Carbon isotopic composition of an isoprenoid-rich oil
and its potential source rock
Richard D. Pancost
a,1, Nils Telnñs
b, Jaap S. Sinninghe DamsteÂ
a,*
aDepartment of Marine Biogeochemistry and Toxicology, Netherlands Institute for Sea Research, PO Box 59,1790AB Den Burg (Texel), The Netherlands
bNorsk Hydro Research Center, Sandsliveien 90, N-5020 Bergen, Norway Received 2 June 2000; accepted 4 October 2000
(returned to author for revision 3 August 2000)
Abstract
Despite the multiple controls on the carbon isotopic composition of organic matter,13C values for saturated and
aromatic hydrocarbon fractions for over 300 Norwegian North Sea (Central Graben) oils range fromÿ26 toÿ31%
and from ÿ24 toÿ30%, respectively. However, for one oil (well 2/2-5),13C values for the saturate and aromatic
fractions areÿ21.5%andÿ22%, signi®cantly enriched in13C relative to the isotopic compositions of all oil fractions
investigated. The saturate hydrocarbon fraction of the 2/2-5 oil is comprised predominantly of low-molecular-weight (<C20), primarily regular, acyclic isoprenoids. Also present are C36±C40irregular acyclic isoprenoids; these compounds
are composed of various combinations of tail-to-tail condensed C15±C20 regular isoprenoid subunits, resulting in
pseudo-homologous series with six to nine methyl groups. The isotopic compositions of both high- and low-molecular-weight isoprenoids are aroundÿ19%. This is in marked contrast ton-alkane13C values (ca.
ÿ31%), and it is clear
that isoprenoid13C values dictate the13C value of the saturate fraction. The same13C-enriched isoprenoids are
pre-sent in a thin (<1 m) immature oil shale facies (hudlestonibiozone) of the Kimmeridge clay formation (KCF; Dorset, UK). Moreover, the relatively immature KCF sediment contains unique high-molecular-weight isoprenoid thiophenes and thianes, whose structures indicate that sulfur has reacted with functional groups in the isoprenoid precursor's interiors. This suggests that these novel high-molecular-weight isoprenoids were probably biosynthesized as such. Reasons for their profound13C-enrichment and predominance are discussed. Although the KCF facies, located in the
southern UK, cannot be a direct source of the North Sea 2/2-5 oil, a related North Sea facies seems plausible since an isotopically enriched TOC zone has been reported in a well also from the Central Graben.#2001 Elsevier Science Ltd. All rights reserved.
Keywords:Carbon isotopes; North Sea oils; Central Graben; Isoprenoids; Kimmeridge clay formation
1. Introduction
Numerous factors govern the carbon-isotopic com-position of algal and bacterial biomass. These include carbon source (Hayes, 1993), nutrient concentrations in
the environment (Freeman and Hayes, 1992; Rau et al., 1992; Laws et al., 1995), and physiological character-istics such as carbon assimilation pathway (Goericke et al., 1994 and references therein) and cell geometry (Popp et al., 1998). Consequently, naturally occurring
13C values can range from >
ÿ6%for certain lacus-trine algal biomarkers (Huang et al., 1997) to <ÿ120%
for the lipids of some methylotrophic archaea (Thiel et al., 1999). Thus, it is likely that factors such as diage-netic conditions that bias the distribution of com-pounds preserved (e.g. Sinninghe Damste et al., 1998) and variations in the biological source of sedimentary
0146-6380/01/$ - see front matter#2001 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 4 9 - 2
Organic Geochemistry 32 (2001) 87±103
www.elsevier.nl/locate/orggeochem
* Corresponding author. Tel.: 222-369550; fax: +31-222-319674.
organic matter (e.g. Hayes, 1993; Revill et al., 1994; Pancost et al., 1999) can strongly in¯uence13C values of
kerogen and oils. Because algal biomass13C values can
range from ÿ15 to ÿ30% depending on physiological
characteristics and growth conditions (Popp et al., 1998), signi®cant variability in 13C values could presumably
occur even without dramatic source variations.
