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Polar and neutral isopranyl glycerol ether lipids as biomarkers of archaea in near-surface sediments from the Nankai Trough
Organic Geochemistry Organic Geochemistry 37 (2006) 1643–1654 www.elsevier.com/locate/orggeochem
Polar and neutral isopranyl glycerol ether lipids as biomarkers of archaea in near-surface sediments from the Nankai Trough Masahiro Oba
a,*
, Susumu Sakata a, Urumu Tsunogai
b
a
Institute for Geo-Resources and Environment, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8567, Japan b Division of Earth and Planetary Sciences, Graduate School of Science, Hokkaido University, N10 W8 Kita-Ku, Sapporo, Hokkaido 060-0810, Japan Available online 9 November 2006
Abstract The molecular and carbon isotopic compositions of polar isopranyl glycerol ether lipids, which are direct indicators of viable archaea, and neutral isopranyl glycerol ether lipids, which are derived from polar lipids via hydrolysis, in near-surface sediments from a methane seep in the Nankai Trough (off central Japan) were investigated. Procedures for extracting, separating and derivatizing polar and neutral ether lipids for detection using gas chromatography were first examined with one sediment sample and a cultivated methanogen. For all sediment samples, archaeol and hydroxyarchaeol were detected in both the polar and neutral ether lipid fractions. Acyclic and cyclic biphytanes were also detected in both types of lipid fractions after treatment with HI/LiAlH4 for ether cleavage and alkylation. The d13C values of archaeol, sn-2-hydroxyarchaeol, and sn-3-hydroxyarchaeol in the sample from 0.82 m below the seafloor were lower than 100& relative to PDB, indicating that diverse living methanotrophic archaea are present in the seep sediments. Biphytanes released from polar ether lipids in the same sample were less depleted in d13C (71& to 36&). The wide range of d13C values suggests that the biphytanes were derived not only from methanotrophic but also from non-methanotrophic archaea, and that the relative contributions of the methanotrophic and non-methanotrophic archaea differed, depending on the biphytane compound. The vertical profiles and d13C values of the neutral ether lipids were similar to those of the intact polar ether lipids, suggesting that neutral ether lipids derived from fossil archaea in the samples had mainly been lost by the time of sampling. 2006 Elsevier Ltd. All rights reserved.
1. Introduction The major constituents of the cell membranes of archaea are isopranyl glycerol diether and/or tetraether lipids, which are distinct from the acyl ester lipids found in bacteria (Kates, 1978; Langworthy *
Corresponding author. Present address: Institute of Geology and Paleontology, Tohoku University, Sendai 980-8578, Japan. Fax: +81 22 795 6668. E-mail address: [email protected] (M. Oba).
et al., 1982). Because isopranyl glycerol ether lipids are unique to archaea, they have been used as biomarkers of such organisms in many studies (e.g., Martz et al., 1983; Pauly and van Vleet, 1986; Nichols et al., 1987; Reed et al., 2002). Notably, molecular and stable carbon isotopic compositions have been measured in recent years to predict archaeal activity in methane hydrate layers and methane seeps, providing information such as magnitude and spatial distribution of biomass, dominant species and the types of activity of the species (e.g.,
0146-6380/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2006.09.002
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Hinrichs et al., 1999; Pancost et al., 2001a; Blumenberg et al., 2004; Orcutt et al., 2005; Elvert et al., 2005). In living archaeal cells, most ether lipids contain polar head groups with phosphate or sugar moieties, or both (Nishihara and Koga, 1987) and are referred to as intact polar lipids (IPLs). By analogy with the polar acyl ester lipids of bacteria, which are known to lose polar head groups via enzymatic hydrolysis upon cell death or cell lysis (White et al., 1979), the detection of intact polar ether lipids is inferred to indicate the presence of living or potentially viable biomass rather than fossil archaeal biomass (Sturt et al., 2004; Biddle et al., 2006). On the other hand, neutral ether lipids, which are hydrolysates of intact polar ether lipids, are considered to be biomarkers of dead or lysed cells. Although many studies of isopranyl glycerol ether lipids in sediments have been reported, none have clearly differentiated intact polar ether lipids from neutral ether lipids. Here, we report methods for the efficient analysis of sediment samples for intact polar and neutral ether lipids using gas chromatography. We then apply the methods to near-surface core sediments
from a cold methane seep in the Nankai Trough and determine the distribution of intact polar and neutral ether lipids near the seafloor. In the Nankai Trough, many cold seeps have been found (Le Pichon et al., 1987, 1992; Ashi et al., 1996), but the information available on lipid biomarkers in the seep sediments has been limited to the presence of acyclic isoprenoid hydrocarbons characteristic of methanogens (Sakata et al., 2004). Therefore, an additional objective was to show the distribution and characteristics of archaea in seep sediments in the Nankai Trough. 2. Materials and methods 2.1. Sediment samples Core samples were collected from the seafloor of the southeastern slope (3404.410 0 N, 13747.511 0 E; 610 m water depth) of Dai-ni Tenryu Knoll in the eastern part of the Nankai Trough off central Japan (Fig. 1) during the 2001 cruise of the R/V Hakureimaru No.2 (operated by the Metal Mining Agency of Japan, now Japan Oil, Gas and Metals National
Fig. 1. Map showing sampling site for core sediments from the seafloor in the Nankai Trough, off central Japan.
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Corporation) conducted by the Japanese Ministry of Economy, Trade and Industry as part of the Deep Sea Survey Technologies for Natural Resources project. In this area, the distribution of cold seep zones has been well investigated. A previous submersible cruise at the Dai-ni Tenryu Knoll showed well developed carbonate crust exposures and large Calyptogena sp. colonies associated with a methane seep (Kuramoto and Joshima, 1998). Methane hydrate bottom-simulating reflectors are widespread in the area and hydrate dissociation and methane seepage are considered to have resulted from fault activity and uplift. A 120 cm long core, consisting largely of clay and clayey silt, was obtained with a gravity corer. After collection, the samples were transported to the laboratory, freeze-dried and pulverized. 2.2. Methanogen cell samples Methanothermobacter thermoautotrophicus DH was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). It was grown in a mineral salts medium. Cultivation was carried out at 55 C for 2 d without agitation in a 1.3-l serum bottle containing 500 ml medium under an atmosphere of 2 atm H2/CO2 (80:20, v/v) as energy and carbon sources. Growth stage was determined by monitoring the increase in optical density at 600 nm (OD600). Whole cells at the stationary phase (OD600 = 0.13) were harvested by centrifugation and immediately freeze-dried. Approximately 30 mg of dry cells were collected from each bottle. 2.3. Extraction, separation and derivatization Total lipids in sediment samples (12–25 g) and cell material (100 mg) were ultrasonically extracted using the method of Bligh and Dyer, modified by Nishihara and Koga (1987) to use trichloroacetic acid-acidified solvent: methanol/chloroform/water with 10% trichloroacetic acid (2:1:0.8, v/v/v), for 30 min in centrifuge tubes, which allows re-extraction of the residue after centrifugation and recovery of supernatant. The supernatant from the first extraction and the following six re-extractions were combined. After phase separation of the combined supernatants by adding chloroform and water to obtain a final methanol/chloroform/water ratio of 1:1:0.9 (v/v/v), the organic phase was recovered
