Steroids in sediments from Zabuye Salt Lake, western Tibet

Organic Geochemistry 35 (2004) 157–168 www.elsevier.com/locate/orggeochem

Steroids in sediments from Zabuye Salt Lake, western Tibet: diagenetic, ecological or climatic signals? R.L. Wanga,*, S.C. Brassellb, S.C. Scarpittaa, M.P. Zhengc, S.C. Zhangd, P.R. Haydea, L.M. Muenchb a

Chemistry Department, Brookhaven National Laboratory, Bldg. 555, Upton, NY 11973, USA b Biogeochemistry Laboratory, Indiana University, Bloomington, IN 47405, USA c R&D Center of Saline Lake and Epithermal Deposits, Chinese Academy of Geological Sciences, Beijing 100037, PR China d Key Laboratory of Petroleum Geochemistry, China National Petroleum Co., Beijing 100083, PR China Received 10 April 2003; accepted 2 October 2003 (returned to author for revision 6 August 2003)

Abstract A 45 cm long core from Zabuye Salt Lake (Tibetan Plateau, S.W China) was studied to reveal the possible interference between diagenesis and climate signals. Steroids, including sterols and sterenes, dominate the soluble organic matter in these cores. The relative abundance of C27 sterol to the C29 sterols decreases with depth, resulting in a predominance of C29 sterols at the bottom section of this core. This change in the relative molecular distribution could be attributed to both environmental/ecological change and diagenetic complication of molecular signals. Sterols in the shallow sediments are relatively enriched in 13C compared to those from lower within the core. This enrichment is possibly associated either with environmental/climatic change (e.g., increase of salinity and global pCO2 level change etc.) or it could be attributed to the biogeochemical change of organic matter during early diagenesis. 4,4-dimethyl spirosterenes and their possible precursors, 4,4-dimethyl sterenes, constitute a major component of the apolar fraction of organic matter. 13C values of the 4,4-dimethyl sterenes indicate that they are derived from phytoplanktonic algae rather than from bacteria. The 13C values of the regular and spiro steroids differ by >2% suggesting either backbone arrangement of steroids might have involved isotopic fractionation or that these steroids are derived separately from different biological sources. Published by Elsevier Ltd.

1. Introduction Steroids occur ubiquitously in eukaryotic organisms from microorganisms to macro algae and vascular plants and are present in most sedimentary organic matter. Thus, steroids are an important group of fossil compounds providing valuable information about sources of organic matter in recent sediments and ancient sedimentary rocks, as well as crude oils. Diagenesis of the organic matter occurring in waters and sediments

* Corresponding author. E-mail address: [email protected] (R.L. Wang). 0146-6380/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.orggeochem.2003.10.003

modifies the structures of precursor steroids in complex ways. Examples of these processes include: oxidation of sterols to sterones, dehydration of the hydroxyl group (of sterols) leading to the formation of steradienes, isomerisation of double bonds of sterenes via tertiary carbon atoms to different positions in the carbon skeletons (e.g., Mackenzie et al., 1982; Brassell et al., 1984; de Leeuw et al., 1989; Sinnighe Damste´ et al., 1999), backbone rearrangement and the formation of spiro steroids (Peakman and Maxwell, 1988), and aromatization on the A,B,C-rings to form aromatic steroids (Brassell et al., 1984). Incorporation and cleavage of sulfur atoms from steroids also play an important role in the early diagenesis of organic matter. Sterols and