Nonetheless, the isotopic compositions of sedimen-tary organic matter generally range fromÿ25 toÿ31%
throughout the last 800 million years of Earth history (Hayes et al., 1999). Likely, this limited range is a con-sequence of the accumulation of organic matter from diverse sources and over extended intervals of time, which homogenizes inputs and averages out sources with extreme end-member 13C values. For example,
kerogen of Upper Devonian age in the Duvernay for-mation (Canada and Williston basins) contains abun-dant, 13C-enriched (ca.
ÿ15%) biomarkers for green sulfur bacteria; nonetheless, the carbon isotopic com-position of bulk organic matter is ÿ29% (Hartgers et
al., 1994) and similar to that observed for typical marine sediments of that age. Similarly, in the Green River shale, 13C-depleted methanogen biomarkers (as low as ÿ80%) are abundant but bulk organic matter 13C
values are ca.ÿ30%(Collister et al., 1992).
However, numerous workers have shown that in some settings, kerogen consists of cell wall material predominantly derived from a single algal species (Tegelaar et al., 1989; Derenne et al., 1991; Gelin et al., 1996; de Leeuw et al., 1991; de Leeuw and Largeau, 1993). In some cases, these organisms apparently have unique physiologies that result in biomass and kerogen with carbon isotopic compositions signi®cantly dierent from those generally observed for most marine or lacustrine oils. Examples include torbanites derived from selectively preservedBotryococcus brauniiremains (Largeau et al., 1984, 1986; Dubreuil et al., 1989), tas-manites possibly derived from prasinophycean algae (Aquino-Neto et al., 1992; Revill et al., 1994), and organic matter-rich deposits of the extinct, organic-wal-led microfossilGloeocapsomorpha prisca(Derenne et al., 1992; Foster et al., 1989; Pancost et al., 1999).
In this paper we present compound distributions and carbon isotope abundances for the Jurassic Kimmeridge Clay Formation (KCF) and a North Sea (Central Gra-ben) oil. This work reveals that the bitumen in a KCF ``oil shale'' facies and the North Sea oil are profoundly enriched in 13C relative to adjacent stratigraphic units
and related oils, respectively. Moreover, both are char-acterized by a predominance of similarly 13C-enriched
isoprenoid compounds. Many of these isoprenoids have not previously been observed, indicating that although the source organism of these compounds is the vastly predominant source of total organic matter in these settings, it has yet to be identi®ed in either modern or other ancient settings.
2. Experimental
2.1. Samples
The 2/2-5 oil from the North Sea, Central Graben, is one of several hundred North Sea oils collected by Norsk Hydro and for which saturate and aromatic13C
values have been determined. The Kimmeridge clay formation (Dorset, UK) sample was collected from one of several narrow ``oil-shale'' intervals in thehudlestoni
biozone (Cox and Gallois, 1981). The particular oil shale (carbonate content is 10%) was investigated by van Kaam-Peters et al. (1998; sample UK4), and throughout this paper, references to the KCF hudlestoni ``oil shale'' facies pertain only to that speci®c interval. This unit is characterized by profound13C-enrichment
(ÿ22.9%) of organic matter and high total organic
car-bon contents (TOC; 22.9%). Similary, the organic sulfur content is elevated relative to adjacent units while the concentration of pyrite is similar to that throughout the KCF (van Kaam-Peters et al., 1998).