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and washed with methanol/distilled water (5:4, v/v) to remove trichloroacetic acid. The extracts were dried over Na2SO4 and concentrated by evaporation under reduced pressure. To separate major compound classes, chromatographic separation on a silica gel column (0.6 g of pre-activated silica, 63–200 mesh) was carried out. Ten fractions were obtained by elution with the following: 6 ml hexane (F1); 3 ml hexane/toluene 3:1 v/v (F2); 3 ml hexane/ethyl acetate 19:1 v/v (F3); 3 ml hexane/ethyl acetate 9:1 v/v (F4); 3 ml hexane/ethyl acetate 17:3 v/v (F5); 3 ml hexane/ethyl acetate 4:1 v/v (F6); 3 ml hexane/ethyl acetate 3:1 v/v (F7); 3 ml hexane/ethyl acetate 1:1 v/v (F8); 3 ml ethyl acetate (F9); 10 ml methanol (F10). Residual organic compounds in the column were ultrasonically extracted twice with methanol/chloroform (1:2, v/v) and combined with the F10 fraction. Each fraction was divided into aliquots for the three different procedures as follows. One aliquot was heated with N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) in pyridine at 75 C for 2 h. In this aliquot (hereafter referred to as the unhydrolyzed aliquot) OH groups were silylated. Another aliquot was heated in 1 ml chloroform/ methanol/concentrated HCl (1:10:1, v/v/v) at 100 C for 2 h, followed by extraction with petroleum ether and chloroform. The organic phase was silylated with BSTFA as above. In this aliquot (hereafter referred to as the hydrolyzed aliquot), IPLs were hydrolyzed to neutral lipids and the OH groups were silylated. The third aliquot of F4–F10 only was refluxed in 56% HI (in H2O by weight) at 100 C for 20 h, followed by extraction with petroleum ether. The organic phase was heated with LiAlH4 at 100 C for 2 h. In this aliquot (hereafter referred to as the alkylated aliquot), ether lipids were converted to alkanes via alkyl iodides so that molecular structures and carbon isotopic compositions of the biphytanes in tetraethers could be determined using gas chromatography (GC). 2.4. Lipid analysis Lipids were identified by using an Agilent 6890N gas chromatograph equipped with an on-column injector and interfaced to an Agilent 5973N mass selective detector mass spectrometer, operated with an ionizing electron energy of 70 eV and scanned from m/z 50–650 with a scan time of 0.8 s. A fused silica CP–Sil 5CB capillary column (60 m, 0.25 mm
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i.d., 0.25 lm film thickness) was used, with helium as carrier gas. The oven temperature was raised from 70 C to 160 C at 20 C/min, then to 310 C at 4 C/min and finally held constant for 50 min. Compound identifications were based on mass spectra and retention times. Individual lipids were quantified by using a Hewlett–Packard 6890 gas chromatograph equipped with on-column injector and flame ionization detector. GC conditions (injector, column, carrier gas, temperature programme) were identical to those for GC-mass spectrometry (GC-MS) analysis. Quantification was based on comparison of peak areas with those of external standards (n-alkanes). Compound-specific carbon isotopic compositions were determined by using a Finnigan MAT 252 mass spectrometer coupled to a Hewlett–Packard 5890 series II. GC conditions were as described above. Values of d13C (&, relative to Pee Dee Belemnite standard, PDB) were established by inserting
multiple peaks of pure CO2 via a separate line so as to prevent coleution with peaks from the GC, and by determining d13C values of the CO2 peaks on separate runs by injecting n-alkane standards into the GC. Carbon added by trimethylsilylation was corrected for, with an error of < ± 1&. 3. Results and discussion 3.1. Methods for separation and detection of ether lipids Fig. 2 shows the total ion chromatogram of the unhydrolyzed aliquot of F4 from one Nankai Trough core sediment sample [0.82 m below seafloor (mbsf)]. Peak Ia was assigned to 2,3-di-O-phytanylsn-glycerol diether (archaeol) by comparing the mass spectrum with literature data (Teixidor et al., 1993). Among the unhydrolyzed aliquots, archaeol was detected only in that from F4 (Table 1). Fig. 3 shows
Fig. 2. Total ion chromatogram of unhydrolyzed aliquot of F4 from a Nankai Trough core sediment (0.82 mbsf).
Table 1 Distribution of isopranyl glycerol ethers in the fractions of total lipid extract from a Nankai Trough sediment (0.82 mbsf) separated by silica gel F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
++ – –
– – –
– – ++
– – +
– – –
– – –
– – –
(2) Free and polar isopranyl glycerol ethers in the hydrolyzed aliquots Archaeol – – – ++ Monophytanyl glycerol ether – – – – Hydroxyarchaeol – – – –
tr tr –
– ++ –
– + –
tr ++ –
tr + –
++ ++ –
(1) Free isopranyl glycerol ethers in the unhydrolyzed aliquots Archaeol – – – Monophytanyl glycerol ether – – – Hydroxyarchaeol – – –
Symbols, ++, +, tr, and – indicate major, minor, trace amounts, and not detected, respectively.
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Fig. 3. Total ion chromatogram of the hydrolyzed aliquot of F10 from a Nankai Trough core sediment (0.82 mbsf).
the total ion chromatogram of the hydrolyzed aliquot of F10 from the same sample. Archaeol was also detected in this hydrolyzed aliquot (peak Ib in Fig. 3). Among the hydrolyzed aliquots, archaeol was detected mostly in the F4 and F10 fractions (Table 1). From these results, we find that neutral archaeol (i.e., archaeol in the form of a neutral lipid) and polar archaeol (i.e., archaeol in the form of IPLs) can be analyzed in the unhydrolyzed aliquot of F4 and the hydrolyzed aliquot of F10, respectively. Fig. 4 shows the total ion chromatogram of the unhydrolyzed aliquot of F6, also from the sediment sample from 0.82 mbsf. A large peak at 72 min was
assigned to hydroxyarchaeol by comparing the mass spectrum with literature data (Hinrichs et al., 2000). Hydroxyarchaeol has the same core structure as archaeol but contains an additional hydroxyl group on the third carbon of the phytanyl moiety etherlinked to either the second (IV; sn-2-hydroxyarchaeol) or third (V; sn-3-hydroxyarchaeol) glycerol carbon. Because mass spectra of these two possible isomers resemble each other, being characterized by dominant fragments at m/z 143, 341, and 517 (Hinrichs et al., 2000), assignment of this peak to one of the isomers on the basis of its mass spectrum alone is difficult. Among the unhydrolyzed aliquots,
Fig. 4. Total ion chromatogram of the unhydrolyzed aliquot of F6 from a Nankai Trough core sediment (0.82 mbsf).
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hydroxyarchaeol was detected in the F6 and F7 fractions (Table 1). On the other hand, hydroxyarchaeol was not detected in the hydrolyzed aliquots of F6, F7, or any other fraction. Remarkably, 1-O-phytanyl glycerolether (peak II in Fig. 3) and 2-O-phytanyl glycerolether (peak III in Fig. 3), which were not detected in any unhydrolyzed fractions, were identified in the hydrolyzed aliquots of F6, F8, and F10 (Table 1). According to Koga et al. (1993), hydroxyarchaeols lose the hydroxy-bearing phytanyl chain by mild acid treatment to yield monophytanyl glycerolethers. Therefore, polar sn-2- and sn-3-hydroxyarchaeol are converted to 1-O- and 2-O-phytanyl glycerolether, respectively, in the process of hydroly-
sis with chloroform/methanol/concentrated HCl. Moreover, the results confirm that, under the hydrolysis conditions used, all hydroxyarchaeols were converted to monophytanyl glycerol ethers, because the total molar concentrations of monophytanyl glycerol ethers in hydrolyzed F6 were approximately equal to the molar concentrations of the neutral hydroxyarchaeols in unhydrolyzed F6. From these results, we find that neutral hydroxyarchaeols can be analyzed in the unhydrolyzed aliquots of F6 and F7, and that polar hydroxyarchaeols (detected as monophytanyl glycerol ethers) can be analyzed in the hydrolyzed aliquots of F8–F10. Fig. 5 shows total ion chromatograms of the alkylated aliquot of lipid fractions F6 and F10 for
Fig. 5. Total ion chromatograms of the alkylated aliquots of F10 and F6 from a Nankai Trough core sediment (0.82 mbsf).