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their diagenetic products contain functionalities such as double bonds, carbonyl and hydroxy groups, that under anoxic conditions are susceptible to incorporation of inorganic sulfur species by natural sulfurization during early diagenesis (e.g., Schouten et al., 1993). Steroids also are valuable tools for determining the complex diagenetic pathways that transform biological precursors into steroid hydrocarbons in all crude oils, their source rocks, and even in the migration pathways of crude oils (e.g., Huang and Meinschein, 1976, 1979; Brassell and Eglinton, 1981; Summons and Capon, 1988, 1991; Summons et al., 1987; Meyers, 1997; Kok et al., 2000; Curiale, 2002). Detailed knowledge about the diagenetic pathways of sterols is important since their diagenetic products (steranes) in crude oils are very useful markers in assessing the geological age of their source rocks, the degree of thermal maturity, and types of organisms present during the deposition of source rocks (e.g., Peters and Moldowan, 1993). Spirosterenes are a group of sedimentary sterenes with a unique molecular backbone rearrangement between C- and D- rings (e.g. Peakman et al., 1984; Peakman and Maxwell, 1988; Summons and Capon, 1988, 1991; Schu¨pfer and Gu¨laçar, 2000; Kok et al., 2000; Rushdi et al., 2003). This family of steroids has dominant mass fragment ions at m/z 206+14n (n=0–2) and a major ion at m/z 121. Spirosterenes are observed in various environments including shales and evaporite sediments (Brassell et al., 1984; Peakman et al., 1984; Peakman and Maxwell, 1988). Beside the natural occurrence of spirosterenes, a mixture of 20R and 20S isomers of 12,14(a’-cyclo-12,13-seco-5a(H)-cholest-b (H)ene (spirosterene) also were synthesized from 5a(H)cholest-7-ene and the corresponding C28 homologues

from (20S)-24-methyl-5a(H)-cholest-7-ene (5a(H)ergost-7-ene) (Peakman et al., 1984). Both natural and synthesized spirosterenes gave major ions at 206 or 220 (from M.+ of C27 or C28 homologues respectively by cleavage through C11–C12 and C8–C14) and m/z 121 (from subsequent cleavage through C20–C22) (Peakman et al., 1984). The mass-spectral features make this group of steroids rather unique and different from regular steroids. The configuration at C20 was assumed by analogy with the acid catalyzed formation of diasteranes (Peakman and Maxwell, 1988) and by the assumption that the presumed 20R isomer was formed first during the rearrangement (Peakman and Maxwell, 1988). Identifying molecular indicators that are controlled primarily by climate and the depositional environment and least impacted by diagenetic processes is an extremely important issue in paleoclimatic study. Our current research involves understanding the Pleistocene/ Holocene climate change on the Tibetan Plateau using isotopic and molecular geochemical techniques on sediments from a core taken from the Zabuye Salt Lake, located in western Tibet Plateau (ZSL, 31.35.N, 84.07.E, Fig. 1). The Zabuye Salt Lake is at 4421 m above sea level (a.s.l.), about 1000 m above the global tree line and is surrounded by high mountains ca 4600–6000 m a.s.l. In such an extreme environment, the influence/pollution from vascular higher plant organic matter could be virtually negligible. The remote ZSL is also far from industrial and anthropogenic perturbation and the impact from allochthonous organic sources to the organic matter is effectively minimized by nature itself. The pristine environment and special geographic location of ZSL made this lake an ideal site for geochemical study of autochthonous biological marker constituents, their

Fig. 1. Maps of the location of Qinghai-Tibet Plateau (A), distribution of saline, brackish and fresh water lakes in the western part of the plateau (B) and the coring site of ZK3 in Zabuye Salt Lake (C) (Based on Zheng et al., 1989 and modified after Wang et al., 2002).


variation with changing environment/climate, and diagenetic/geochemical change with time. In this study, we report on the distributions of sterols, sterenes and their carbon isotope composition from the ZSL and their relationship to climate, source input and early diagenetic process. In particular, we report the presence of two isomers of the unusual spirosterenes with two methyl groups attached on the A-ring, and discuss the possible biological and diagenetic precursors and pathways of the formation for these steroids according to their carbon isotope compositions. These types of spirosteroid structures are found normally with sediments or crude oils associated with saline and hypersaline environments. It was anticipated that this study would help clarify the molecular and isotopic characteristics of this group of unusual steroids, and that a better understanding of steroid diagenetic processes will yield molecular fossils that more accurately reflect climate changes.

