Fossil bacterial ecosystem at methane seeps

Geochimica et Cosmochimica Acta, Vol. 66, No. 23, pp. 4085– 4101, 2002 Copyright © 2002 Elsevier Science Ltd Printed in the USA. All rights reserved 0016-7037/02 $22.00 ⫹ .00

Pergamon

PII S0016-7037(00)00979-1

Fossil bacterial ecosystem at methane seeps: Origin of organic matter from Be’eri sulfur deposit, Israel R. Y. P. BURHAN,1,† J. M. TRENDEL,1 P. ADAM,1 P. WEHRUNG,1 P. ALBRECHT,1,* and A. NISSENBAUM2 1

Laboratoire de Ge´ochimie Bioorganique, Ecole Europe´enne de Chimie, Polyme`res et Mate´riauv, Universite´ Louis Pasteur, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France 2 Department of Environmental Sciences and Energy Research, The Weizmann Institute of Science, Rehovot, 76100, Israel (Received July 18, 2001; accepted in revised form June 6, 2002)

Abstract—The Be’eri sulfur mine (Israel) is a unique deposit mainly composed of sandstone intercalated with biogenic mats and possessing organic matter exceptionally depleted in 13C. Molecular and isotopic studies of free and bound biomarkers were performed to unravel the source of the organic matter co-occurring with sulfur in this deposit and to propose a paleoenvironmental model of bacterial life in a type of extreme environment. They showed that the biomarkers are all extremely 13C-depleted and almost exclusively composed of hopanoids and biphytane derivatives of bacterial origin, notably methanotrophic bacteria and acidophilic archaea. ␦13C values of individual components and of bulk organic carbon are in the ⫺80% to ⫺90% range and are among the lowest values ever measured for hopanoids. Organic matter in the sandstone and the mats differ mainly by the occurrence of 3-methylated hopanoids in the mats, which may reflect either different bacterial populations or different conditions of growth. These data demonstrate that the complete biomass of this deposit primarily derives from methanotrophic hopanoid-synthesizing bacteria consuming methane having seeped toward the surface, and that all other organisms—apparently only archaea and bacteria—must have been thriving on methane-derived carbon (methane, CO2, biomass of methanotrophic bacteria). Unambiguous evidence for photosynthetic organisms in the environment of deposition could not be found. The Be’eri sulfur deposit is thus a fossil remain of an exclusively bacterial ecosystem fueled by methane as sole carbon source and having developed in an interstitial aqueous medium within the sandstone. Elemental sulfur from the deposit probably originates from the oxidation of hydrogen sulfide seeping along with methane, which could have been oxidized either abiotically or biologically by sulfur-oxidizing Beggiatoa-like bacteria and archaea. Further oxidation of elemental sulfur might explain the high acidity of the deposit. The oxidizing conditions now prevailing in the Be’eri deposit were revealed by the occurrence of degraded, oxidized, or thiophenic hopanoid structures. Some of them, unambiguously characterized by synthesis, were also obtained by heating hopenes with elemental sulfur, thus suggesting that the latter could play a role, as dehydrogenating and oxidizing agent, in the transformations undergone by organic matter in the Be’eri deposit. Copyright © 2002 Elsevier Science Ltd posed an alternative model in which sulfur was thought to originate from the bacterial oxidation of H2S migrating from the underlying Messinian evaporites at a depth of 400 to 200 m, where it had been formed by bacterial reduction of sulfates. The biomass would, according to these authors, mainly originate from sulfur-oxidizing bacteria. The aim of the present work was to discriminate between the earlier hypotheses for the genesis of this unique deposit on the basis of a more detailed molecular and isotopic study, or eventually to propose a new paleoenvironmental model. We therefore investigated the source of the organic matter cooccurring with sulfur, as well as the particular transformations undergone by this organic matter. For this purpose, free biologic markers, as well as those released by chemical degradation methods from macromolecular organic matter (LiA/H4, RuO4, nickel boride, and Raney nickel) were analyzed by gas chromatography (GC)–mass spectrometry (MS) and gas chromatography–isotope ratio monitoring MS (GC-irmMS). The identification by synthesis of novel hopanoid hydrocarbons, ketones, and thiophenes reflecting the diagenetic transformations undergone by hopanoids in this deposit is also reported.

1. INTRODUCTION

The Be’eri sulfur deposit, located in the Pleistocene sandstones of the Southwestern Mediterranean Coastal Plain of Israel, has been the subject of several investigations as a result of the unusually depleted ␦13C values of the total organic matter (␦13C ⬇ ⫺85‰), which is associated with elemental sulfur (Kaplan and Nissenbaum, 1966; Nissenbaum and Kaplan, 1966; Druckman et al., 1992, 1994). Nissenbaum and Kaplan, (1966) proposed a model involving deposition of the ore in a shallow lagoon fed by Mediterranean water in which the sulfate was reduced to H2S in the anaerobic zone and the sulfide being microbially oxidized to sulfur in the aerobic areas. The source of the extremely light carbon was assumed to be light biogenic methane, which percolated upward to the lagoon and was oxidized to carbon dioxide, which was eventually fixed photosynthetically by algae. Druckman et al., 1992; 1994 pro-

* Author to whom correspondence should be addressed (albrecht@ chimie.u-strasbg.fr). †Present address: Department of Chemistry, Sepuluh Nopember Institute of Technology, Kampus ITS Keputih, Surabaya 60111, Indonesia. 4085

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2.1. Instrumentation 2.1.1. NMR spectroscopy 1 H-NMR spectra were taken on a Bruker AM-400 spectrometer operating at observation frequencies of 400 MHz, and data were recorded at 300 K. Chemical shifts (␦) are reported (ppm) from tetramethylsilane, with the solvent (CDCl3: ␦1H 7.26) used as internal reference. 13C broad band, 1H decoupled and Distortionless Enhancement by Polarization Transfer (DEPT) spectra, homonuclear 1H-1H COSY and NOESY, heteronuclear HSQC, and HMBC experiments were performed on a Bruker ARX-500 spectrometer operating at observation frequencies of 500 MHz for 1H and 125 MHz for 13C. These experiments were performed in CDCl3 (␦1H 7.26, ␦13C 77.16).

2.1.2. GC

to 0.5 (reference: lupenone); alcohols: Rf 0.5 to 0.05 (lupeol). The apolar fraction was further fractionated by TLC (SiO2/hexane), yielding saturated hydrocarbons: Rf 1.0 to 0.9, (reference: elemental sulfur) and aromatics Rf 0.9 to 0.1, (reference: 1,2,5,6-dibenzanthracene). 2.3. Preparation of Kerogen An aliquot of the extracted samples (⬇ 60 g) was treated with 100 mL of a concentrated HCl solution for 4 h. The residue obtained was thoroughly washed with distilled water. In the case of the mats, the sample was further treated with 200 mL of HCl/HF 1:1 solution for 20 h. The black sandstone was treated with HF for 20 h. The kerogens recovered were washed with distilled water until pH 7 was attained. In the case of the sandstone, all the minerals, in particular sand, could not be completely removed by treatment with acids. Organic material was separated from the sand with tweezers. The kerogens obtained were dried in an oven (100°C).

GC was performed on a Carlo Erba 4160 gas chromatograph equipped with an on-column injector, a FID detector, and a DB5 J&W fused silica column (30 m by 0.25 mm, 0.1-␮m film thickness). Hydrogen was used as carrier gas. The temperature program was as follows: (a) 60°C (1 min), 60 to 100°C (10°C/min), 100 to 300°C (4°C/min), 300°C isothermal; (b) 40°C (1 min), 40 to 100°C (10°C/ min), 100 to 300°C (4°C/min), 300°C isothermal; or (c) 80°C (1 min), 80 to 100°C (10°C/min), 100 to 300°C (4°C/min), 300°C isothermal.