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the same sediment sample. Peaks VIa and VIb were assigned to phytane, which was derived from the isopranyl moieties of archaeol and hydroxyarchaeol (Thiel et al., 2001; Blumenberg et al., 2004). Peaks VII (a and b) to IX (a and b) were assigned to biphytanes containing 0–2 cyclopentane rings, on the basis of interpretation of their mass spectra and comparison with literature data (Schouten et al., 1998; DeLong et al., 1998). Peak X (a and b) was assigned to a tricyclic biphytane by comparing the mass spectrum with literature data (Schouten et al., 1998; DeLong et al., 1998). This compound, initially inferred to have a molecular structure with three cyclopentane rings (Schouten et al., 1998; DeLong et al., 1998), was later proved to have two cyclopentane rings and one cyclohexane ring (Sinninghe Damste´ et al., 2002). All of these biphytanes were derived from the alkyl moieties of neutral and polar glycerol tetraethers (glycerol dialkyl glycerol tetraethers; GDGTs). Acyclic and cyclic biphytanes were detected mainly from the aliquots of F6, F8, and F10 (Table 2). To clarify how neutral GDGTs are separated on the silica gel column, we applied the separation procedures to the total lipid extract from M. thermoautotrophicus DH, which synthesizes dibiphytanyl diglycerol tetraether. Before separation on silica gel, the lipid sample was treated with acid to hydrolyze IPLs completely. Treatment of the separated fractions with HI/LiAlH4 followed by analysis with GC–MS revealed that acyclic biphytane was detected mostly in F6 (Table 2). From these results, we find that neutral and polar GDGTs can be analyzed in the alkylated aliquots of F6 and of F8–F10, respectively. Knowing how each isopranyl glycerol ether lipid is separated on the silica gel column, we can simplify the procedures by combining certain sets of fractions before derivatization. For the analysis of sediment samples we therefore adopted the procedures summarized schematically in Fig. 6.
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3.2. Concentration and carbon isotopic composition of isopranyl glycerol ethers in Nankai Trough sediments 3.2.1. Polar archaeol and hydroxyarchaeol Concentrations of polar archaeol in the IPL fraction (Fig. 6) extracted from the Nankai Trough core sediments ranged from 57.6 to 300 ng g1 dry sediment and tended to decrease with increasing depth, except for an anomalously high value at 0.82 mbsf (Fig. 7). The d13C values (relative to PDB) of polar archaeol at 0.82 mbsf were 110&. Concentrations of polar sn-2-hydroxyarchaeol and sn-3-hydroxyarchaeol in the IPL fraction ranged from 52.0 to 210 and from 23.4 to 132 ng g1 dry sediment, respectively; sn-2-hydroxyarchaeol was always more abundant than sn-3-hydroxyarchaeol. The depth profiles of these hydroxyarchaeols showed a similar trend to that of polar archaeol. Maximum concentrations of hydroxyarchaeols were accompanied by extreme depletion in 13C (compound II, 117&; compound III, 113&). Archaeol has been reported in halophiles, thermophiles and methanogens and is the most common and ubiquitous compound among the archaeal lipids (Koga et al., 1998a,b); sn-2-hydroxyarchaeol is found predominantly in methanogenic archaea of the order Methanosarcinales (Sprott et al., 1993; Koga et al., 1998a). The occurrence of these intact polar ether lipids in all the sediment samples indicates, therefore, the presence of living cells of archaea. Recent investigations have shown that archaeol and sn-2-hydroxyarchaeol extremely depleted in 13 C are widely observed in sediments at marine methane seep sites, for example, Eel River Basin (Hinrichs et al., 1999, 2000; Orphan et al., 2001), Santa Barbara Basin (Hinrichs et al., 2000), Hydrate Ridge (Boetius et al., 2000; Elvert et al., 2005) and Mediterranean Sea mud volcanoes (Pancost et al., 2000, 2001a), as well as in a microbial
Table 2 Distribution of GDGTsa (detected as biphytanes) in fractions of two lipid samples separated with silica gel F4
F5
F6
(1) Total lipid extract from a Nankai Trough sediment (0.82 mbsf) Biphytanes Tr Tr ++
F7
F8
F9
F10
+
++
+
++
(2) Total lipid extract from cells of Methanothermobacter thermoautotrophicus, treated with acid in advance of hydrolyzing polar GDGTs Biphytanes – – ++ Tr – – – a
Glycerol dialkyl glycerol tetraether.
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Fig. 6. Procedures for measuring neutral and polar isopranyl glycerol ethers in sediment samples. F1–F10 indicate fractions from total lipid extracts separated on a silica gel column. IPL, intact polar lipid; BSTFA, N,O-bis(trimethylsilyl)trifluoracetamide.
Fig. 7. Depth profiles of concentrations of diether lipids in the Nankai Trough core sediments; d13C values (vs. PDB) of ether lipids at 0.82 mbsf are shown.
mat from the Black Sea (Michaelis et al., 2002; Blumenberg et al., 2004). These diether lipids are understood to be the products of archaea living symbiotically with sulfate reducing bacteria and anaerobically oxidizing methane. Thus, the occur-
rence of extremely 13C-depleted polar sn-2-hydroxyarchaeol at 0.82 mbsf suggests the presence of methanotrophic archaea at this site. Organisms known to contain sn-3-hydroxyarchaeol include methanogens such as Methanosaeta concilii
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(Ferrante et al., 1988) and Methanococcus voltae (Sprott et al., 1993). Although sn-3-hydroxyarchaeol has been found in sediments from some methane seep sites (Pancost et al., 2000, 2001b; Elvert et al., 2005) it appears to be much less common. Thus, the presence of highly 13C-depleted polar sn-3-hydroxyarchaeol in our samples suggests that methanotrophic archaea distinct from Methanosarcina spp. are present. 3.2.2. Ether lipids detected as hydrocarbon derivatives HI/LiAlH4 treatment of the IPL fraction extracted from seven sediment samples yielded various concentrations of phytane (VIb) and acyclic (VIIb), monocyclic (VIIIb), bicyclic (IXb) and tricyclic (Xb) biphytanes (Fig. 8). The d13C value of phytane at 0.82 mbsf was 106&, close to that of archaeol and hydroxyarchaeol in the IPL fraction.
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Moreover, the depth profile of phytane was similar to the profiles of the diether lipids. These results support the idea that phytane was derived mostly from diether lipids. Acyclic VIIb was the most abundant biphytane, ranging from 337 to 428 ng g1 dry sediment, followed by bicyclic IX and tricyclic X. Monocyclic VIII was the least abundant of the biphytanes at all depths except 0.82 mbsf. These biphytanes were derived from polar GDGTs. GDGTs are diagnostic for, and common in, archaea and, like diether lipids, indicate the presence of viable archaea. The concentrations of VIIb, IXb, and Xb were about the same regardless of depth (Fig. 8). On the other hand, that of VIIIb was relatively high at 0.82 mbsf. Moreover, the d13C value of VIIIb at this depth was 71&, which was lower than that of the other biphytanes, although VIIIb was enriched in 13C relative to the polar diether lipids.
Fig. 8. Depth profiles of concentrations of phytane and biphytanes released by HI/LiAlH4 treatment of polar and neutral lipids in Nankai Trough core sediments; d13C values (vs. PDB) of ether lipids at 0.82 mbsf are shown.