2. Geological setting Samples were taken from the northern basin of ZSL (Core ZK3) located in western Tibetan Plateau, S.W. China. ZSL is the terminal basin of the Taro-Zabuye lake chain and has a current area of 243 km2. Due to the enhanced evaporation and extremely arid climate condition, the lake size has been decreasing every year. A large area of playa was exposed around the lake, with mirabilite and halite currently being deposited in the lake. In the northern basin of the lake, where core ZK3 was taken, waters are recharged mainly from melted ice/ snow (Zheng et al., 1989). Due to its exceptional high altitude ( 1000 m above tree-line), the predominant organic source can be attributed to the autochthonous production of the lake system dominated by halophilic algae (Dunaliella salina, Zheng et al., 1985). In addition to algae, other halophilic organisms contain halophilic bacteria as well as halophilic archaea at zones with higher salinity (salt playa) were found. These halophilic organisms and their decomposition products color this salt lake reddish during the blooming season of summer.

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homogenized samples using a LECO C/S Analyzer using the previously published methodologies (Wang and Zheng, 1998; Wang et al., 2002). 3.2. GC and GC–MS analyses of biomarkers Freeze-dried sediment was extracted by CH3OH/ CH2Cl2 (1:1) in a Soxhlet extractor. The total extract was separated using silica gel column chromatography by eluting with hexane, toluene and dichloromethane. The polar fraction was treated with BF3/CH3OH (Aldrich, esterification of carboxylic acids) and BSTFA (bis(trimethylsilyl)trifluoroacetamide, Aldrich) and then purified using the same column chromatography procedure. The collected hexane-dissolved fractions (apolar) were analyzed by gas chromatography and gas chromatography–mass spectrometry (GC–MS). The gas chromatography column was an Ultra 1 (Crosslinked Methyl Silicone Gum, 60 m0.32 mm i.d.0.52 mm film thickness). Oven temperatures were programmed from 60 to 320 C at 4 C/min and held isothermal at 320 C for 30 min. GC–MS analyses were carried out initially on a Finnigan MAT TSQ-700 system using electron impact ionization (70 eV). Then the structure confirmation was repeated on a HP 5970 mass selective detector at Brookhaven National Laboratory using the same GC–MS conditions. Helium was the carrier gas for all analyses. 3.3. Isotope measurements of individual biomarkers by irm-GC–MS An isotope-ratio-monitoring GC–MS system (irmGC–MS) (Matthews and Hayes, 1978; Merritt et al., 1995) was employed to obtain the 13C values of the steroid compounds. Carbon isotope values are expressed in the traditional notation of values: sample ð%Þ ¼ Rsample Rstandard =ðRstandard Þ 1000;

3. Experimental

where R is the abundance ratio of 13C /12C in the samples or in the standard. Isotope ratios are reported relative to the PDB (Pee Dee Belemnite) standard via a secondary standard (VPDB) for carbon isotope measurements on the individual molecular CO2 (irmGC–MS) and the bulk CO2 (algae sample).

3.1. Measurement of elemental carbon and sulfur concentrations

4. Results and discussions

The core, 45 cm long, was separated into 10 sections and frozen in dry ice before shipment to the laboratory. Measurements of total carbon (TC), total organic carbon (TOC), total inorganic carbon (TIC) and total sulfur (TS) were performed at the Biogeochemical Lab of Indiana University on dried, ground, and

4.1. Elemental carbon and sulfur distributions of core ZK3 Carbon and sulfur elemental concentrations are plotted against the depth of the core in Fig. 2. From the bottom section of the core (i) to top section (v) (Fig. 2),

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Fig. 2. Distribution of total organic and inorganic carbon (TOC, TIC), total organic and inorganic sulfur (TOS, TIS) and total sulfur (TS) in the core ZK3, Zabuye Salt Lake, western China, plotted versus burial depth (all in % of dry sediment weight; i–v: core sections used in the text).

