of methane fixed as authigenic carbonate rock by microbial process and the
volume of methane that vents into the atmosphere. Such
insight is necessary to better understand the roles that
microbes play in mediating carbon cycling in methanerich environments and perhaps will shed light on the coevolution of microbial and geological processes through
the Earth’s history.
C.L. Zhang et al. / Organic Geochemistry 34 (2003) 827–836
5. Conclusions
Archaeal lipid biomarkers in the Gulf of Mexico
hydrate samples are dominated by archaeol and sn-2
hydroxyarchaeol and have extremely low 13C values
(69 to 99%). These results are consistent with
13
C-depleted lipid biomarkers of sulfate-reducing bacteria and suggest that AOM is mediated by consortia of
archaea and sulfate-reducing bacteria. The distribution
of archaeal lipids in the Gulf of Mexico is similar to that
observed in California Margin and Hydrate Ridge;
however, different isotopic fractionations between substrate methane and archaeal and bacterial lipids suggest a
diversity of microorganisms, reaction kinetics and pathways. Extensive oxidation of oil hydrocarbons also
occurs in the Gulf of Mexico, which not only adds to the
accumulation of enormous volumes of carbonate from
AOM but also contributes to the complexity of carbon
cycling mediated by different microbial processes.
Acknowledgements
Comments from two anonymous reviewers, Dr. KaiUwe Hinrichs, and the Editor enhanced the quality of
the manuscript. We thank Dr. Marcus Elvert for sharing unpublished data of archaeal biomarker abundance
from the Hydrate Ridge and comments to improve the
manuscript. Support for this research was provided by
the National Science Foundation Biocomplexity Program (CLZ), the Petroleum Research Fund (CLZ), the
University of Missouri Prime Fund (CLZ), and the
Royal Society of the United Kingdom (RDP).
Associate Editor— G. Wolff
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