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Similar isotopic distributions have been observed for Mediterranean Sea methane seep sediments (Pancost et al., 2001a). According to Pancost et al. (2001a) this isotopic distribution suggests that biphytane VIIIb best reflects the carbon isotopic composition of GDGTs generated by methanotrophic archaea. The d13C value of both IXb and Xb was 36&; thus, they were highly enriched in 13 C relative to the polar diether lipids and other biphytanes. These findings suggest that the biphytanes IXb and Xb derived from some non-methanotrophic archaea sp(p). Component X is known to be a constituent of the carbon skeleton of crenarchaeol and has been proposed as a taxonomically specific biomarker for non-thermophilic crenarchaeota (Sinninghe Damste´ et al., 2002). Similarly high d13C values have been observed for Mediterranean Sea methane seep sediments (Pancost et al., 2001a) and the Black Sea water column (Schouten et al., 2001; Wakeham et al., 2003), where a crenarchaeota origin was inferred. The d13C values of the acyclic biphytane VIIb are intermediate between those of VIIIb and Xb, indicating that VIIb likely derived from both methanotrophic archaea and non-methanotrophic archaea. 3.2.3. Neutral isopranyl glycerol ether lipids The vertical profiles of the concentrations of neutral diether lipids and biphytanes released from neutral GDGTs in neutral lipid fractions A and B are shown in Figs. 7 and 8, respectively. The profiles and d13C values of the neutral diether lipids were similar to those of the polar diether lipids (Fig. 7). Biphytanes released from neutral GDGTs also exhibited similar trends to both the vertical profiles and d13C values of biphytanes derived from polar GDGTs (Fig. 8). These results show that, in many respects, information on the activity of archaea inferred from neutral ether lipids in the sediments was similar to that inferred from intact polar ether lipids. This was different from what we had expected, because the sediments are relatively old (> 0.16 Ma from foraminifera and nannofossil data; written communication, Japan Oil, Gas and Metals National Corporation) and Peckmann and Thiel (2004) demonstrated that neutral archaeol could be preserved in carbonate rocks up to the Eocene. In truth, it is likely that neutral ether lipids derived from fossil archaea in the samples had mostly been lost by the time of sampling, probably by processes such as microbial degradation and incorporation into kerogen.
The concentrations of neutral diether lipids and biphytanes released from neutral GDGTs in neutral lipid fractions A and B were of the same level as those of the IPLs at each depth (Figs. 7 and 8). It is, therefore, essential to determine the concentration of intact polar ether lipids for a precise estimation of viable archaeal biomass. Analytical methods that do not precisely distinguish neutral ether lipids from intact polar ether lipids can cause error in the estimation of the viable archaeal biomass. This error may be still more significant when deep sediments are analyzed. By analogy with the findings of other investigations showing that the relative abundance of diglyceride fatty acids to phospholipid fatty acids increases exponentially with depth (Ringelberg et al., 1997; Kieft et al., 1998), we suggest that the ratio of neutral ether lipids to intact polar ether lipids also increases with depth. Moreover, it is quite likely that the stability of ether bonds during diagenesis contributes to a relative increase in the concentrations of neutral ether lipids. 4. Conclusions • We established analytical procedures for detecting polar and neutral isopranyl glycerol ether lipids in a sediment for detection using GC. • We applied the procedures to near-surface core sediments from the Nankai Trough and obtained the following results: • Archaeol and hydroxyarchaeol were detected for both IPL and neutral lipid fractions in all the sediments. Acyclic and cyclic biphytanes were also detected in both IPL and neutral lipid fractions treated with HI/LiAlH4. • Polar archaeol, sn-2-hydroxyarchaeol and sn-3hydroxyarchaeol at 0.82 mbsf were highly depleted in 13C, reflecting the prominent activity of living methanotrophic archaea in the seep sediments. The d13C values of biphytanes released from polar GDGTs at this depth varied, suggesting that these biphytanes derived not only from methanotrophic archaea but also from nonmethanotrophic archaea, and the relative contributions of methanotrophic and nonmethanotrophic archaea differed depending on the biphytane compound. • Vertical profiles and d13C values of the neutral ether lipids were similar to those of the intact polar ether lipids, suggesting that neutral ether lipids derived from fossil archaea in the samples had mostly been lost by the time of sampling.
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Acknowledgements We express our sincere thanks to A. Stadnitskaia, Royal Netherlands Institute for Sea Research (Netherlands) and an anonymous reviewer for constructive reviewing comments, which helped us improve the manuscript. We are grateful to the Ministry of Economy, Trade and Industry for permitting us to publish the data in this study. We thank M. Tanahashi and J. Ashi for help in collecting sediment samples, and Y. Kamagata, N. Shinzato and H. Yoshioka for help in growing the methanogen culture. We also thank Y. Koga for valuable information on ether lipids in archaeal cells. Discussions with T. Treude and H. Niemann helped us interpret the sediment lipid signatures. The work was supported by Research Grant for Fellowship No. 00001622 from the Japanese Society for the Promotion of Science (JSPS) to M.O. Guest Associate Editor—R.D. Pancost References Ashi, J., Segawa, J., Le Pichon, X., Lallemant, S., Kobayashi, K., Hattori, M., Mazzotti, S., Aoike, K., 1996. Distribution of cold seepage at the Ryuyo Canyon off Tokai: the 1995 KAIKO-Tokai ‘‘Shinkai 2000’’ Dives. JAMSTEC Journal of Deep Sea Research 12, 159–166. Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., Fredricks, H.F., Elvert, M., Kelly, T.J., Schrag, D.P., Sogin, M.L., Brenchley, J.E., Teske, A., House, C.H., Hinrichs, K.-U., 2006. Heterotrophic archaea dominate sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy of Sciences USA 103, 3846–3851. Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the National Academy of Sciences USA 97, 14421–14426. Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Gieseke, A., Amann, R., Jørgensen, B.B., Witte, U., Pfannkuche, O., 2000. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626. DeLong, E.F., King, L.L., Massana, R., Cittone, H., Murray, A., Schleper, C., Wakeham, S.G., 1998. Dibiphytanyl ether lipids in non-thermophilic crenarchaeotes. Applied and Environmental Microbiology 64, 1133–1138. Elvert, M., Hopmans, E.C., Treude, T., Boetius, A., Suess, E., 2005. Spatial variations of methanotrophic consortia at cold methane seeps: implications from a high-resolution molecular and isotopic approach. Geobiology 3, 195–209. Ferrante, G., Ekiel, I., Patel, G.B., Sprott, G.D., 1988. A novel core lipid isolated from the aceticlastic methanogen, Methanothrix concilii GP6. Biochimica et Biophysica Acta 753, 249– 256.