TOC and TIC decreased from about 0.6 to 0.3% and 7.5 to 6.5%, respectively. The gradual decrease of organic carbon from section ‘i’ to ‘v’ (Fig. 2) is, perhaps, associated with the increase of salinity (evaporation/ precipitation ratio increase, Zheng et al., 1989) and the decline of productivity (as TOC) in the lake. The gradual decrease of carbonate carbon (TIC) and increase of sulfate as indicated by the increased total sulfur (TS) in the core also are indications of increasing salinity of the lake. Fluctuations of salinity occurred as indicated by the variability of total sulfur contents of the core. The rapid drop of sulfur in the topmost section (section v, Fig. 2) could be associated with the further salinization of the lake, such that the sulfate was largely replaced by halite, marking a hypersaline salt lake environment during interval ‘v’. Total organic sulfur content remained rather low in the top sections above 30 cm, but increases significantly with depth below 30 cm, indicating that active sulfate reduction and formation of organic sulfur species could be taking place in the sediment below this depth level ( 30 cm). 4.2. Distribution of sterols in core ZK3 Sterols are found to be the predominant components in the total organic extract of these salt lake sediments.

The most abundant steroids are cholesterol (C27), C28 5,22-sterol, C29 5,22-24-ethyl sterol and C29 5 sterol (Fig. 3). Other components, such as the normal-chain moieties mainly contributed from terrestrial vascular plant input are low in these cores (Fig. 3). The relative abundance of C27–C29 sterols shows a steady decrease in the cores from surface (No. 1) to the bottom (No. 10) (Figs. 3 and 4). The relative percentage of both C29:1 sterol and C29:2 sterol in the total neutral lipid fraction decreases slightly in shallow sediments but increases steadily with increasing depth below 18 cm in the core. On the other hand, C27:1 sterol abundance decreases markedly with increasing depth at the top section (v) (Fig. 4). The ratio of C27/C29 (see Fig. 4 for details) decreases steadily from about 0.8 at the surface to about 0.3 at the bottom of the core (Fig. 4). These changes in steroid distributions may be due to an ecological change in the phytoplankton community that occurred during the time represented by this 45 cm core. Alternatively, the variation in the molecular distribution of these steroids could result from a differentiation in the diagenetic transformation of different steroids. Recent studies of early steroid sulfurization in Ace Lake sediments showed that incorporation of sulfur is biased toward C27 sterols (Kok et al., 2000). In the surface sediments from Ace Lake in Antarctica, a predominance


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Fig. 3. Gas chromatograms of alcohols and sterols (as thiolated fractions) extracted from sediment core ZK3 in Zabuye Salt Lake, Tibet (distributions of the relative abundance of compounds A,B,C are shown in Fig. 4 and 13C values of compounds A and B are shown in Fig. 5).

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Fig. 4. Depth profiles of relative percentages of sterols and their molecular ratio in core ZK3 (chromatograms of compounds A,B and C are shown in Fig. 3; sections i–v same as in Fig. 2).

of sulfurized C27 steroid contrasted with the distribution of free sterols with a strong predominance of C29 sterols (Kok et al., 2000). Therefore, diagenetic differentiation could be responsible for the observed change of molecular distribution of sterols in these cores. In the case of ZSL, both organic carbon and sulfur contents in these cores are rather high (Fig. 2). In the southern basin of ZSL, sulfur content is even higher, with TS as high as 14% (Wang et al., 2002). Under hypersaline conditions, incorporation of sulfur into the steroid molecules can be an important process especially at burial depths where sulfate reduction is significant. 4.3. Carbon isotopic composition of individual sterols The 13C values of C29 24-ethyl-5,22-sterenol and C29 ster-5-enol, the two major sterols extracted from core ZK3, are plotted vs. burial depth in Fig. 5. Both compounds have 13C values between 25 and 28% except for the two samples in section ‘v’. The 13C values of sterols in this section are much lower, 31 to 32% for both compounds. The dramatic shift of carbon isotope values (up to 4%) at the top section of the core (‘iv’ to ‘v’) is possibly due to either environmental/ecological change (e.g., salinity change) during deposition and/or early diagenesis. Due to the markedly increased salinity

Fig. 5. Distribution of molecular carbon isotope composition (13C) of C29-24-ethyl-5,22-sterenol (A) and C29ster-5-enol (B) vs. burial depth sediments in core ZK3 from northern basin of Zabuye Salt Lake in Tibet (both peaks shown in Fig. 3; 13C values in % VPDB).