2.2. Extraction and fractionation
For the KCF sample, powdered rock (ca. 70 g) was Soxhlet extracted with methanol (MeOH)/dichloro-methane (DCM) (1:7.5 v/v) for 24 h. Asphaltenes were removed from the extracts by precipitation in heptane. An aliquot of the maltene fraction (ca. 200 mg), to which a mixture of four standards was added for quan-titative analysis (Kohnen et al., 1990), was separated into apolar and polar fractions using a column packed with alumina (van Kaam-Peters et al., 1998). An aliquot of the maltene fraction of the 2/2-5 oil was similarly prepared but during column chromatography was split into three fractions using hexane (saturate fraction), hexane:DCM (9:1 v/v; aromatic fraction), and DCM/ MeOH (1:1 v/v; stripping) as eluents. Saturated hydro-carbon and aromatic hydrohydro-carbon fractions were ana-lyzed by GC and GC/MS. n-Alkanes were removed from aliquots (ca. 4 mg) of the saturated hydrocarbon fractions by molecular sieve adduction (van Kaam-Peters et al., 1998). An aliquot (ca. 10 mg) of the KCF apolar fraction was further separated by argentation TLC (thin layer chromatography) into four fractions, which were scraped o the TLC plate and ultrasonically extracted with ethyl acetate (van Kaam-Peters et al., 1998). Aliquots (ca. 15 mg) of the polar fraction and the A2 (thiophenes) and A4 (thianes) TLC fractions of the KCF were desulfurized using Raney nickel and subse-quently hydrogenated (Sinninghe Damste et al., 1988).
2.3. O-line pyrolysis and fractionation
extracted rock sample, equivalent to ca. 100 mg organic carbon to which 100mg standard (3-methyl-6-dideutero-henicosane) was added. The sample was heated (400C)
for 1.5 h and the volatile products generated were trap-ped in cold traps and fractionated by column chroma-tography according to the methods described in van Kaam-Peters et al. (1998).
2.4. Analysis of biomarkers
Biomarker fractions were analyzed by gas chromato-graphy (GC) and gas chromatochromato-graphy/mass spectro-metry (GC/MS) for identi®cation using previously described conditions. GC/MS with chemical ionization was performed using an HP 6890 gas chromatograph coupled to a HP 5973 mass spectrometer with a mass range m/zof 50±800 and a cycle time of 0.5 s. Curie-point pyrolysis-gas chromatography (Py-GC) was per-formed with a Hewlett-Packard 5890 gas chromato-graph using a FOM-3LX unit for pyrolysis and according to the conditions published in HoÈld et al. (1998).
2.5. Isotope-ratio-monitoring gas chromatography-mass spectrometry (irm-GC/MS)
Irm-GC/MS was performed on a Finnigan Delta C and used to determine compound-speci®c 13C values
(Hayes et al., 1990; Merrit et al., 1994). The GC condi-tions are the same as those used during GC/MS ana-lyses. 13C values are expressed against the Vienna
Peedee Belemnite standard and have an error of less than 0.4% unless otherwise noted (error based on
analytical accuracy and precision of measurements of co-injected standards ÿC20 and C24 perdeuterated n
-alkanes). For high-molecular weight isoprenoids, the errors are typically1.0%due to co-elution.
Carbon isotope abundances of the saturate and aro-matic fractions of the North Sea oils were determined using the methods described by Bjorùy et al. (1994).
3. Results
3.1. Geochemical analyses of the 2/2-5 North Sea oil
Extensive analyses of over 300 North Sea oils reveal a remarkable similarity of saturate and aromatic fraction
13C values. Despite age or inferred source rock facies,
relatively narrow ranges ofÿ26 toÿ31% andÿ24 to
ÿ30% were obtained for all saturate and aromatic
fractions, respectively (Fig. 1). This is essentially an extension and con®rmation of previously published analyses of 72 Central Graben oils (Bjorùy et al., 1993). The sole exception is an oil from the 2/2-5 well, for which the saturate fraction has a13C value of
ÿ21.5%
and the aromatic fraction has a 13C value of ÿ22%.