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surface sediments as records for microbial consumption or production of methane. In: Thirty-second International Geological Congress, electronic version posted on-line on July 20, 2004, Abstracts Volume, abs. 315-6. Schouten, S., Hoefs, M.J.L., Koopmans, M.P., Bosch, H.J., Sinninghe Damste´, J.S., 1998. Structural characterization, occurrence, and fate of archaeal ether-bound acyclic and cyclic biphytanes and corresponding diols in sediments. Organic Geochemistry 29, 1305–1319. Schouten, S., Wakeham, S.G., Sinninghe Damste´, J.S., 2001. Evidence for anaerobic methane oxidation by archaea in euxinic waters of the Black Sea. Organic Geochemistry 32, 1277–1281. Sinninghe Damste´, J.S., Schouten, S., Hopmans, E.C., van Duin, A.C.T., Geenevasen, J.A.J., 2002. Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenarchaeota. Journal of Lipid Research 43, 1641–1651. Sprott, G.D., Dicaire, C.J., Choquet, C.G., Patel, G.B., Ekeil, I., 1993. Hydroxydiether lipid structures in Methanosarcina spp. and Methanococcus voltae. Applied and Environmental Microbiology 59, 912–914. Sturt, H.F., Summons, R.E., Smith, K., Elvert, M., Hinrichs, K.U., 2004. Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometrynew biomarkers for biogeochemistry and microbial ecology. Rapid Communications in Mass Spectrometry 18, 617–628. Teixidor, P., Grimalt, J.O., Pueyo, J.J., Rodrigeuez-Valera, F., 1993. Isopranylglycerol diethers in non-alkaline evaporitic environments. Geochimica et Cosmochimica Acta 57, 4479– 4489. Thiel, V., Peckmann, J., Richnow, H.-H., Luth, U., Reitner, J., Michaelis, W., 2001. Molecular signals for anaerobic methane oxidation in Black Sea seep carbonates and a microbial mat. Marine Chemistry 73, 97–112. Wakeham, S.G., Lewis, C.M., Hopmans, E.C., Schouten, S., Sinninghe Damste´, J.S., 2003. Archaea mediate anaerobic oxidation of methane in deep euxinic waters of the Black Sea. Geochimica et Cosmochimica Acta 67, 1359–1374. White, D.C., Davis, W.M., Nickels, J.S., King, J.D., Bobbie, R.J., 1979. Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40, 51– 62.
Polar and neutral isopranyl glycerol ether lipids as biomarkers of archaea in near-surface sediments from the Nankai Trough Masahiro Oba
a,*
, Susumu Sakata a, Urumu Tsunogai
b
a
Institute for Geo-Resources and Environment, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8567, Japan b Division of Earth and Planetary Sciences, Graduate School of Science, Hokkaido University, N10 W8 Kita-Ku, Sapporo, Hokkaido 060-0810, Japan Available online 9 November 2006
Abstract The molecular and carbon isotopic compositions of polar isopranyl glycerol ether lipids, which are direct indicators of viable archaea, and neutral isopranyl glycerol ether lipids, which are derived from polar lipids via hydrolysis, in near-surface sediments from a methane seep in the Nankai Trough (off central Japan) were investigated. Procedures for extracting, separating and derivatizing polar and neutral ether lipids for detection using gas chromatography were first examined with one sediment sample and a cultivated methanogen. For all sediment samples, archaeol and hydroxyarchaeol were detected in both the polar and neutral ether lipid fractions. Acyclic and cyclic biphytanes were also detected in both types of lipid fractions after treatment with HI/LiAlH4 for ether cleavage and alkylation. The d13C values of archaeol, sn-2-hydroxyarchaeol, and sn-3-hydroxyarchaeol in the sample from 0.82 m below the seafloor were lower than 100& relative to PDB, indicating that diverse living methanotrophic archaea are present in the seep sediments. Biphytanes released from polar ether lipids in the same sample were less depleted in d13C (71& to 36&). The wide range of d13C values suggests that the biphytanes were derived not only from methanotrophic but also from non-methanotrophic archaea, and that the relative contributions of the methanotrophic and non-methanotrophic archaea differed, depending on the biphytane compound. The vertical profiles and d13C values of the neutral ether lipids were similar to those of the intact polar ether lipids, suggesting that neutral ether lipids derived from fossil archaea in the samples had mainly been lost by the time of sampling. 2006 Elsevier Ltd. All rights reserved.
1. Introduction The major constituents of the cell membranes of archaea are isopranyl glycerol diether and/or tetraether lipids, which are distinct from the acyl ester lipids found in bacteria (Kates, 1978; Langworthy *
Corresponding author. Present address: Institute of Geology and Paleontology, Tohoku University, Sendai 980-8578, Japan. Fax: +81 22 795 6668. E-mail address: [email protected] (M. Oba).
et al., 1982). Because isopranyl glycerol ether lipids are unique to archaea, they have been used as biomarkers of such organisms in many studies (e.g., Martz et al., 1983; Pauly and van Vleet, 1986; Nichols et al., 1987; Reed et al., 2002). Notably, molecular and stable carbon isotopic compositions have been measured in recent years to predict archaeal activity in methane hydrate layers and methane seeps, providing information such as magnitude and spatial distribution of biomass, dominant species and the types of activity of the species (e.g.,
0146-6380/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2006.09.002
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Hinrichs et al., 1999; Pancost et al., 2001a; Blumenberg et al., 2004; Orcutt et al., 2005; Elvert et al., 2005). In living archaeal cells, most ether lipids contain polar head groups with phosphate or sugar moieties, or both (Nishihara and Koga, 1987) and are referred to as intact polar lipids (IPLs). By analogy with the polar acyl ester lipids of bacteria, which are known to lose polar head groups via enzymatic hydrolysis upon cell death or cell lysis (White et al., 1979), the detection of intact polar ether lipids is inferred to indicate the presence of living or potentially viable biomass rather than fossil archaeal biomass (Sturt et al., 2004; Biddle et al., 2006). On the other hand, neutral ether lipids, which are hydrolysates of intact polar ether lipids, are considered to be biomarkers of dead or lysed cells. Although many studies of isopranyl glycerol ether lipids in sediments have been reported, none have clearly differentiated intact polar ether lipids from neutral ether lipids. Here, we report methods for the efficient analysis of sediment samples for intact polar and neutral ether lipids using gas chromatography. We then apply the methods to near-surface core sediments
from a cold methane seep in the Nankai Trough and determine the distribution of intact polar and neutral ether lipids near the seafloor. In the Nankai Trough, many cold seeps have been found (Le Pichon et al., 1987, 1992; Ashi et al., 1996), but the information available on lipid biomarkers in the seep sediments has been limited to the presence of acyclic isoprenoid hydrocarbons characteristic of methanogens (Sakata et al., 2004). Therefore, an additional objective was to show the distribution and characteristics of archaea in seep sediments in the Nankai Trough. 2. Materials and methods 2.1. Sediment samples Core samples were collected from the seafloor of the southeastern slope (3404.410 0 N, 13747.511 0 E; 610 m water depth) of Dai-ni Tenryu Knoll in the eastern part of the Nankai Trough off central Japan (Fig. 1) during the 2001 cruise of the R/V Hakureimaru No.2 (operated by the Metal Mining Agency of Japan, now Japan Oil, Gas and Metals National
Fig. 1. Map showing sampling site for core sediments from the seafloor in the Nankai Trough, off central Japan.