2.4. Raney Nickel Hydrogenolysis

2.1.3. GC-MS

Desulfurization of polar fractions with deuterated nickel boride was performed on polar fractions P1 following the procedure described by Poinsot et al. (1995). Saturated hydrocarbons were obtained in ⬇ 2.5% yield in the case of the bacterial mats and in ⬇ 3.0% yield in the case of the black sandstone.

GC-MS analyses were performed either on a Finnigan MAT INCOS 50 spectrometer connected to a Varian 3400 gas chromatograph equipped with an on-column injector and with a J&W DB-5 column (30 m by 0.25 mm, 0.1-␮m film thickness), or on a Finnigan MAT TSQ 700 mass spectrometer connected to a Varian 3400 gas chromatograph (on-column injector, J&W DB-5 column, 60 m by 0.25 mm, 0.1-mm film thickness). Mass spectra were produced at 70 eV and helium was used as carrier gas. Coinjection experiments with the synthetic standards were performed via both an apolar (J&W; DB5 60 m by 0.25 mm; 0.1-␮m film thickness) and a more polar column (J&W, DB17, 60 m by 0.25 mm; 0.1-␮m film thickness).

Desulfurization with Raney nickel was performed on polar fractions P1 following the procedure described by Adam et al. (1993). Saturated hydrocarbons were obtained in ⬇ 1.0% yield in the case of the bacterial mats and in ⬇ 2.0% yield in the case of the black sandstone. 2.5. Deuterated Nickel Boride Desulfurization

2.6. Ruthenium Tetroxide Degradation of Kerogen Oxidation with ruthenium tetroxide of kerogens was performed following the procedure described by Reiss et al. (1997). Monoacids were obtained in ⬇ 1.5% yield in the case of the bacterial mats and in ⬇ 0.4% yield in the case of the black sandstone. 2.7. LiA/H4 Degradation of Macromolecular Organic Matter

2.1.4. GC-irmMS Isotope values (␦13C relative to V-PDB standard) were obtained by a Finnigan MAT 252 spectrometer coupled to a Varian 3400 gas chromatograph fitted with an on column injector and a NiO/Pt combustion reactor set at 1050°C. A DB1 J&W column (60 m by 0.25 mm, 0.1-␮m film thickness) was used with helium as carrier gas (25 cm/s). The temperature program was 80°C to 310°C at 2.5°C min⫺1 (40 min isothermal). The ␦13C values (vs. V-PDB) were calculated by integrating the 44, 45, and 46 ion currents, and the values reported are averages of at least two analyses. ␦13C ⫽ 103 [(Rx ⫺ Rs)/Rs], where R is 13 C/12C, x is the sample, s is the V-PDB standard, and Rs ⫽ 0.0112372. 2.2. Extraction and Fractionation The crushed samples were extracted sequentially with CHCl3/MeOH (3:1 v/v) and toluene/MeOH (3:1 v/v) at 60°C under stirring for 2 h. The organic extracts recovered by centrifugation were combined. After removal of the solvent under reduced pressure, the acids were separated on silica gel impregnated with potassium hydroxide following the method described by McCarthy and Duthie (1962), yielding a fraction of neutral compounds (elution with diethylether) and a fraction of acids (elution with 2 and 5% solutions of formic acid in diethylether). Elution with CHCl3/MeOH/H2O (65/25/4) affords the polars (P1). The acids were esterified with diazomethane and the esters separated into three fractions by thin-layer chromatography (TLC) (SiO2/CH2Cl2) by use of synthetic references, as follows. Monoesters: Rf 0.9 to 0.5 (reference: stearic acid methyl ester); diesters: Rf 0.5 to 0.25 (reference: phtalic acid methyl ester); and polyesters: Rf 0.25 to 0.05 (reference: pyromellitic acid methyl ester). The neutral fraction was further fractionated by TLC (SiO2/CH2Cl2 and SiO2/hexane, respectively) by use of references. SiO2/CH2Cl2; Apolar compounds: Rf 1.0 to 0.8; ketones: Rf 0.8

HI/LiALH4 degradation has been performed on polar fractions P1 (see 2.2.) isolated from the mats and from the black sandstone and, also, directly on the extracted sandstone. An aliquot (⬇ 60 mg) of the polar fraction and 10 mL of hydroiodic acid (58% aqueous) were heated in a glass tube under argon at 110°C. After 6 h, the mixture was poured into distilled water and extracted with CH2Cl2. Iodides were separated from the crude extract by TLC (SiO2/hexane; Rf ⬎ 0.6). Iodides dissolved in 15 mL anhydrous THF were refluxed under argon with a large excess of LiAlH4. After 1 h, the reaction mixture was slowly poured into distilled water and the formed alkanes were extracted with diethylether (bacterial mats: 6% yield; black sandstone: 5% yield). An aliquot of the extracted sandstone (50 g) was covered with hydroiodic acid (58% aqueous) and the mixture was refluxed for 3 h. The mixture was poured into water and extracted with CH2Cl2. Iodides were isolated and reduced to alkanes following the procedure described above. 2.8. Synthesis of 17␣,18-dimethyl-des-E-hopane 17␣,18-Dimethyl-des-E-hopane 11 (bold numbers refer to structures in the Appendix) was synthesized from 17 ␣-formyl-18-vinyl-des-Ehopane 12 (Trendel and Albrecht, 1984) (Fig. 1). Wolff-Kishner reduction of 17 ␣-formyl-18-vinyl-des-E-hopane 12 (Huang-Minlon, 1946) yielded 17 ␣-methyl-18-vinyl-des-E-hopane in 58% yield. The latter was treated with ozone affording an aldehyde (81% yield), which gave 17␣,18-dimethyl-des-E-hopane 11 in 90% yield by Wolff-Kishner reduction. All synthetic intermediates gave satisfactory analytical data (MS, NMR). 17␣,18-Dimethyl-des-E-hopane 11: 1H-NMR (CDCl3, 400 MHz) 0.640 (3H, s), 0.775 (3H, s), 0.788 (3H, d, J ⫽ 6.5 Hz, H-21), 0.806 (3H, s), 0.826 (3H, s), 0.832 (3H, s), 0.921 (3H, s), 0.942 (3H, s).

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2.10. Geological Setting

Fig. 1. Synthesis of tetracyclic hydrocarbon 11 from tetracyclic aldehyde 12.

GC-MS (EI, 70 eV): 358 [M⫹] (18%), 343(8), 205(5), 191(100), 137(62).