The reason for this dierence is unclear and is exacer-bated by uncertainty in the age of the 2/2-5 oil. The aromatic fraction contains isorenieratene alteration products (Koopmans et al., 1996a), which are bio-markers for green sulfur bacteria (Chlorobiaceae; Liaaen-Jensen, 1979). These compounds are enriched in
13C in the 2/2-5 oil, as is commonly observed for
iso-renieratene derivatives (Sinninghe Damste et al., 1993; Koopmans et al., 1996a) due to this organism's utiliza-tion of the reverse tricarboxylic acid cycle (SirevaÊg and Ormerod, 1970; Quandt et al., 1977; SirevaÊg et al., 1977). While this could explain the 13C-enrichment of
the aromatic fraction, it cannot explain the enrichment observed for saturated hydrocarbons, and so we exam-ined this fraction in greater detail.
3.1.1. Biomarker distributions of the 2/2-5 oil
The saturate hydrocarbon fraction of the 2/2-5 oil contains typical marine oil biomarkers, including ster-anes, hopster-anes, andn-alkanes with a distribution exhi-biting no odd-over-even predominance and a maximum at n-C17. However, these compounds are of relatively
low abundance compared to the profound predominance of acyclic isoprenoids (compounds a to l; Fig. 2a and structures are shown in the Appendix). The distribution of the isoprenoids ranges from C13to C40and is
bimo-dal with the most abundant compounds containing 16± 20 carbon atoms. The second maximum in the iso-prenoid distribution pro®le occurs in the C36ÿ40 range
and is considerably more complex than that of the low-molecular-weight isoprenoids (Fig. 2a).
3.1.2. Identi®cation of high-molecular-weight irregular isoprenoids
Chemical ionization (CI) MS was used to ascertain the molecular weight of the compounds eluting in the high-molecular-weight range and revealed that they consist of multiple isomers of C36ÿ40acyclic compounds.
In all compounds, pronounced enhancements of them/z
113 and 183 fragments in their electron impact (EI) mass spectra indicate that these compounds are iso-prenoids. With the exception of lycopane (h±h; see Appendix for structures) and g±h (Philp, 1985), these are previously unreported compounds and thus, neither previously published mass spectra nor authentic stan-dards are available. On the basis of molecular weights determined by GC/CI-MS and fragmentation patterns obtained by GC/EI-MS, structures were tentatively assigned.
These assignments were further con®rmed by com-parison to calculated retention (Kovats) indices (Table 1). Retention indices were calculated according to the method of Kissin et al. (1986) and are system-atically oset from measured values by 20±30 units. Because the identi®cations of g±h and, particularly,
lycopane have been established, this oset is likely a consequence of our particular GC conditions. Correct-ing for this oset reveals that all measured retention indices are within 5 units of those calculated and sup-ports our structural assignments.
In general, the compounds are C36ÿ40irregular acyclic
isoprenoids composed of various combinations of C15±
C20 regular isoprenoid subunits, resulting in
pseudo-homologous series with six to nine methyl groups. For example, compound f±g is characterized by enhanced
m/z 113, 183, and 253 fragments consistent with an acyclic isoprenoid structure (Fig. 3a). The larger mass fragments do not extend this pattern but instead exhibit enhanced fragments atm/z295, 365, 435 indicating that the structure is an irregular isoprenoid. The speci®c frag-mentation is consistent with a C37isoprenoid containing
an extendedn-alkyl portion in the core of the molecule. In all cases, the high-molecular-weight isoprenoids have terminal head groups, indicating that two C15ÿ20
isoprenoid units have been joined together in a tail-to-tail linkage. Indeed all structures are consistent with a tail-to-tail condensation of two C15ÿ20isoprenoid units,
and in most cases represent the joining of the two term-inal carbon atoms. The sole exception is h±h*, a C40
compound that appears to be composed of a terminal carbon on one C20 isoprenoid unit joined to the o-1
carbon of another. This compound, in particular, gen-erates highly diagnostic fragments during GC/MS ana-lyses. The preferred cleavage of the bond linking two tertiary carbon atoms results in pronounced m/z 252 and 308 peaks (Fig. 3b). Such cleavages are expected to generate the atypical even mass fragments (cf. Kohnen et al., 1990).