M. Oba et al. / Organic Geochemistry 37 (2006) 1643–1654
Corporation) conducted by the Japanese Ministry of Economy, Trade and Industry as part of the Deep Sea Survey Technologies for Natural Resources project. In this area, the distribution of cold seep zones has been well investigated. A previous submersible cruise at the Dai-ni Tenryu Knoll showed well developed carbonate crust exposures and large Calyptogena sp. colonies associated with a methane seep (Kuramoto and Joshima, 1998). Methane hydrate bottom-simulating reflectors are widespread in the area and hydrate dissociation and methane seepage are considered to have resulted from fault activity and uplift. A 120 cm long core, consisting largely of clay and clayey silt, was obtained with a gravity corer. After collection, the samples were transported to the laboratory, freeze-dried and pulverized. 2.2. Methanogen cell samples Methanothermobacter thermoautotrophicus DH was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). It was grown in a mineral salts medium. Cultivation was carried out at 55 C for 2 d without agitation in a 1.3-l serum bottle containing 500 ml medium under an atmosphere of 2 atm H2/CO2 (80:20, v/v) as energy and carbon sources. Growth stage was determined by monitoring the increase in optical density at 600 nm (OD600). Whole cells at the stationary phase (OD600 = 0.13) were harvested by centrifugation and immediately freeze-dried. Approximately 30 mg of dry cells were collected from each bottle. 2.3. Extraction, separation and derivatization Total lipids in sediment samples (12–25 g) and cell material (100 mg) were ultrasonically extracted using the method of Bligh and Dyer, modified by Nishihara and Koga (1987) to use trichloroacetic acid-acidified solvent: methanol/chloroform/water with 10% trichloroacetic acid (2:1:0.8, v/v/v), for 30 min in centrifuge tubes, which allows re-extraction of the residue after centrifugation and recovery of supernatant. The supernatant from the first extraction and the following six re-extractions were combined. After phase separation of the combined supernatants by adding chloroform and water to obtain a final methanol/chloroform/water ratio of 1:1:0.9 (v/v/v), the organic phase was recovered
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and washed with methanol/distilled water (5:4, v/v) to remove trichloroacetic acid. The extracts were dried over Na2SO4 and concentrated by evaporation under reduced pressure. To separate major compound classes, chromatographic separation on a silica gel column (0.6 g of pre-activated silica, 63–200 mesh) was carried out. Ten fractions were obtained by elution with the following: 6 ml hexane (F1); 3 ml hexane/toluene 3:1 v/v (F2); 3 ml hexane/ethyl acetate 19:1 v/v (F3); 3 ml hexane/ethyl acetate 9:1 v/v (F4); 3 ml hexane/ethyl acetate 17:3 v/v (F5); 3 ml hexane/ethyl acetate 4:1 v/v (F6); 3 ml hexane/ethyl acetate 3:1 v/v (F7); 3 ml hexane/ethyl acetate 1:1 v/v (F8); 3 ml ethyl acetate (F9); 10 ml methanol (F10). Residual organic compounds in the column were ultrasonically extracted twice with methanol/chloroform (1:2, v/v) and combined with the F10 fraction. Each fraction was divided into aliquots for the three different procedures as follows. One aliquot was heated with N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) in pyridine at 75 C for 2 h. In this aliquot (hereafter referred to as the unhydrolyzed aliquot) OH groups were silylated. Another aliquot was heated in 1 ml chloroform/ methanol/concentrated HCl (1:10:1, v/v/v) at 100 C for 2 h, followed by extraction with petroleum ether and chloroform. The organic phase was silylated with BSTFA as above. In this aliquot (hereafter referred to as the hydrolyzed aliquot), IPLs were hydrolyzed to neutral lipids and the OH groups were silylated. The third aliquot of F4–F10 only was refluxed in 56% HI (in H2O by weight) at 100 C for 20 h, followed by extraction with petroleum ether. The organic phase was heated with LiAlH4 at 100 C for 2 h. In this aliquot (hereafter referred to as the alkylated aliquot), ether lipids were converted to alkanes via alkyl iodides so that molecular structures and carbon isotopic compositions of the biphytanes in tetraethers could be determined using gas chromatography (GC). 2.4. Lipid analysis Lipids were identified by using an Agilent 6890N gas chromatograph equipped with an on-column injector and interfaced to an Agilent 5973N mass selective detector mass spectrometer, operated with an ionizing electron energy of 70 eV and scanned from m/z 50–650 with a scan time of 0.8 s. A fused silica CP–Sil 5CB capillary column (60 m, 0.25 mm
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i.d., 0.25 lm film thickness) was used, with helium as carrier gas. The oven temperature was raised from 70 C to 160 C at 20 C/min, then to 310 C at 4 C/min and finally held constant for 50 min. Compound identifications were based on mass spectra and retention times. Individual lipids were quantified by using a Hewlett–Packard 6890 gas chromatograph equipped with on-column injector and flame ionization detector. GC conditions (injector, column, carrier gas, temperature programme) were identical to those for GC-mass spectrometry (GC-MS) analysis. Quantification was based on comparison of peak areas with those of external standards (n-alkanes). Compound-specific carbon isotopic compositions were determined by using a Finnigan MAT 252 mass spectrometer coupled to a Hewlett–Packard 5890 series II. GC conditions were as described above. Values of d13C (&, relative to Pee Dee Belemnite standard, PDB) were established by inserting
multiple peaks of pure CO2 via a separate line so as to prevent coleution with peaks from the GC, and by determining d13C values of the CO2 peaks on separate runs by injecting n-alkane standards into the GC. Carbon added by trimethylsilylation was corrected for, with an error of < ± 1&. 3. Results and discussion 3.1. Methods for separation and detection of ether lipids Fig. 2 shows the total ion chromatogram of the unhydrolyzed aliquot of F4 from one Nankai Trough core sediment sample [0.82 m below seafloor (mbsf)]. Peak Ia was assigned to 2,3-di-O-phytanylsn-glycerol diether (archaeol) by comparing the mass spectrum with literature data (Teixidor et al., 1993). Among the unhydrolyzed aliquots, archaeol was detected only in that from F4 (Table 1). Fig. 3 shows
Fig. 2. Total ion chromatogram of unhydrolyzed aliquot of F4 from a Nankai Trough core sediment (0.82 mbsf).
Table 1 Distribution of isopranyl glycerol ethers in the fractions of total lipid extract from a Nankai Trough sediment (0.82 mbsf) separated by silica gel F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
++ – –
– – –
– – ++
– – +
– – –
– – –
– – –
(2) Free and polar isopranyl glycerol ethers in the hydrolyzed aliquots Archaeol – – – ++ Monophytanyl glycerol ether – – – – Hydroxyarchaeol – – – –
tr tr –
– ++ –
– + –
tr ++ –
tr + –
++ ++ –
(1) Free isopranyl glycerol ethers in the unhydrolyzed aliquots Archaeol – – – Monophytanyl glycerol ether – – – Hydroxyarchaeol – – –
Symbols, ++, +, tr, and – indicate major, minor, trace amounts, and not detected, respectively.
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Fig. 3. Total ion chromatogram of the hydrolyzed aliquot of F10 from a Nankai Trough core sediment (0.82 mbsf).
the total ion chromatogram of the hydrolyzed aliquot of F10 from the same sample. Archaeol was also detected in this hydrolyzed aliquot (peak Ib in Fig. 3). Among the hydrolyzed aliquots, archaeol was detected mostly in the F4 and F10 fractions (Table 1). From these results, we find that neutral archaeol (i.e., archaeol in the form of a neutral lipid) and polar archaeol (i.e., archaeol in the form of IPLs) can be analyzed in the unhydrolyzed aliquot of F4 and the hydrolyzed aliquot of F10, respectively. Fig. 4 shows the total ion chromatogram of the unhydrolyzed aliquot of F6, also from the sediment sample from 0.82 mbsf. A large peak at 72 min was
assigned to hydroxyarchaeol by comparing the mass spectrum with literature data (Hinrichs et al., 2000). Hydroxyarchaeol has the same core structure as archaeol but contains an additional hydroxyl group on the third carbon of the phytanyl moiety etherlinked to either the second (IV; sn-2-hydroxyarchaeol) or third (V; sn-3-hydroxyarchaeol) glycerol carbon. Because mass spectra of these two possible isomers resemble each other, being characterized by dominant fragments at m/z 143, 341, and 517 (Hinrichs et al., 2000), assignment of this peak to one of the isomers on the basis of its mass spectrum alone is difficult. Among the unhydrolyzed aliquots,
Fig. 4. Total ion chromatogram of the unhydrolyzed aliquot of F6 from a Nankai Trough core sediment (0.82 mbsf).