2.9. Preparation of hop-17(21)-en-20-one 25a and hopa15,17(21)-dien-20-one 27a Hop-22(29)-ene (15 mg) and elemental sulfur (30 mg) were heated at 200°C for 1 h in a glass tube sealed under vacuum. The mixture obtained was fractionated by TLC (SiO2/hexane) yielding a polar fraction Rf ⬍ 0.05 containing compounds 25a and 27a. Further fractionation of the latter by TLC (SiO2/CH2Cl2: hexane) and high-performance liquid chromatography (HPLC; Du Pont 250⫻4.6 mm, Zorbax ODS, 5 ␮m, 80 Å; MeOH; 1 mL/min) led to the isolation of compounds 25a and 27a (⬇ 2mg each) with a purity of 95% (GC). These compounds were characterized by NMR studies including homonuclear (1H-1H, COSY, and NOESY) and heteronuclear (1H-13C, HSQC and HMBC) correlation experiments, which allowed assignment of the signals of all the protons and all the carbon atoms. Hop-17(21)-en-20-one 25a. 1H-NMR (CDCl3, 500 MHz) 0.74 (H-5), 0.79 (H␣-1), 0.799 (3H, s, H-24), 0.822 (3H, s, H-25), 0.859 (3H, s, H-23), 0.905 (3H, s, H-26), 1.088 (3H, s, H-28), 1.14 (H␣-3), 1.158 (3H, s, H-27), 1.158 (3H, d, 7.5 Hz, H-30), 1.173 (3H, d, 7.5 Hz, H-29), 1.28 (H␤-11), 1.32 (2H, H␤-6, H-9), 1.36 (H␤-3), 1.39 (H␣-2), 1.40 (H␤-12), 1.49 (2H, H-7), 1.50 (3H, H-13, H␣ ⫺15, H␤-15), 1.51 (H␣-12), 1.52 (H␣-11), 1.53 (H␣-6), 1.58 (H␤-2), 1.66 (H␤-1), 2.08 (2H, H-19), 2.35 (H␣-16), 2.71 (H␤-16), 2.74 (sept, H-22). 13C-NMR (CDCl3, 125 MHz) 15.56 (C-27), 16.07 (C-25), 16.39 (C-26), 18.65 (C-2), 18.69 (C-6), 20.87 (C-29), 21.02 (C-30), 21.05 (C-11), 21.58 (C-24), 22.29 (C-16), 22.48 (C-28), 24.40 (C-22), 24.72 (C-12), 33.19 (C-7), 33.20 (C-15), 33.31 (C-4), 33.41 (C-23), 37.50 (C-10), 40.43 (C-1), 41.63 (C-8), 42.10 (C-3), 42.24 (C-14), 44.67 (C-18), 48.30 (C-13), 50.75 (C-9), 53.84 (C-19), 56.28 (C-5), 140.18 (C-21), 180.51 (C-17), 208.06 (C-20). GC-MS (EI, 70 eV): 424 [M⫹] (6%), 409(4), 231(5), 191(29), 152(100), 107(16). Hop-15,17(21)-dien-20-one 27a. 1H-NMR (CDCl3, 500 MHz) 0.79 (H-5), 0.81 (H␣-1), 0.807 (s, H-25), 0.812 (3H, s, H-24), 0.873 (3H, s, H-23), 0.901 (3H, s, H-26), 1.065 (3H, s, H-28), 1.14 (H␣-3), 1.160 (3H, d, 7.0 Hz, H-29), 1.172 (3H, d, 7.0 Hz, H-30), 1.205 (3H, s, H-27), 1.31 (H␣-11), 1.35 (H-9), 1.38 (2H, H␣-2, H␤-3), 1.40 (H␤-6), 1.50 (H␤-7), 1.52 (2H, H-12), 1.58 (H␤-2), 1.61 (2H, H␣-6, H␣-7), 1.65 (H␤-1), 1.70 (H␣-11), 1.90 (H-13), 2.08 (H␤-19), 2.22 (H␣-19), 2.77 (sept, 7.0 Hz, H-22), 6.14 (H-15), 6.59 (d, 10.5 Hz, H-16). 13C-NMR (CDCl3, 125 MHz) 15.83 (C-27), 15.62 (C-25), 19.38 (C-26), 18.69 (C-2), 18.69 (C-6), 20.69 (C-29), 21.26 (C-30), 21.47 (C-11), 21.53 (C-24), 23.36 (C-12), 24.39 (C-22), 25.15 (C-28), 32.98 (C-7), 33.34 (C-23), 33.41 (C-4), 37.80 (C-10), 40.18 (C-1), 41.81 (C-8), 42.07 (C-3), 42.56 (C-18), 44.88 (C-13), 46.36 (C-14), 50.38 (C-9), 52.96 (C-19), 56.48 (C-5), 119.87 (C-16), 139.09 (C-21), 144.94 (C-15), 170.75 (C-17), 207.70 (C-20). GC-MS (EI, 70 eV): 422 [M⫹] (30%), 286(3), 243(12), 203 (79), 202(100), 173(34).

Location and geology of the Be’eri sulfur deposit are described in detail by Nissenbaum and Kaplan (1966) and Druckman et al. (1992, 1994). The Be’eri sulfur quarries are situated ⬃2.5 km southwest of Kibbutz Be’eri on top of an 80-m-high eolianite ridge, which belongs to the upper part of the Pleshet Formation and is of Upper Pleistocene age. The stratigraphy of the quarries is highly variable, both vertically and laterally. Nissenbaum and Kaplan (1966) and Druckman et al. (1994) recognized five units that overlie the eolianite, including a unit of black sandstone, rich in humic material, which laterally interfingers with brown sandstones with biogenic mats. These units are overlain with a more clayey zone with limonite horizons, which is in turn covered by friable sandstone bleached and decarbonated by sulfuric acid formed by oxidation of elemental sulfur. The gypsum observed in this unit has therefore probably a secondary origin (Druckman et al., 1994). The top layer is an eolic loess. The unit containing the biogenic mats is 0 to 3 m thick and is composed of brown, silty sandstone. This unit is characterized by black, brittle organic mats, ⬃2 to 3 mm thick. In thin sections, no structures other than parallel layering were observed. The Pleistocene eolianite sequence is underlain by a thick column of the clayey Neogene Saquie group. This sequence contains the Mavqi’im Formation of Miocene age, which is composed of anhydrite and carbonates. Sulfur from Be’eri is thought to originate from these evaporites (Druckman et al., 1994). Hydrogen sulfide gas associated with methane (␦13C ⫽ ⫺70%, Nissenbaum and Goldberg, 1988) and probably resulting from the reduction of the underlying sulfates (Druckman et al., 1994) has indeed been observed in a borehole near Be’eri. The sulfur isotopic composition of elemental sulfur from the Be’eri sulfur quarries shows a slight enrichment in 34S (␦34S ⫽ ⫹27%) relative to the Messinian sulfates (␦34S ⫽ ⫹22 to 24%; Nissenbaum and Kaplan, 1966). The black sandstone (TOC 1.3%) and the mats (TOC 15.7%) investigated were sampled in the more organic rich layers in the sulfur-rich horizon. 3. RESULTS AND DISCUSSION

3.1. Saturated Hydrocarbons 3.1.1. Hopanes Analysis of the saturated hydrocarbons from the black sandstone and the mats (Fig. 2) shows that these fractions are exclusively composed of hopanoid hydrocarbons. The distribution of the regular hopanes (1, 2, 3; bold numbers refer to structures in the Appendix) extends from C27 to C33. The 17␤(H), 21␤(H) isomers 1 indicative of the immaturity of the samples are largely predominant (e.g., Seifert and Moldowan, 1980). All the other isomers 17␣(H), 21␤(H) 2 and 17␤(H), 21␣(H) 3 are present to limited extent. The C27 17␤(H)hopane 1a and the C29 17␤(H), 21␤(H) hopane 1b dominate the distribution. Predominance of the short-chain homologues can be interpreted as indicative of rather oxidizing paleoconditions but may also indicate that the precursor hopanoid lipids were functionalized at position 29 such as aminobacteriohopanepentol 4 abundant in methanotrophic bacteria (e.g., Methylococcus capsulatus or Methylomonas methanica; Neunlist and Rohmer, 1985; Zundel and Rohmer, 1985a). This particular hopanoid, functionalized at position 29, cannot, however, be considered as exclusive precursor of C29 hopanes, considering the high sensitivity of side chains from biologic hopanoids (Peiseler, 1992). 3␤-Methylhopanes (5, 6, 7) have been detected in the biogenic mat sample. Their distribution perfectly mimics that of the regular hopanes. Because the samples investigated are very immature, hopanes and methylhopanes likely derive from ho-

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Fig. 3. Mass spectrum (EI, 70 eV) of tetracyclic hopanoid hydrocarbon 11 present in the black sandstone and the bacterial mats from the Be’eri deposit.