With the exception of lycopane and g±h, these are novel compounds. Regular isoprenoids extending up to C45were reported by Albaiges (1980) and head-to-head
isoprenoids ranging from C28 to C40 are speci®c
bio-markers for archaea and are found in diverse oils and
sediments (Moldowan and Seifert, 1979; Chappe et al., 1980; Petrov et al., 1990). Lycopane and its unsaturated homologues, typically attributed to archaea but also observed in many microorganisms and higher plants (Schmidt, 1979), are the only previously reported high molecular weight (>C35) tail-to-tail isoprenoids.
In addition, C36ÿ40 isoprenoids with aromatic
moi-eties near the center of the molecule are also present. Probably, because of the extended alkyl components, these compounds eluted in the saturate fraction during our preparation. These isoprenoids are present in abun-dances about one order of magnitude lower than that of
Fig. 2. Gas chromatograms of the saturate hydrocarbon fractions of the 2/2-5 oil (a) and the KCFhudlestoni``oil shale'' facies (b). The insets show the distributions of high-molecular-weight C35±C40isoprenoids as revealed by anm/z183 mass chromatogram. Iso-prenoid compounds of interest are labeled (a±l; selected mass spectra are shown in Fig. 3 and structures are shown in the Appendix) and squares denoten-alkanes.
the acyclic isoprenoids and comprised of a very complex distribution of structural isomers. Nonetheless, their distribution and chain lengths are consistent with a genetic link to the acyclic isoprenoids.
3.1.3. Low-molecular-weight isoprenoids
In addition to the abundant regular isoprenoids, we also observe C19(g0) and C20(h0) isoprenoid compounds
characterized by dierent retention times and fragmen-tation patterns than regular isoprenoids (Fig. 2a; struc-tures shown in Appendix). Mass spectra suggest that these compounds are composed of three acyclic iso-prenoid units with an additional four and ®ve methylene units, respectively. The mass spectrum of compound g0
has been previously published (McCarthy and Calvin, 1967) and is in excellent agreement with our observa-tions. Such compounds could have been generated by thermal cracking of the previously described high-mole-cular-weight isoprenoids. Speci®cally, cleavage at branch points inf±gandh±cwill generate the C19
iso-prenoid with an extendedn-alkyl tail and cleavage ofh± d and h±f will generate the C20 compound. Based on
such considerations, higher molecular weight iso-prenoids (C21ÿ25) with extended n-alkyl tails are also
expected (e.g. 2,6,10,14 tetramethylicosane [l0] would
also be generated by cleavage of f±g). Indeed, careful inspection reveals that such compounds probably are present in the saturate hydrocarbon fraction of the 2/2-5 oil; however, their abundances are too low relative to background to obtain unambiguous mass spectra.
3.1.4. Compound-speci®cd13C values
All acyclic isoprenoids, including both low and high-molecular-weight components, have13C values ranging
from ÿ17 to ÿ20% (Fig. 4a). The similarity of 13C
values for both high and low-molecular-weight iso-prenoids suggests that they derive from the same source.
These values are also identical to that of the C19
iso-prenoid with an extendedn-alkyl tail (g0). In contrast, 13C values of co-occurringn-alkanes range from
ÿ28 to
ÿ31%, profoundly depleted in13C relative to all acyclic
isoprenoids.
3.2. Geochemical analyses of the KCFhudlestonioil shale facies
3.2.1. Saturate hydrocarbons
During previous molecular and isotopic investigations of the KCF, van Kaam-Peters et al. (1998) observed that an interval of thehudlestoni``oil shale'' facies (Cox and Gallois, 1981) is characterized by a profound abundance of13C-enriched isoprenoids similar to that observed in
the 2/2-5 oil. These investigations revealed that low-molecular-weight isoprenoids with a predominance of the C16ÿ20homologues are the most abundant components of
the saturate hydrocarbon fraction (Fig. 2b). In contrast to adjacent KCF facies in which phytane concentrations are always <350mg/g TOC and typically lower, in the
hudlestoni``oil shale'' facies the phytane concentration is >1200 mg/g TOC. n-Alkane concentrations, on the other hand, do not vary among the hudlestoni ``oil shale'' and adjacent facies, suggesting that inputs of these compounds did not vary substantially.