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hydroxyarchaeol was detected in the F6 and F7 fractions (Table 1). On the other hand, hydroxyarchaeol was not detected in the hydrolyzed aliquots of F6, F7, or any other fraction. Remarkably, 1-O-phytanyl glycerolether (peak II in Fig. 3) and 2-O-phytanyl glycerolether (peak III in Fig. 3), which were not detected in any unhydrolyzed fractions, were identified in the hydrolyzed aliquots of F6, F8, and F10 (Table 1). According to Koga et al. (1993), hydroxyarchaeols lose the hydroxy-bearing phytanyl chain by mild acid treatment to yield monophytanyl glycerolethers. Therefore, polar sn-2- and sn-3-hydroxyarchaeol are converted to 1-O- and 2-O-phytanyl glycerolether, respectively, in the process of hydroly-
sis with chloroform/methanol/concentrated HCl. Moreover, the results confirm that, under the hydrolysis conditions used, all hydroxyarchaeols were converted to monophytanyl glycerol ethers, because the total molar concentrations of monophytanyl glycerol ethers in hydrolyzed F6 were approximately equal to the molar concentrations of the neutral hydroxyarchaeols in unhydrolyzed F6. From these results, we find that neutral hydroxyarchaeols can be analyzed in the unhydrolyzed aliquots of F6 and F7, and that polar hydroxyarchaeols (detected as monophytanyl glycerol ethers) can be analyzed in the hydrolyzed aliquots of F8–F10. Fig. 5 shows total ion chromatograms of the alkylated aliquot of lipid fractions F6 and F10 for
Fig. 5. Total ion chromatograms of the alkylated aliquots of F10 and F6 from a Nankai Trough core sediment (0.82 mbsf).
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the same sediment sample. Peaks VIa and VIb were assigned to phytane, which was derived from the isopranyl moieties of archaeol and hydroxyarchaeol (Thiel et al., 2001; Blumenberg et al., 2004). Peaks VII (a and b) to IX (a and b) were assigned to biphytanes containing 0–2 cyclopentane rings, on the basis of interpretation of their mass spectra and comparison with literature data (Schouten et al., 1998; DeLong et al., 1998). Peak X (a and b) was assigned to a tricyclic biphytane by comparing the mass spectrum with literature data (Schouten et al., 1998; DeLong et al., 1998). This compound, initially inferred to have a molecular structure with three cyclopentane rings (Schouten et al., 1998; DeLong et al., 1998), was later proved to have two cyclopentane rings and one cyclohexane ring (Sinninghe Damste´ et al., 2002). All of these biphytanes were derived from the alkyl moieties of neutral and polar glycerol tetraethers (glycerol dialkyl glycerol tetraethers; GDGTs). Acyclic and cyclic biphytanes were detected mainly from the aliquots of F6, F8, and F10 (Table 2). To clarify how neutral GDGTs are separated on the silica gel column, we applied the separation procedures to the total lipid extract from M. thermoautotrophicus DH, which synthesizes dibiphytanyl diglycerol tetraether. Before separation on silica gel, the lipid sample was treated with acid to hydrolyze IPLs completely. Treatment of the separated fractions with HI/LiAlH4 followed by analysis with GC–MS revealed that acyclic biphytane was detected mostly in F6 (Table 2). From these results, we find that neutral and polar GDGTs can be analyzed in the alkylated aliquots of F6 and of F8–F10, respectively. Knowing how each isopranyl glycerol ether lipid is separated on the silica gel column, we can simplify the procedures by combining certain sets of fractions before derivatization. For the analysis of sediment samples we therefore adopted the procedures summarized schematically in Fig. 6.
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3.2. Concentration and carbon isotopic composition of isopranyl glycerol ethers in Nankai Trough sediments 3.2.1. Polar archaeol and hydroxyarchaeol Concentrations of polar archaeol in the IPL fraction (Fig. 6) extracted from the Nankai Trough core sediments ranged from 57.6 to 300 ng g1 dry sediment and tended to decrease with increasing depth, except for an anomalously high value at 0.82 mbsf (Fig. 7). The d13C values (relative to PDB) of polar archaeol at 0.82 mbsf were 110&. Concentrations of polar sn-2-hydroxyarchaeol and sn-3-hydroxyarchaeol in the IPL fraction ranged from 52.0 to 210 and from 23.4 to 132 ng g1 dry sediment, respectively; sn-2-hydroxyarchaeol was always more abundant than sn-3-hydroxyarchaeol. The depth profiles of these hydroxyarchaeols showed a similar trend to that of polar archaeol. Maximum concentrations of hydroxyarchaeols were accompanied by extreme depletion in 13C (compound II, 117&; compound III, 113&). Archaeol has been reported in halophiles, thermophiles and methanogens and is the most common and ubiquitous compound among the archaeal lipids (Koga et al., 1998a,b); sn-2-hydroxyarchaeol is found predominantly in methanogenic archaea of the order Methanosarcinales (Sprott et al., 1993; Koga et al., 1998a). The occurrence of these intact polar ether lipids in all the sediment samples indicates, therefore, the presence of living cells of archaea. Recent investigations have shown that archaeol and sn-2-hydroxyarchaeol extremely depleted in 13 C are widely observed in sediments at marine methane seep sites, for example, Eel River Basin (Hinrichs et al., 1999, 2000; Orphan et al., 2001), Santa Barbara Basin (Hinrichs et al., 2000), Hydrate Ridge (Boetius et al., 2000; Elvert et al., 2005) and Mediterranean Sea mud volcanoes (Pancost et al., 2000, 2001a), as well as in a microbial
Table 2 Distribution of GDGTsa (detected as biphytanes) in fractions of two lipid samples separated with silica gel F4
F5
F6
(1) Total lipid extract from a Nankai Trough sediment (0.82 mbsf) Biphytanes Tr Tr ++
F7
F8
F9
F10
+
++
+
++
(2) Total lipid extract from cells of Methanothermobacter thermoautotrophicus, treated with acid in advance of hydrolyzing polar GDGTs Biphytanes – – ++ Tr – – – a
Glycerol dialkyl glycerol tetraether.
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Fig. 6. Procedures for measuring neutral and polar isopranyl glycerol ethers in sediment samples. F1–F10 indicate fractions from total lipid extracts separated on a silica gel column. IPL, intact polar lipid; BSTFA, N,O-bis(trimethylsilyl)trifluoracetamide.
Fig. 7. Depth profiles of concentrations of diether lipids in the Nankai Trough core sediments; d13C values (vs. PDB) of ether lipids at 0.82 mbsf are shown.
mat from the Black Sea (Michaelis et al., 2002; Blumenberg et al., 2004). These diether lipids are understood to be the products of archaea living symbiotically with sulfate reducing bacteria and anaerobically oxidizing methane. Thus, the occur-
rence of extremely 13C-depleted polar sn-2-hydroxyarchaeol at 0.82 mbsf suggests the presence of methanotrophic archaea at this site. Organisms known to contain sn-3-hydroxyarchaeol include methanogens such as Methanosaeta concilii
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(Ferrante et al., 1988) and Methanococcus voltae (Sprott et al., 1993). Although sn-3-hydroxyarchaeol has been found in sediments from some methane seep sites (Pancost et al., 2000, 2001b; Elvert et al., 2005) it appears to be much less common. Thus, the presence of highly 13C-depleted polar sn-3-hydroxyarchaeol in our samples suggests that methanotrophic archaea distinct from Methanosarcina spp. are present. 3.2.2. Ether lipids detected as hydrocarbon derivatives HI/LiAlH4 treatment of the IPL fraction extracted from seven sediment samples yielded various concentrations of phytane (VIb) and acyclic (VIIb), monocyclic (VIIIb), bicyclic (IXb) and tricyclic (Xb) biphytanes (Fig. 8). The d13C value of phytane at 0.82 mbsf was 106&, close to that of archaeol and hydroxyarchaeol in the IPL fraction.