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Fig. 6. Partial mass chromatograms of m/z 220, 234, 412 and partial RIC showing the co-occurrence of 4,4,24-trimethyl-12,14a-cyclo12,13-seco-5a(H)-13(17)ene (black, #1 and 2) and 4,4,24-trimethyl-5a(H)-cholest-7-ene (#3 and 4). Mass spectra of compounds 1 and 2 are shown in Fig. 7.

Fig. 7. Mass spectra of 4,4,24-trimethyl-12,14a-cyclo-12,13-seco-5a(H)-13(17)enes and their tentative structure assignment based on GC–MS data (for conditions of GC–MS operation please refer to the text).

and lower productivity, a larger portion of the sterols in the shallow sediments (top section of the core) might be contributed by higher plant input from the allochthonous source, for example, wind blown pollen/spore and

other plant debris from aerosol precipitation into the lake. Another possibility is that such molecular isotope shift might be related to climate/environmental change in a rather larger scale or even global pCO2 level change.

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However, as we discussed earlier, section ‘v’ represents precipitation under higher salinity than that of section ‘iv’ (Fig. 2). Organic molecules synthesized in waters with higher salinity tend to be isotopically heavier rather than being lighter. For example, studies of the halophytes Salicornia europaea subsp. Rubra and Puccinellia nuttalliana, show that increasing salt concentrations result in decreased isotope discrimination in both field samples and growth chamber samples resulting in 13C enriched lipids (O’Leary, 1981). Therefore, it is likely that the dramatic shift of carbon isotope values of sterols (up to 4%) in this core section is associated mainly with climate/environmental/ecological change, although a ‘diagenetic’ isotope fractionation between precursors and products in the sediment cannot be excluded completely. 4.4. Occurrence of 4,4-dimethyl spirosterenes Partial mass chromatograms of m/z 220, 234 and 412 illustrate the 20R and 20S isomers of 4,4,24-trimethyl12,14a-cyclo-12,13-seco-5a(H)-cholest-13(17)ene (4,4dimethyl spirosterene) observed in the sediment cores of ZSL (Fig. 6). Mass spectra of 20R and 20S isomers are virtually identical (Fig. 7a and b respectively). This group of sterenes is a predominant component in the hexane-dissolved fraction of the total extract of all core samples. However, they were not observed in the extract of algae samples. The mass spectrometric characteristics are quite similar to those of the C28 spirosterene homologues reported previously (Peakman et al., 1984). Spirosterenes were reported previously in a variety of sediments including the Cretaceous black shales (Leg 50,

Fig. 8. Down-core profile of the ratio of C20R/C20S isomers of 4,4,24-trimethyl-12,14a-cyclo-12,13-seco-5a(H)-13(17)ene in the later Holocene core ZK3 from northern basin of Zabuye Salt Lake, western Tibet.

Moroccan Basin; Leg 71, Falkland Plateau; Leg 75, Angola Basin) (Brassell et al., 1984), Miocene DSDP sediments from Southern California Bight, and Cretaceous Black shale from Northern Italy (Brassell et al., 1984; van Graas et al., 1982). These compounds show dominant fragment ions at m/z 206+14n (n=0–2) and a major ion at m/z 121. Peakman et al. (1984) also synthesized a mixture of 20R and 20S isomers of 12,14acyclo-12,13-seco-5a(H)-cholest-b(H) ene from 5a(H)cholest-7-ene and the corresponding C28 homologues from (20S)-24-methyl-5a(H)-cholest-7-ene (5a(H)ergost-7-ene). A C27 spirosterene with one methyl group on the A-ring was observed as a significant component in hydrous pyrolytic products (Abbott et al., 1995). Both natural and synthesized spirosterenes yield major ions at 206 or 220 (from M.+ of C27 or C28 homologues respectively by cleavage through C11–C12 and C8–C14) and m/z 121 (from subsequent cleavage through C20– C22. The configuration at C20 was assumed by analogy with the acid catalyzed formation of diasteranes (Peakman and Maxwell, 1988) and by the fact that the presumed 20R isomer was formed first during the rearrangement. The compounds extracted from ZSL sediment are rather similar to those published previously but with two methyl groups on the A-ring (Fig. 7). The molecular ions (M+.) of these methyl steroids in ZSL are at m/z 412 (15%), rather than 384 as shown by Peakman et al. (1984). Both 20S and 20R isomers have base fragment ions at m/z 220 (100%, by cleavage through C11–C12

Fig. 9. Carbon isotope values (13C, %) of sterenes plotted against the burial depth of sediments from core ZK3 [1,2: 20R and 20S isomers of 4,4,24-trimethyl-12,14a-cyclo-12,13-seco5a(H)-13(17)enes, respectively; 3: 20R isomer of 4,4,24-trimethyl-5a(H)-cholest-7-ene (No. 4 in Fig. 6)].