13C values of the isoprenoids in the hudlestoni ``oil
shale'' facies range fromÿ17 to ÿ21.5% and are enri-ched in13C relative to isoprenoids in all other analyzed
KCF facies. In one facies, the mean isoprenoid 13C
value isÿ22.5%, in another it isÿ25%, and in all
oth-ers, it is ca.ÿ30%(van Kaam-Peters et al., 1998). The acyclic isoprenoids in the hudlestoni ``oil shale'' facies are also profoundly enriched in13C relative to
co-occur-ringn-alkanes, which are generallyÿ28%(Fig. 4b). The
onlyn-alkanes with13C values comparable to those of
the isoprenoids aren-C21andn-C23, but these enriched n-alkanes are characteristic of all other KCF facies (van Kaam-Peters et al., 1997, 1998).
Further investigation revealed that the high-mole-cular-weight isoprenoids observed in the 2/2-5 oil are also present in the KCF hudlestoni ``oil shale'' facies (Fig. 2b). Although there are some minor dierences in compound distributions between the KCF and the 2/2-5 oil, the same compounds are present and the overall distribution is the same. These compounds are also enriched in13C (Fig. 4b), although low signal to
back-ground ratios resulted in large accuracy errors for these determinations (1.5%). The KCF hudlestoni ``oil shale'' facies also contains cyclisized and aromatized isoprenoids similar to those observed in the 2/2-5 oil.
3.2.2. Sulfur-containing compounds
In addition to the aromatic and acyclic isoprenoids, we also observed thiophenes, thianes, and thiolanes with C36ÿ40isoprenoid skeletons (Figs. 5a and 6a). Although Table 1
Calculated and measured Kovats indices for high-molecular-weight isoprenoids in the 2/2-5 oil
some low-molecular-weight sulfur-containing iso-prenoids are present, the high-molecular-weight com-ponents are dominant and, based on mass spectra, appear to be directly related to the acyclic high-mole-cular-weight isoprenoids. The structural relationship between the acyclic isoprenoids and the thiophenes, thianes, and thiolanes was proven by desulfurization of the former compounds (Figs. 5b and 6b). In both cases, the previously described acyclic isoprenoids were
gener-ated. Based on mass spectra of the intact thiophenes, thianes, and thiolanes, sulfur was incorporated into the interior of the aforementioned high-molecular-weight isoprenoids (Fig. 7) Ð similar to the patterns expressed by the isoprenoids with aromatic moieties.
Although both the thiophenes and thianes/thiolanes are clearly structurally related to the high-molecular-weight isoprenoids, their distributions dier. In the case of isoprenoid thiophenes, the distribution of compounds
Fig. 3. Mass spectra (subtracted for background) for compounds f±g (a) and h±h* (b).
generated upon desulfurization is similar to the dis-tribution of sulfur-free compounds. In contrast, the thiane/thiolane fraction contains predominantly C39and
C40 isoprenoids Ð speci®cally those compounds in
which the thiane or thiolane moiety is joined to one of
the isoprenoid chains via a quaternary carbon. It seems likely that the dierences in distribution re¯ect the age and thermal maturity of the KCF. A more complex distribution of thianes and thiolanes was probably originally present; however, with thermal maturity
Fig. 4. Compound-speci®c13C values (in standard%notation relative to VPDB) of hydrocarbons in the 2/2-5 oil (a) and KCF
compounds lacking a quaternary carbon were trans-formed into more thermodynamically stable thiophenes (Sinninghe Damste and de Leeuw, 1990).
In addition to isoprenoid compounds with inter-molecular sulfur moieties, the presence of intramole-cular sulfur incorporation was shown by desulfurization of the polar fraction (Fig. 8). Both low and mole-cular-weight isoprenoids were generated and the high-molecular-weight compounds have structures identical to those observed in the saturated hydrocarbon fraction and generated from the thiophene and thiane/thiolane fractions. The distribution of high-molecular-weight isoprenoids is similar to that observed in the free hydrocarbon fraction, although with somewhat elevated abundances of the C39and C40components.