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Moreover, the depth profile of phytane was similar to the profiles of the diether lipids. These results support the idea that phytane was derived mostly from diether lipids. Acyclic VIIb was the most abundant biphytane, ranging from 337 to 428 ng g1 dry sediment, followed by bicyclic IX and tricyclic X. Monocyclic VIII was the least abundant of the biphytanes at all depths except 0.82 mbsf. These biphytanes were derived from polar GDGTs. GDGTs are diagnostic for, and common in, archaea and, like diether lipids, indicate the presence of viable archaea. The concentrations of VIIb, IXb, and Xb were about the same regardless of depth (Fig. 8). On the other hand, that of VIIIb was relatively high at 0.82 mbsf. Moreover, the d13C value of VIIIb at this depth was 71&, which was lower than that of the other biphytanes, although VIIIb was enriched in 13C relative to the polar diether lipids.
Fig. 8. Depth profiles of concentrations of phytane and biphytanes released by HI/LiAlH4 treatment of polar and neutral lipids in Nankai Trough core sediments; d13C values (vs. PDB) of ether lipids at 0.82 mbsf are shown.
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Similar isotopic distributions have been observed for Mediterranean Sea methane seep sediments (Pancost et al., 2001a). According to Pancost et al. (2001a) this isotopic distribution suggests that biphytane VIIIb best reflects the carbon isotopic composition of GDGTs generated by methanotrophic archaea. The d13C value of both IXb and Xb was 36&; thus, they were highly enriched in 13 C relative to the polar diether lipids and other biphytanes. These findings suggest that the biphytanes IXb and Xb derived from some non-methanotrophic archaea sp(p). Component X is known to be a constituent of the carbon skeleton of crenarchaeol and has been proposed as a taxonomically specific biomarker for non-thermophilic crenarchaeota (Sinninghe Damste´ et al., 2002). Similarly high d13C values have been observed for Mediterranean Sea methane seep sediments (Pancost et al., 2001a) and the Black Sea water column (Schouten et al., 2001; Wakeham et al., 2003), where a crenarchaeota origin was inferred. The d13C values of the acyclic biphytane VIIb are intermediate between those of VIIIb and Xb, indicating that VIIb likely derived from both methanotrophic archaea and non-methanotrophic archaea. 3.2.3. Neutral isopranyl glycerol ether lipids The vertical profiles of the concentrations of neutral diether lipids and biphytanes released from neutral GDGTs in neutral lipid fractions A and B are shown in Figs. 7 and 8, respectively. The profiles and d13C values of the neutral diether lipids were similar to those of the polar diether lipids (Fig. 7). Biphytanes released from neutral GDGTs also exhibited similar trends to both the vertical profiles and d13C values of biphytanes derived from polar GDGTs (Fig. 8). These results show that, in many respects, information on the activity of archaea inferred from neutral ether lipids in the sediments was similar to that inferred from intact polar ether lipids. This was different from what we had expected, because the sediments are relatively old (> 0.16 Ma from foraminifera and nannofossil data; written communication, Japan Oil, Gas and Metals National Corporation) and Peckmann and Thiel (2004) demonstrated that neutral archaeol could be preserved in carbonate rocks up to the Eocene. In truth, it is likely that neutral ether lipids derived from fossil archaea in the samples had mostly been lost by the time of sampling, probably by processes such as microbial degradation and incorporation into kerogen.
The concentrations of neutral diether lipids and biphytanes released from neutral GDGTs in neutral lipid fractions A and B were of the same level as those of the IPLs at each depth (Figs. 7 and 8). It is, therefore, essential to determine the concentration of intact polar ether lipids for a precise estimation of viable archaeal biomass. Analytical methods that do not precisely distinguish neutral ether lipids from intact polar ether lipids can cause error in the estimation of the viable archaeal biomass. This error may be still more significant when deep sediments are analyzed. By analogy with the findings of other investigations showing that the relative abundance of diglyceride fatty acids to phospholipid fatty acids increases exponentially with depth (Ringelberg et al., 1997; Kieft et al., 1998), we suggest that the ratio of neutral ether lipids to intact polar ether lipids also increases with depth. Moreover, it is quite likely that the stability of ether bonds during diagenesis contributes to a relative increase in the concentrations of neutral ether lipids. 4. Conclusions • We established analytical procedures for detecting polar and neutral isopranyl glycerol ether lipids in a sediment for detection using GC. • We applied the procedures to near-surface core sediments from the Nankai Trough and obtained the following results: • Archaeol and hydroxyarchaeol were detected for both IPL and neutral lipid fractions in all the sediments. Acyclic and cyclic biphytanes were also detected in both IPL and neutral lipid fractions treated with HI/LiAlH4. • Polar archaeol, sn-2-hydroxyarchaeol and sn-3hydroxyarchaeol at 0.82 mbsf were highly depleted in 13C, reflecting the prominent activity of living methanotrophic archaea in the seep sediments. The d13C values of biphytanes released from polar GDGTs at this depth varied, suggesting that these biphytanes derived not only from methanotrophic archaea but also from nonmethanotrophic archaea, and the relative contributions of methanotrophic and nonmethanotrophic archaea differed depending on the biphytane compound. • Vertical profiles and d13C values of the neutral ether lipids were similar to those of the intact polar ether lipids, suggesting that neutral ether lipids derived from fossil archaea in the samples had mostly been lost by the time of sampling.
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Acknowledgements We express our sincere thanks to A. Stadnitskaia, Royal Netherlands Institute for Sea Research (Netherlands) and an anonymous reviewer for constructive reviewing comments, which helped us improve the manuscript. We are grateful to the Ministry of Economy, Trade and Industry for permitting us to publish the data in this study. We thank M. Tanahashi and J. Ashi for help in collecting sediment samples, and Y. Kamagata, N. Shinzato and H. Yoshioka for help in growing the methanogen culture. We also thank Y. Koga for valuable information on ether lipids in archaeal cells. Discussions with T. Treude and H. Niemann helped us interpret the sediment lipid signatures. The work was supported by Research Grant for Fellowship No. 00001622 from the Japanese Society for the Promotion of Science (JSPS) to M.O. Guest Associate Editor—R.D. Pancost References Ashi, J., Segawa, J., Le Pichon, X., Lallemant, S., Kobayashi, K., Hattori, M., Mazzotti, S., Aoike, K., 1996. Distribution of cold seepage at the Ryuyo Canyon off Tokai: the 1995 KAIKO-Tokai ‘‘Shinkai 2000’’ Dives. JAMSTEC Journal of Deep Sea Research 12, 159–166. Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., Fredricks, H.F., Elvert, M., Kelly, T.J., Schrag, D.P., Sogin, M.L., Brenchley, J.E., Teske, A., House, C.H., Hinrichs, K.-U., 2006. Heterotrophic archaea dominate sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy of Sciences USA 103, 3846–3851. Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the National Academy of Sciences USA 97, 14421–14426. Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Gieseke, A., Amann, R., Jørgensen, B.B., Witte, U., Pfannkuche, O., 2000. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626. DeLong, E.F., King, L.L., Massana, R., Cittone, H., Murray, A., Schleper, C., Wakeham, S.G., 1998. Dibiphytanyl ether lipids in non-thermophilic crenarchaeotes. Applied and Environmental Microbiology 64, 1133–1138. Elvert, M., Hopmans, E.C., Treude, T., Boetius, A., Suess, E., 2005. Spatial variations of methanotrophic consortia at cold methane seeps: implications from a high-resolution molecular and isotopic approach. Geobiology 3, 195–209. Ferrante, G., Ekiel, I., Patel, G.B., Sprott, G.D., 1988. A novel core lipid isolated from the aceticlastic methanogen, Methanothrix concilii GP6. Biochimica et Biophysica Acta 753, 249– 256.
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