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Fig. 10. Possible pathways of the formation of spiro sterenes in the early diagenesis of sediments as observed in hypersaline ZSL sediments; the backbone rearrangement from Nos. 3 and 4 to the spiro steroids (Nos. 1 and 2, Fig. 6) are believed to be catalyzed by acidic minerals in peat (Peakman et al., 1984) (R=C7H15).

and C8-C14 from M+., Fig. 7) and a major ion at 121 (40%, by a secondary cleavage through C20–C22). Other major diagnostic ions are m/z 107 (15%, m/z 121– 14), m/z 135 (10%, m/z 121+14), m/z 233/234 (15%, m/ z 220+14) and m/z 397 (M+.–CH3). The structures (4,4,24-dimethyl-12,14a-cyclo-12,13-seco-5a(H)-cholest13(17)-ene) are assigned tentatively based primarily on the GC–MS features compared to previously published data on spirosterenes (e.g., Peakman et al., 1984; Peakman and Maxwell, 1988; Abbott et al., 1995). While these compounds are not observed in the extract from a modern algae sampled from this lake, they may be formed from macro-molecular lipid precursors. If so, further study using pyrolytic degradation of this algae

sample might yield more supportive evidence. Unfortunately such data are not available at this time. Spirosterenes detected in deep-sea sediments were thought to be formed by an acid catalyzed rearrangement involving clay minerals (Peakman et al., 1984). Their precursors could be 7-8(14) sterenes (van Graas et al., 1982; Peakman et al., 1984; Peakman and Maxwell, 1988) although these alkenes are not commonly identified in sediments. Pyrolysis studies showed that under acidic and aqueous conditions (e.g., in hot water), regular 5a(H)-cholestane also can undergo backbone rearrangement yielding a C27 spirosterene (Abbott et al., 1995). The reaction mechanism for the spirosteroids observed in saline sediments could be much more

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complicated. The abundance of 20R and 20S isomers shows a gradual change through the burial depth of sediments (Fig. 8). The ratio of 20R/20S decreases quickly in the surface sediment until it finally reaches a steady-state ratio of 20R/20S.0.5 at about 30 cm burial depth in the core. This indicates that 20R could be the initial configuration inherited from its biologically synthesized precursors, perhaps a 4,4-dimethylsterol with an OH group on C-3 (Fig. 7). The isomerization occurring at C-20, therefore, can be used as an indicator of early biogeochemical transformation of organic matter under hypersaline conditions, although such rapid isomerization would limit the potential usage in the geological time scale. 4.5. Carbon isotope compositions of spiro steroids 13C values of 20R and 20S isomers of 4,4,24-trimethyl-12,14a-cyclo-12,13-seco-5a(H)-cholest-13(17)ene are 24.0 26.7% (Fig. 9). The 13C values of 4,4dimethyl sterenes in these samples are in the same range, i.e., 24 27% (Fig. 9) suggesting that the carbon skeletons of both of these A-ring alkylated sterenes were biosynthesized originally by the same organism. The relatively consistent difference of 13C values between these two groups of steroids (13C 2%) further suggest that these compounds are related either by biosynthesis or by diagenetic transformation. The isotopic change (enrichment) observed in the down-core profile could reflect biochemical response to environmental change, locally (e.g. salinity change) and even globally (e.g., global atmospheric CO2 level change). Alternatively, isotopic fractionation may be occurring during early diagenesis via a multiple-step mechanism (Fig. 10). Such a mechanism would require the formation of unidentified, isotopically lighter compounds during the skeletal rearrangement of the 4,4-dimethysterenes. It is also possible that the gradual increase in 13C values of both spiro and regular 4,4-dimethyl steroids is fortuitous and the 4,4-dimethylsteroids are not the precursors of the 4,4-dimethyl spirosteroids. Biological sources of 4,4-dimethyl steroids could either be algae (Goodwin et al., 1988), particularly prymnesiophyte microalgae (Volkman et al., 1990), or methanotrophic bacterium (Bird et al., 1971; Bouvier et al., 1976). Methylococcus capsulatus is known to biosynthesize many hopanoids and steroids (Bird et al., 1971; Bouvier et al., 1976), the latter being dominated by the 4,4,14-trimethyl (lanosterol), 3-keto-4-methyl, and 4-methyl sterols. This group of bacteria has the ability to use simple C1 compounds such as methane, methanol or methylamine as the sole source of carbon for growth. Generally, methanotrophic bacteria enrich 12 C in their biosynthesis causing their lipids to be depleted in 13C by up to 50% relative to the starting materials due to a strong isotopic fractionation effect (e.g. Freeman et al., 1990; Hayes, 1992). However, the