Methylococcus capsulatus, 3␤-methylated hopanoids were predominantly biosynthesized in the stationary phase of cell growth, whereas their nonmethylated counterparts appeared mainly in the exponential growth phase. This clearly suggests that cell density and availability of methane may influence extent of biosynthesis of methylhopanoids in a given bacterium. The strong similarities between the distributions of the hopane homologues in the two samples suggest that the biologic lipids have undergone similar diagenetic transformations under rather oxidizing conditions. 3.1.2. Secohopanes

Fig. 2. Gas chromatogram of saturated hydrocarbon fraction from (a) the black sandstone and (b) the bacterial mats from the Be’eri deposit.
, 17␤(H), 21␤(H) hopanes 1; 䊐, 17␣(H), 21␤(H) hopanes 2; 䡬, 17␤(H), 21␣(H) hopanes 3; 䢇, 17␤(H), 21␤(H) 3␤-Me-hopanes 5. ■, 17,21-secohopanes 8. Œ, 3␤-Me-17,21-secohopanes 9. ⽧,17␤(H), 21␣(H) 3␤-Me-hopanes 7. ␦13C values are indicated above each peak. Temperature program, condition a (see section 2.1).

panoid precursors bearing the same functionalities in the side chain. 3-Methylated hopanoids, precursors of 3␤-methylhopanes, are biosynthesized by various microorganisms such as methylotrophic bacteria (Methylococcus capsulatus or Methylomonas methanica; Neunlist and Rohmer, 1985; Zundel and Rohmer, 1985b) or Acetobacter species (Zundel and Rohmer, 1985b,c). Respective contributions of regular and 3␤-methylhopanes (1–3, 5–7) are of the same order of magnitude in the mats, whereas the latter are absent in the black sandstone. This difference in the hopane distribution may be due to differences in the bacterial populations, although the possibility that the microorganisms thriving in the sandstone and in the mat are similar cannot be excluded. Indeed, the presence of methylated hopanes (5–7) in the mats may be due to differences in the paleoenvironmental conditions favoring biosynthesis of methylated hopanoids. Such a hypothesis is supported by an observation made by Summons et al. (1994). Indeed, in cultures of

Besides the predominant pentacyclic hopanes (1–3, 5–7), minor 17(21)-secohopanes 8 (C24-C26) could also be detected (Trendel et al., 1982). Position 3 methylated homologues 9 also occur in the mats. These secohopanes are supposed to derive from pentacyclic hopanoids by thermal degradation of ring E or by (microbial or abiotic) oxidation of hop-17,21-enes 10 (e.g., Aquino Neto et al., 1983; Tritz et al., 1999). The latter hypothesis is more likely in the case of our immature samples, which did not undergo high thermal stress. 17(21)-Secohopanes 8 are accompanied by a minor C26 tetracyclic alkane 11, to which the structure of 17␣,18-dimethyl-des-E-hopane could be ascribed on the basis of mass spectral data (Fig. 3). This compound could be conclusively identified by comparison of mass spectrum and chromatographic behavior (coinjection on two GC columns coated with two different phases) with those of a standard obtained from 17␣-formyl-18vinyl-des-E-hopane 12 (Trendel and Albrecht, 1984) following the synthetic scheme shown in Figure 1. The 3␤-methylated homologue 13 has also been detected in the mats (Fig. 2). Formation of these compounds are clearly the result of cleavage reactions affecting the C(20)-C(21) bond in ring E of pentacyclic hopanoids. Such a transformation affecting hopanoids in sediments has, up to now, never been reported. 3.1.3. Carbon isotopic composition of hopanes The carbon isotopic composition of the hopanes (1–3, 5–7) and 17(21)-secohopanes (8, 9) measured by GC-irmMS shows an exceptional depletion in 13C. Indeed, the ␦13C values, between ⫺79% and ⫺93%, are among the lowest ever measured

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Table 1. Carbon isotopic composition of selected hopanoid saturated hydrocarbons from the sandstone and the bacterial mats from Be’eri deposit

␦ Hopanes C25 (18␣H)-3␤-methyl-des-E-hopane C25 18-methyl-des-E-hopane C26 17␣,18-dimethyl-des-E-hopane C27 3␤,17␣,18-trimethyl-des-E-hopane C26 18-ethyl-des-E-hopane C27 18-ethyl-3␤-methyl-des-E-hopane C27 (17␣H)-22,29,30-trinorhopane C28 (17␣H)-3␤-methyl-22,29,30trinorhopane C27 (17␤H)-22,29,30-trinorhopane C28 (17␤H)-3␤-methyl-22,29,30trinorhopane C29 (17␣H,21␤H)-30-norhopane C30 (17␣H,21␤H)-3␤-methyl-30-norhopane C29 (17␤H,21␣H)-30-norhopane C30 (17␤H,21␣H)-3␤-methyl-30-norhopane C29 (17␤H,21␤H)-30-norhopane C30 (17␤H,21␤H)-3␤-methyl-30-norhopane C30 (17␤H,21␤H)-hopane C31 (17␤H,21␤H,22R)-29-methylhopane C32 (17␤H,21␤H,22R)-29-ethylhopane

for hopanoids (Table 1), which clearly indicates that the hopanoid-synthesizing bacteria are directly or indirectly living on a 13C-depleted carbon source such as methane. Thus, part of the hopanoids most likely derives from aerobic methanotrophic bacteria such as Methylococcus capsulatus or Methylomonas methanica (Neunlist and Rohmer, 1985; Zundel and Rohmer, 1985b), as was already suggested by the distribution of the various homologues (see above). This constitutes an exceptional situation. Indeed, 13C-depleted hopanes deriving from methanotrophic bacteria are frequently encountered in sediments, but they generally co-occur along with less 13C-depleted biomarkers from various origins (e.g., Freeman et al., 1990; Collister et al., 1992), indicating that all the hopanes do generally not originate from methanotrophic bacteria. Frequently, only the C29 homologue shows an important depletion in 13C (Freeman et al., 1990). Summons et al. (1994) showed that assimilation of methane by methanotrophic bacteria is accompanied by an isotopic fractionation of approximately ⫺30% for the biosynthesis of hopanoids when they are cultivated under methane nonlimited conditions. Thus, the ␦13C value of approximately ⫺70% measured for methane from the underlying Neogene Saquie formation (Nissenbaum and Goldberg, 1988) suggests that the methanotrophic bacteria from Be’eri are probably growing under methane-limited conditions. It is noteworthy that hopanes are slightly less depleted in the mats than in the sandstone, which may be linked to the occurrence of other bacterial species (Table 1). It is, however, also possible that the observed reduction of the carbon isotopic fractionation is due to a limitation of methane (which must have the same origin in the sandstone and in the mats) at high cell densities in the bacterial mats. Moreover, 3␤-methylated hopanes from the mats appear to be less depleted (⫹4%) than nonmethylated hopanes (Table 1), similar to the situation observed in a culture of Methylococcus capsulatus (Summons et

Sansdstone

13

C (%␦) Mats

— ⫺90.0 ⫺87.2 — ⫺87.6 — ⫺93.0 —

⫺83.0 ⫺87.5 ⫺86.2 ⫺81.3 ⫺87.9 ⫺80.6 ⫺86.6 ⫺82.8

⫺91.9 —

⫺89.0 ⫺85.0

⫺87.4 — ⫺91.2 — ⬃⫺92.0 — ⬃⫺90.0 ⫺91.5 ⫺91.4

— ⫺85.1 ⫺88.9 ⫺85.3 ⬃⫺89.5 ⫺84.9 ⬃⫺92.3 ⫺84.9 —

al., 1994). On the average, differences of approximately 5 to 10% of the ␦13C values were observed depending on conditions and on stage of growth. This was interpreted as being possibly due to the carbon isotopic composition of the 3␤-methyl group (Summons et al., 1994), which is thought to originate from a methionine methyl group (Zundel and Rohmer, 1985c). Alternatively, Summons et al. (1994) envisaged the possibility that methylated and nonmethylated hopanoids derive from isotopically and spatially distinct squalene pools, although they considered this possibility as unlikely. As mentioned above, Summons et al. (1994) observed that in cultures of Methylococcus capsulatus biosynthesis of 3␤-methylated hopanoids is controlled by the growth conditions. Thus, the concomitant occurrence of 3␤-methylhopanoids, as well as the slightly reduced isotopic fractionation in the mats, may be due to different growth conditions in the mats and in the sandstone. These differences may reflect denser bacterial populations (bacterial mats) resulting in limitation of methane rather than occurrence of different bacterial populations. In conclusion, the fact that the saturated hydrocarbon fraction is exclusively composed of 13C-depleted hopanes that have a carbon isotopic composition similar to that of the bulk organic matter from the Be’eri deposit suggests that methanotrophic bacteria are the primary producers of the whole paleoecosystem that may, however, contain other (heterotrophic), eventually hopanoid synthesizing, bacteria. The similarity of the carbon isotopic composition of saturated hydrocarbons and of bulk organic matter is surprising because the carbon isotopic composition of the bulk is generally more representative of that of the biomass (comprising sugar, amino acid– derived carbon) and is thus generally less 13 C-depleted than the hydrocarbons, which are more representative of carbon isotopic composition of the lipids. This may be due to an unusual aliphatic character of the kerogen. Indeed, as observed by Nissenbaum and Kaplan (1966), the kerogen in the

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Be’eri deposit is solely composed of humic substances (separated from the sandstone matrix by 0.1 N NaOH extraction), which are characterized by an unusually high aliphaticity. This aliphaticity is clearly evidenced by bulk spectrometric analyses (13C-NMR and infrared spectroscopy; Nissenbaum et al., 2001) and is probably related to the particular origin of the kerogen, which apparently exclusively derives from a bacterial and archaeal biomass (see below). The oxidizing conditions, as well as the low pH now prevailing in this deposit, may have enhanced the aliphatic character of the kerogen by favoring hydrolysis of biopolymers based on sugars and amino acids followed by oxidation of the most sensitive monomers. This particular point is further supported by the almost complete absence of spectrometric evidence (13C-NMR) for sugars in the humic substances. The high solubility in alkaline solutions of the major part of the (highly aliphatic) organic material from Be’eri might be ascribed to the occurrence of sulfonate functions formed by oxidation of S-bridges present in the macromolecular structures, a hypothesis that we are now examining more closely. Occurrence of mats constituted of a biomass predominantly deriving from methanotrophic bacteria, as suggested by the abundance and carbon isotopic composition of hopanes, has been reported from Tanzania (Mpanju and Philp, 1994). A similar situation has also been observed in Kuwait, where methanotrophic bacteria developed at oil seepages (Connan et al., 1999). At these seepages, organic matter is composed of a mixture containing mature bitumen that has seeped to the surface and biogenic mats. These observations show that ecosystems such as Be’eri are more widely occurring and might thus play a significant role in the consumption of methane coming from the subsurface. 3.2. Alkenes and Aromatic Compounds Alkenes are dominated by C27 and C30 hop-17(21)-enes ([M⫹] ⫽ 368 ⫹ nx14, fragment at m/z 231) 10. On the basis of MS investigation, several unknown hopanoid mono- ([M⫹] ⫽ 368 ⫹ nx14) and dienes ([M⫹] ⫽ 366 ⫹ nx14) were also detected. Among the aromatic hydrocarbons, two series of benzohopanes—those cyclized at C-20 14 ([M⫹] ⫽ 446 ⫹ nx14; C32-C35, Hussler et al., 1984a,b) and those cyclized at C-16 15 ([M⫹] ⫽ 432 ⫹ nx14; C31-C35, Schaeffer et al., 1995a)—are the predominant constituents. Their 3␤-methylated homologues (16, 17) are present in the mats. Thiophenic hopanoids extending up to C35 ([M⫹] ⫽ 508 for C35 homologue) are present in traces in the black sandstone and are absent in the mats. We have identified hopanoids bearing the thiophenic ring in the side chain (e.g., 18, Valisolalao et al., 1984; Sinninghe Damste´ and de Leeuw, 1990) as well as compounds 19 and 20 having a thiophenic ring fused to ring E of the hopanoid skeleton ([M⫹] ⫽ 438 ⫹ nx14 and [M⫹] ⫽ 436 ⫹ nx14, respectively). These hopanoids were obtained by Bisseret and Rohmer (1990, 1993) in experiments involving hopanoid hydrocarbons and elemental sulfur. Compounds 19 and 20 (R⫽H) could be identified in our sample by comparison of mass spectra and chromatographic behavior (coelution on two columns coated with two different phases) with those of reference compounds synthesized following the procedure described by Bisseret and Rohmer (1990, 1993). This is the first

report of these compounds from a sedimentary source. Their presence in our samples may be related to the particular sulfurization processes likely to have occurred in the sulfur Be’eri deposit and involving oxidation of organic matter by elemental sulfur (Alexander et al., 1987). 3.3. Ketones The ketones, in fact hopanoid ketones (21–23), are very similar in the black sandstone and in the mats (Fig. 4). Again, distributions in both samples differ mainly by the presence of significant amount of 3␤-methylated homologues in the mats. Their distribution extends from C27 to C33 (C28-C34 in the case of the 3␤-methylated counterparts). Some minor tetracyclic ketones (e.g., 24) have also been detected, and on the basis of mass spectral data ([M⫹] ⫽ 344 ⫹ nx14; fragments at m/z 191, m/z 343), they were thought to result from the degradation of ring E of hopanoids. A more polar fraction (Fig. 5), “alcohol” fraction; see Experimental Methods) contains a major series of hopanoid derivatives that, on the bases of their mass spectra (Fig. 6) and on their retention factor on silica gel, were ascribed to ␣,␤-unsaturated hopanoid ketones bearing the carbonyl and the double bond on ring D or E. Their mass spectra (Fig. 6a) display a molecular ion at m/z 382 ⫹ nx14 (m/z 396 for 3␤-methyl homologues), an intense fragment at m/z 191 (m/z 205 for 3␤-methyl homologues), which indicates that the functionality is located on rings D or E as well as an even fragment at m/z 110 ⫹ nx14, which increases with the molecular ion. A minor series of hopanoids could also be detected, and on the basis of mass spectral data and polarity, this series was supposed to correspond to diunsaturated ketones. Mass spectra of these compounds (Fig. 6b, [M]⫹380 ⫹ nx14) show intense even fragments at m/z 160 ⫹ nx14 increasing with the molecular ion. The structure of the hopadienones was thought to differ from that of the hopenones described above by the presence of an additional conjugated double bond. The C30 homologues from both series 25a and 27a occurring in Be’eri samples were obtained in small amounts by heating hop-22(29)-ene with elemental sulfur at 200°C for 1 h (Burhan, 1996). These ketones 25a and 27a could be isolated by reverse phase HPLC and characterized by NMR studies, which allowed us to establish unambiguously the structures of the isolated compounds as, respectively, hop-17(21)-en-20-one 25a and hopa-15,17(21)-dien-20-one 27a. Comparison of mass spectra, and of chromatographic behaviors confirmed that synthetic and naturally occurring unsaturated ketones are identical. Formation of ketones 25a and 27a by heating hop-22(29)-ene with elemental sulfur may have resulted from allylic oxidation of hop-17(21)-ene 10a into unsaturated thioketones, further hydrolyzed to the corresponding ketones. These compounds could have been formed in the Be’eri deposit by oxidation of the highly sensitive allylic position of hop-17(21)-enes 10 present in our samples and resulting either from elimination of an alcohol group on hydroxyhopanoids followed by migration of the double-bond (favored by the low pH prevailing in the Be’eri deposit; Nissenbaum and Kaplan, 1966) or by bacterial dehydrogenation of saturated hopane skeletons (Tritz et al., 1999). The oxidation process leading to incorporation of the carbonyl group at position 20 may be

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Fig. 4. Gas chromatograms of the ketone fractions from (a) the black sandstone and (b) the bacterial mat from the Be’eri deposit. 䡬: tetracyclic hopanoid ketones, 䢇: unsaturated hopanoid ketones. A prime indicates that hopanoid ketone is 3-methylated. Temperature program: condition a (see section 2.1).

abiotically induced and may involve oxygen or even elemental sulfur as suggested by the heating experiment performed with hop-17(21)-ene 10a. Presence of these unsaturated hopanoid ketones clearly reflects the oxidizing conditions prevailing in the Be’eri deposit.