13C values of 4,4-dimethyl steroids compounds in the salt lake sediments indicate that these compounds must have originated from phytoplankton algae, rather than methanotrophic bacteria. Algae, particularly the halophilic species Chlamydomnas and Dunaliella salina, are abundant during summer blooming seasons in ZSL (Zheng et al., 1985). The bulk algal 13C value is as high as 18.6% (VPDB). This bulk isotope value of the algal biomass also suggests that these algae (or generally, phytoplankton) are likely the biological source of the abundant steroids we observed in the sediments. Under the hypersaline environments at ZSL, extended residence time of the ZSL lake waters have caused enrichment of 13C not only in the carbonate (Wang et al., 2002), but also in the organic carbon of algae lipids, including the regular and spirosteroids in this hypersaline lacustrine environment.

5. Conclusions Unsaturated sterols are the predominant component of the total extract of the sediment cores from Zabuye Salt Lake. Abundance of C27 sterol relative to the C29 sterols decreases with depth resulting in a predominance of C29 sterols at the bottom section of this core. The change in the relative molecular distribution could be attributed to environmental change (drought, salinization, etc.) of the region, and perhaps to certain degree it could also be associated with the possible differential sulfurization rate of C27 and C29 sterols. The obvious enrichment of 13C in sterols was seen in samples below 6–10 cm, possibly associated with either environmental change (e.g. increase of salinity, pCO2 level change etc.) or more or less, it might be associated with some unknown process during early diagenesis of organic matter. 4,4-dimethyl spirosterenes and their possible precursors, 4,4-dimethyl sterenes were seen as a major component of the apolar fraction of organic matter extracted from the sediment core from ZSL. 13C values of these compounds indicate that these steroids were likely derived from phytoplanktonic algae rather than from bacteria. The rearrangement of 4,4,-dimethyl 4 and 5 steroids yielding 4,4-dimethyl spirosteroids starts from the early diagenesis of organic matter in the sediment. If these 4,4,-dimethyl 4 and 5 steroids are really the precursors of the spirosteroids, the >2% difference of 13C values between these two types of compounds suggest that a multiple-step mechanism may be involved in this backbone structural rearrangement in the saline sediments.

Acknowledgements We would like to thank our colleagues at Brookhaven National Lab (BNL), Indiana University and Chinese


Academy of Geological Sciences (CAGS) for their support and contribution to this research. Dr. W. Qi (CAGS) are particularly acknowledged for assistance in the core sampling. We thank Dr. Arndt Schimmelmann, Mr. J. Fong and Mr. S. Studley (all at IU) for their assistance during the irm-GC–MS analysis of steroids and other part of the project. Finally we would like to thank Dr. Clifford Walters (ExxonMobil), Dr. J. M. Moldowan (Stanford) and Dr. R. Pancost (Bristol) for many valuable comments and suggestions. Initial research of this project was supported by a Geochemistry Fellowship from Indiana University (RLW) and also partially supported by the National Natural Science Foundation of China grant 49833010 (MPZ). Associate Editor—C. Walters

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