3.4. Acids The monoacid fraction from the sandstone (Fig. 7) is exclusively composed of hopanoid acids (31–34). Analysis of the acids from the mats shows occurrence of linear acids beside the

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Fig. 5. Gas chromatograms of the “alcohol” fractions from (a) the black sandstone and (b) the bacterial mat from the Be’eri deposit. A prime indicates that hopanoid is 3-methylated. Unlabeled peaks are unknown hopanoid oxygenated derivatives as suggested by occurrence of fragment m/z 191 in their mass spectra. Temperature program: condition b (see section 2.1).

hopanoids. Their distribution is dominated by the C16 isomer of unknown origin. On the basis of mass spectral data ([M⫹] 416 ⫹ nx14, fragments at m/z 191 and 343), tetracyclic acids 34 could be related to the 20, 21 secohopanoid hydrocarbon 11 detected in the saturated hydrocarbon fractions.

3.5. Intact Biohopanoids Intact biohopanoids have only rarely been reported from sedimentary sources, mainly because these compounds are highly sensitive to degradation and hence rapidly transformed.

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Fig. 6. Mass spectra (EI, 70eV) of (a) hop-17(21)-en-20-one 25a and (b) hopa-15,17(21)-dien-20-one 27a.

Detection of bacteriohopanetetrol 29, for example, was therefore restricted to some recent sediments (e.g., Rohmer et al., 1980; Boon et al., 1983; Ries-Kautt and Albrecht, 1989; Innes et al., 1997, 1998). It has also been obtained by chemical degradation of the kerogen from the immature Messel Shale (Eocene, Germany) to which it was covalently bound (Mycke et al., 1987). MS-MS investigation of an acetylated polar fraction isolated from the mats specifically searching for acetylated aminobacteriohopanetriol 30a (daughter ions of [M⫹] 713) clearly revealed the presence of a hopanoid having a mass spectrum identical to that of a standard (Fig. 8), thus confirming its presence in the mats. Aminobacteriohopanetriol 30 occurs in various bacteria and has, notably, been shown to be biosynthesized by methanotrophs (Neunlist and Rohmer, 1985). On the other hand, specific search by the same approach for aminobacteriohopanepentol 4, an ideal precursor for short chain hopanoids (C27 and C29), was unsuccessful. Because the hopanoids detected in the sandstone and in the mats are rather degraded reflecting the oxidizing conditions prevailing in the Be’eri deposit, the occurrence of labile intact biohopanoids is surprising. Preservation of aminobacteriohopanetriol might be explained by its sequestration in intact bacterial structures such as those evidenced by microscopy by Druckman et al. (1994).

Fig. 7. Gas chromatograms of the fractions of the acids (as methylesters) from (a) the black sandstone and (b) the bacterial mats from the Be’eri deposit. 䡬, linear acids; ■, 17␤(H), 21␤(H) hopanoic acids; 䊐, 17␤(H), 21␤(H) 3␤-Me-hopanoic acids;
, 17␤(H), 21␣(H) hopanoic acids. Temperature program: condition b (see section 2.1).

isomers 1) and thus differs slightly from that of the free hydrocarbons that culminates at the C32 homologue. 17,21 secohopanes (C24-C26) 8, as well as 17␣,18-dimethyl-des-Ehopane 11 are also present. In the bacterial mat, all the hopanes are accompanied by their 3␤-methylated (5 to 7) homologues. Steroids and carotenoids, which would be indicative of contributions from photosynthetic organisms and frequently occur as bound subunits in sulfur cross-linked macromolecules (Schmid, 1986; Sinninghe Damste´ and de Leeuw, 1990; Kohnen et al., 1991; Adam et al., 1993; Richnow et al., 1993; Schaeffer et al., 1995b; Hartgers et al., 1996), are absent. Desulfurization with deuterated reagents (nickel boride; Back et al., 1993; Schouten et al., 1993; Schaeffer et al., 1995b) resulted in the release of a mixture of hopanes, similar to that obtained with Raney nickel, and bearing several deuterium atoms in the side-chain (with traces of up to 10 deuterium atoms for the C35 homologue). Although part of the hopanes obtained upon Nickel boride treatment are probably the result of the release of S-bound hopanoids, it cannot be excluded that another (major?) part results from deuterolysis of polyfunctionalized (oxygenated, unsaturated) hopanoids (see section 3.5) (e.g., Hartgers et al., 1996) occurring in our immature samples.

3.6. Desulfurization of Polar Fractions The saturated hydrocarbons obtained by Raney nickel desulfurization (Fig. 9) of polar fractions from the black sandstone are exclusively composed of hopanes (1 to 3). Their distribution extends from C27 to C35 (predominantly 17␤(H), 21␤(H)

3.7. Chemical Degradation of Polar Fraction and Kerogen with HI/LiAlH4 Degradation with HI/LiAlH4 of polar fractions isolated from the sandstone and the mats, as well as of the kerogen from the

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Fig. 8. MS-MS mass spectra (daughter ions of [M⫹] 713, CID, ⫺15 eV, argon) of (a) acetylated aminobacteriohopanetriol 30 present in the black sandstone from the Be’eri deposit and (b) an acetylated standard.

sandstone has been performed to track biomarkers attesting for the presence of organisms in the Be’eri deposit different from those evidenced by the analysis of the free lipids. This reagent is able to release ether- and ester-bound subunits from macromolecular substances as saturated hydrocarbons. Hopanes (1–3) that derive most probably from lipids from methylotrophic bacteria, as suggested by their low ␦13C values (⬇ ⫺85%), were the predominant hydrocarbons released (Fig. 10). Besides, phytane 35, one monocyclic 36 and two bicyclic derivatives 37 of biphytane were recognized on the basis of comparison with published mass spectra (De Rosa et al., 1977a; Chappe et al., 1980). Phytane 35 may derive from glycerol diethers 38 present in almost all types of archaea: methanogens, halophiles, thermophiles and acidophiles (Kates, 1993). Glycerol diethers such as 38 could, however, not be detected by GC-MS suggesting that these compounds are mainly occurring in Be’eri as bound constituents of macromolecular substances. The mono- and bicyclic biphytane derivatives (36 and 37) most probably correspond to the lipidic part of glycerol tetraethers present in archaea (e.g., 39). The occurrence of the bicyclic biphytanes as a mixture of two isomers with identical mass spectra is uncommon. Indeed, bicyclic biphytane has up to now only been detected in sediments as a single compound (e.g., Chappe et al., 1980; Schouten et al., 1998). Comparison of chromatographic behavior (coelution in GC) and of mass spectra of a bicyclic biphytane obtained from Sulfolobus solfataricus (De Rosa et al., 1983) and of the bicyclic biphytane derivatives obtained from the Be’eri samples shows that the last eluting isomer (Fig. 10) corresponds to that occurring in thermoacidophilic archaea. Because mass spectra of the two bicyclic biphytane isomers 37 are almost identical, we infer that the

Fig. 9. Gas chromatograms of the saturated hydrocarbons obtained upon Raney nickel desulfurization of a polar fraction from (a) the black sandstone and (b) the bacterial mats from the Be’eri deposit.
, 17␤(H), 21␤(H) hopanes 1; 䊐, 17␣(H), 21␤(H) hopanes 2; 䡬, 17␤(H), 21␣(H) hopanes 3; 䢇, 17␤(H), 21␤(H) 3␤-Me-hopanes 5;⽧, 17␤(H), 21␣(H) 3␤-Me-hopanes 7; ■, 17,21-secohopanes 8; Œ, 3␤-Me-17,21secohopanes 9. Temperature program: condition c (see section 2.1).

early eluting isomer from the Be’eri samples differs by configurations at asymmetric centers located either on the rings or at the methyl-bearing carbon atoms. Because of the immaturity of the Be’eri deposit, presence of isomers is probably not the result of thermal isomerization but rather reflects the presence of a mixture of isomeric bicyclic C40 compounds in the precursor organism. Although mass spectra of the two bicyclic biphytane isomers 37 are almost identical, it cannot be completely excluded that the rings on the early eluting bicyclic compound from Be’eri are located at other positions. Moreover, a C41 bicyclic component 40 that, on the basis of mass spectral data (Fig. 11), is clearly related to cyclized biphytanes 37 has also been detected and has, to the best of our knowledge, never been reported in the literature. Interpretation of its mass spectrum suggests that there is an additional methyl group located between the two rings (Fig. 11). It is noteworthy that Arigoni (personal communication) has characterized an acyclic biphytane–related C41 isoprenoid that bears an additional methyl group at position 13 in a methanogenic bacterium. Because acyclic biphytane 41 was not detected, it can be

Fossil bacterial ecosystem at methane seeps

Fig. 10. (a) Gas chromatogram of the saturated hydrocarbons obtained upon HI/LiAlH4 treatment of a polar fraction from the black sandstone of the Be’eri deposit. (b) Partial gas chromatogram showing the distribution of the biphytane derivatives.
, 17␤(H), 21␤(H) hopanes 1; ●, 17␤(H), 21␣(H) hopanes 2. Temperature program: condition c (see section 2.1).

inferred that methanogens were probably not present in the environment of deposition although the occurrence of cyclized counterparts in methanogens has been envisaged on the basis of geochemical data (Chappe et al., 1982). Hence, the origin of the cyclized C40 biphytanes is difficult to assess. These skeletons have up to now only been reported from thermophilic or thermoacidophilic archaea (De Rosa et al., 1977a– c, 1983; De Rosa and Gambacorta, 1988; Kates, 1993; Koga et al., 1993), but geological studies (Nissenbaum and Kaplan, 1966; Druckman et al., 1994) indicate that conditions favorable for the development of thermophilic bacteria never prevailed in the Be’eri deposit. Thus, the archaea from which these cyclized biphytane derivatives originate must have been tolerant to low temperature conditions. More recently, however, Hoefs et al. (1997) and Schouten et al. (1998) brought evidence for the existence of “planktonic” archaea that live at low temperature in the marine water column and are able to biosynthesize cyclized biphytane derivatives. On the basis of their carbon isotopic composition, Hoefs et al. (1997) and Schouten et al. (1998) concluded that these organisms might be chemoautotrophs. In the case of the Be’eri samples, the extremely low ␦13C values measured for phytane and the cyclized biphytane derivatives (␦13C ⬇ ⫺83 to ⫺88%), which are in the same

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Fig. 11. Mass spectra (EI, 70 eV) of (a) C40 bicyclic biphytane derivative 37 and (b) C41 bicyclic biphytane derivative 40 obtained by HI/LiAlH4 treatment of a polar fraction (P1) from the black sandstone of the Be’eri deposit.

range as those measured for hopanoids indicate that the carbon source for these archaea is methane-derived carbon—for example, methane itself, CO2 resulting from methane oxidation or carbon derived from the biomass of methanotrophic bacteria. Thiel et al. (2001), Pancost et al. (2001), and Schouten et al. (2001) reported 13C-depleted cyclized biphytane derivatives in the Black Sea and at Mediterranean methane seeps in which anaerobic methane oxidation, a consortium of archaea and sulfate-reducing bacteria, is taking place (e.g., Elvert et al., 1999, 2000; Hinrichs et al., 1999; Thiel et al., 1999; Boetius et al., 2000; Pancost et al., 2000). However, the distribution of the cyclized biphytane derivatives from Be’eri (occurrence of two isomeric bicyclic derivatives, presence of a C41 homologue) is clearly different from that of the biphytanes identified in the Black sea or at Mediterranean vents. We would therefore rather exclude the possibility that the biphytane-synthesizing archaea from Be’eri have been involved in anaerobic methane oxidation. In addition, there is no evidence that an anoxic zone developed in the insterstitial medium of the sandstone at the depth where the samples investigated have been collected. Indeed, we were not able to detect any biomarker related to anaerobic organisms and, in particular, to organisms involved in anaerobic methane oxidation such as, for instance, crocetane or pentamethyleicosane (e.g., Hinrichs et al., 1999; Thiel et al., 1999; Pancost et al., 2000). Furthermore, the predominance of typical hopanoids related to lipids of methanotrophic bacteria further points to an oxic environment. Because the geochemical

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context rather supports the hypothesis that these archaea are aerobic species, one cannot exclude the existence of yet unknown aerobic methanotrophic archaea. In another alternative hypothesis, the archaea from Be’eri may have lived on organic matter (as heterotrophs) deriving from the bacterial biomass or on CO2 (as chemotrophs) formed by biologic oxidation of methane or resulting from the biologic or abiotic oxidation of the organic matter fixed by the methanotrophs. Thus, these archaea may, with respect to their metabolism, be sulfide-oxidizing bacteria or be related to thermoacidophilic archaea, such as those of the genus Sulfolobus, which oxidize elemental sulfur to sulfuric acid and use either CO2 or organic matter as their carbon source (Brock and Madigan, 1988). Occurrence of such organisms would be compatible with the acidic conditions prevailing now in the Be’eri deposit (pH ⫽ 1) and the abundance of sulfur (Nissenbaum and Kaplan, 1966). It is possible that such organisms were not present in the original paleoenvironment but have appeared later when the environment became strongly acidic as a result of progressive oxidation of elemental sulfur. It cannot be excluded that in addition to heterotrophic or chemotrophic sulfuroxidizing archaea, nonarchaeal equivalents (Beggiatoa-like bacteria, Larkin et al., 1994) have contributed to the biomass as proposed by Druckman et al. (1994). 3.8. Chemical Degradation of Kerogens with Ruthenium Tetroxide Degradation with ruthenium tetroxide is a further mean for the study of macromolecular organic matter from sediments. This reagent oxidizes in particular double bonds, aromatic subunits and ethers to release bound-biomarkers as carboxylic acids or ketones (Stock and Tse, 1983; Choi et al., 1988; Rullko¨ tter and Michaelis, 1990; Trifilieff et al., 1992; Reiss et al., 1997). The main objective of the use of this severe chemical degradation method was the release of bound biomarkers attesting of the presence in the paleoenvironment of organisms (notably photosynthetic or anaerobic) different from those evidenced by the analysis of free lipids or of lipids obtained by more selective chemical degradation methods (HI/LiAlH4; Nickel boride). The acids obtained upon degradation of kerogens isolated from the sandstone and the mats (GC not shown) are again characterized by the occurrence of hopanoid (31 to 34) and isoprenoid (biphytane-related) skeletons (e.g., 42 to 44). Attribution of structures for the biphytane related diacids is based on comparison with published mass spectra (Meunier-Christmann, 1988; De Lemos Scofield, 1990). These results confirm the predominant contribution of the biomass from methanotrophic bacteria and archaea. Part of the hopanoid acids dominated by the C30 acid presumably results from the oxidation of biologic hopanoid aminopolyols (see section 3.5) rather than from degradation of macromolecules. A significant contribution of linear skeletons (monoacids) could also be detected. In the bacterial mats their distribution was restricted to the lower homologues and mimics that of the free linear acids (C11-C18; predominant C16) whereas, in the sandstone, their distribution extends from C8 to C25 and is dominated by the C15, C23 and C25 homologues, thus reflecting contributions of different organisms. These distributions could,

Fig. 12. Paleoenvironmental model of the Be’eri deposit (adapted from Druckman et al., 1994).

however, not be related to the contribution of specific organisms. Investigation of the carbon isotopic composition (␦13C in the ⫺80%/⫺90% range) showed that these acids derive from organisms living on methane-derived carbon. 4. CONCLUSIONS

Analysis of free and bound biomarkers from the Be’eri sulfur quarry shows the occurrence of essentially two types of lipids: hopanoids, phytane, and biphytane derivatives, along with small amounts of linear components. The predominance of these lipids, especially of the hopanoids, as well as the absence of steroids, demonstrates the essentially bacterial or archaeal origin of the organic matter from the Be’eri deposit. Furthermore, our molecular and isotopic study shows the absence of unambiguous markers for photosynthesis (absence of algal steroids and carotenoids). Consequently, it appears that the initial hypothesis of Nissenbaum and Kaplan (1966), which proposed a model involving deposition of the ore in a shallow lagoon in which algae were thriving on CO2 formed by oxidation of extremely light biogenic methane, must be revisited. Consequently, our model (Fig. 12) presents some clear similarities with that proposed by Druckman et al. (1992, 1994), because similarly to these authors, we envisage that the organic matter associated with sulfur in the sandstone and the mats derives from a bacterial biomass that has developed in the interstitial aqueous medium of the sandstone and was fueled by gases (predominantly methane accompanied by H2S) percolating upward from deeper zones under the Be’eri deposit. Our model differs, however, from that of Druckman et al. (1992, 1994) concerning the main biologic origin of the biomass. Indeed, these authors did not take into account the carbon isotopic composition of the biomass and envisaged that the

Fossil bacterial ecosystem at methane seeps

biomass was mainly formed by chemotrophic sulfide-oxidizing Beggiatoa-like bacteria. Because the carbon isotopic composition of all bacterial and archaeal biomarkers shows an extreme 13C depletion (in the ⫺80%/⫺90% range), independently of their origin, it appears clearly that the exclusive source of carbon for the ecosystem was methane that was primarily fixed by methanotrophic hopanoid-synthesizing bacteria developing in the intersitial waters of the sandstone. This result also demonstrates that oxic conditions were prevailing in the upper part of the ecosystem. In addition, our study reveals the occurrence of archaea (synthesizing phytane and bicyclic biphytanes) living on 13C-depleted carbon source deriving from methane. The most likely hypothesis is that these archaea are sulfur or sulfide oxidizers utilizing H2S, which accompanies methane. These archaea were thus living as chemotrophs on 13C-depleted CO2 or heterotrophically on the bacterial biomass. Isotopically light CO2 might have originated either by oxidation of methane performed by the methanotrophic bacteria or been formed by anaerobic methane oxidation likely to have occurred in deeper zones under Be’eri (Messinian sulfates, see below), as suggested by data from Druckman et al. (1994). Because it has been established that oxic conditions were prevailing in the Be’eri deposit and that methane seeping from deeper zones contains H2S, we cannot exclude that Beggiatoa-like sulfide oxidizers have contributed to the biomass. We consider, however, contrary to Druckman et al. (1992, 1994), that the contribution of these organisms was probably not predominant. Sulfur from the Be’eri deposit is probably formed by oxidation within the sandstone of H2S present in methane. This oxidation may have proceeded either abiotically or biologically by Beggiatoa-like bacteria or by sulfide-oxidizing archaea. H2S results most probably from the bacterial reduction of underlying Messinian sulfates in a process involving concomitant anaerobic oxidation of methane (Druckman et al., 1994). Investigation of isotopic composition of the elemental sulfur by Nissenbaum and Kaplan (1966) shows that its 34S content is in the same range as that of the Messinian sulfates. This observation is in apparent contradiction with the postulated origin of H2S (from which elemental sulfur derives), which is thought to result from the biologic sulfate reduction processes because it would be expected to be much lighter than the sulfates. It is worth to be mentioned that it is almost impossible to make a complete mass balance of all the sulfur species occurring in the Be’eri quarry because some of them have progressively been lost in the atmosphere. The isotopic composition of elemental sulfur from Be’eri might thus, for instance, be explained by the loss of “light” sulfur species such as SO2. Because abiotic or biologic oxidation of H2S to elemental sulfur requires molecular oxygen, it is likely that the approximately 3m thick sulfur-rich horizon where our samples have been collected was located within the oxic part of the sandstone. It is most probable that the deeper layers beneath were anoxic, explaining that they are devoid of elemental sulfur. Could the oxic-anoxic interface have moved over time with the sulfur-rich horizon being temporarily subjected to anoxic conditions? Because all the biomarkers identified in our samples and collected from the sulfur-rich layers apparently derive from lipids of aerobic organisms, and because we did not find any evidence that anaerobic organisms have contributed to the

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organic matter of the samples investigated, it is likely that anaerobic conditions never prevailed at least at the depth where our samples have been collected. The oxidizing conditions now prevailing in the Be’eri deposit were revealed by the occurrence of several degraded, oxidized, or thiophenic hopanoid structures, although even under these circumstances, an apparently intact biohopanoid (aminobacteriohopanetriol) has been preserved. Some of these compounds were obtained by heating hopenes with elemental sulfur, thus suggesting that the latter could play a role as dehydrogenating and oxidizing agent, in the transformations undergone by organic matter in the Be’eri deposit. The acidic and oxidizing conditions, as well as the exclusively bacterial and archaeal origin of the biomass, may also explain the high lipid enrichment of the humic substances from Be’eri. Acknowledgments—We thank Estelle Motsch and Marie-Claude Schweigert, Universite´ Louis Pasteur, Strasbourg, for mass spectral analyses; and Roland Graff, Universite´ Louis Pasteur, Strasbourg, for NMR measurements. Partial support for this study was provided by the Earth Science Directorate of the Ministry of National Infrastructure, Israel. Associate editor: N. E. Ostrom REFERENCES Adam P., Schmid J. C., Mycke B., Strazielle C., Connan J., Huc A., Riva A., and Albrecht P. (1993) Structural investigation of nonpolar sulfur cross-linked macromolecules in petroleum. Geochim. Cosmochim. Acta 57, 3395–3419. Aquino Neto F. R., Trendel J. M., Restle´ A., Connan J., and Albrecht P. (1983) Occurrence and formation of tricyclic and tetracyclic terpanes in sediments and petroleums. In Advances in Organic Geochemistry 1981 (eds M. Bjorøy et al.), pp. 659 – 667. Wiley. Alexander G., Hazai I., Grimalt J., and Albaige´ s J. (1987) Occurrence and transformation of phyllocladanes in brown coals from Nograd Basin, Hungary. Geochim. Cosmochim. Acta 51, 2065–2074. Back T. G., Baron D. L., and Yang K. (1993) Desulfurization with nickel and cobalt boride: Scope, selectivity, stereochemistry, and deuterium-labelling studies. J. Org. Chem. 58, 2407–2413. Bisseret P. and Rohmer M. (1990) Bromine, N-bromosuccinimide and sulphur induced isomerization in the hopane series. Tetrahedron Lett. 31, 7445–7448. Bisseret P. and Rohmer M. (1993) Heating of hop-17(21)-ene in molten sulphur: A route to new sedimentary biomarkers of the hopane series? Tetrahedron Lett. 34, 5295–5298. Boetius A., Ravenschlag K., Schubert C. J., Rickert D., Widdel F., Gleseke A., Amann R., Jørgensen B. B., Witte U., and Pfannkuche O. (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623– 626. Boon J. J., Hines H., Burlingame A. L., Klok J., Rijpstra W. I. C., de Leeuw J. W., Edmunds K. E., and Eglinton G. (1983) Organic geochemical studies of Solar Lake laminated cyanobacterial sediments. In Advances in Organic Geochemistry 1981 (eds. M. Bjorøy et al.), pp. 207–227. Wiley. Brock T. D. and Madigan M. T. Biology of Microorganisms. 5th ed. Prentice-Hall. Burhan R. Y. P. (1996) Origine et e´ volution de la matie`re organique du de´ poˆ t de soufre de Be’eri (Israe¨ l). Aspects structuraux et isotopiques. Ph.D. thesis. Universite´ Louis Pasteur, Strasbourg. Chappe B., Michaelis W., and Albrecht P. (1980) Molecular fossils of Archaebacteria as selective degradation products of kerogen. In Advances in Organic Geochemistry 1979 (eds. A. G. Douglas and J. R. Maxwell), pp. 265–274. Pergamon Press. Chappe B., Albrecht P., and Michaelis W. (1982) Polar lipids of Archaebacteria in sediments and petroleum. Science 217, 65– 66. Choi C. Y., Wang S. H., and Stock L. M. (1988) Ruthenium tetroxide catalyzed oxidation of maceral groups. Energy Fuels 2, 37– 48.

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