13C values for thiophenes and thianes were not
determined. However,13C values of the
high-molecular-weight isoprenoids generated by desulfurization of the polar fraction have values ranging fromÿ18.5 toÿ20%
and are consistent with those determined for all other isoprenoids in the KCFhudlestoni``oil shale'' facies.
3.2.3. Kerogen pyrolysis
GC/MS traces clearly show that the kerogen pyroly-sate of the KCF hudlestoni ``oil shale'' facies is
dominated by isoprenoids (Fig. 9) (HoÈld et al., 1998). Van Kaam-Peters et al. (1998) performed o-line pyr-olysis to examine the 13C values of these and other
kerogen-bound compounds (Fig. 4b). In general, the kerogen-bound components exhibit the same isotope patterns as those expressed by the free lipids. Speci®-cally, isoprenoids in thehudlestoni``oil shale'' facies are enriched relative to isoprenoids in other facies and are enriched in 13C relative to n-alkanes in both the same
and other facies.
4. Discussion
4.1. Relationship between the KCFhudlestoni``oil shale'' facies and 2/2-5 oil
The KCF hudlestoni ``oil shale'' facies cannot be a direct source for the 2/2-5 oil. The location of the KCF is too far south to have served as a source rock for the Central Graben oil. Additionally, this southern UK KCF site is not suciently thermally mature to have generated oil. This is apparent from burial history and biomarker ratios in the KCF. In this study, the presence of abundant sulfur-containing compounds indicate that
Fig. 5. Chromatograms showing thiophenes in the KCFhudlestoni``oil shale'' facies: (a) shows the Total Ion Current (TIC) trace of the A2 (thiophene and aromatic) fraction, and (b) shows them/z183 mass chromatogram for the desulfurized and hydrogenated A2 fraction. Labels denote structures shown in the Appendix and Fig. 7.
the KCF is not even of sucient thermal maturity for all C±S bonds to have been cleaved and further indicates that it is not a petroleum source rock.
Nonetheless, the bitumen in the KCFhudlestoni``oil shale'' facies and the 2/2-5 oil share several marked similarities that clearly suggest a relationship between the organic matter source for each and distinguish them from other oils and bitumens. In both, 13C-enriched
isoprenoids are the dominant components in the satu-rate hydrocarbon fraction. Moreover, both fractions contain13C-enriched high-molecular-weight isoprenoids
with structures that have not previously been reported for any other oil or source rock. Nor have possible pre-cursor compounds been observed in any modern envir-onment or extant organism. Other biomolecular characteristics, such as the presence of isorenieratane in both, reinforce this similarity.
There is a thin zone of source rocks with a carbon isotopic enrichment (ÿ24%versusÿ28%on average) in
both the kerogen and the kerogen pyrolysate of the Kimmeridgian of the Central Graben (Bailey et al., 1990). Thus, this zone could be the source rock of the Central Graben 2/2-5 oil. If this is indeed true, then a
thin isotopically enriched zone in the middle of several hundred meters of potential source rock has generated and expelled hydrocarbons, whereas no signi®cant con-tribution from the rest of the source rock section is noted in the 2/2-5 oil. This could be explained by the fact that, like the KCF oil shale facies, the Central Graben facies originally contained abundant sulfur-bound isoprenoids, resulting in a much earlier oil gen-eration. Arti®cial maturation experiments of organic sulfur-rich source rocks in combination with the analy-sis of sulfur-bound biomarkers have demonstrated that generation of hydrocarbons can indeed occur at low levels of thermal stress in rocks where organic sulfur contents are high (Koopmans et al., 1996b, 1998)
4.2. Relationship amongst isoprenoid components
There are relatively few cases in which isoprenoids comprise a signi®cant component of either an organ-ism's biosynthesized or preserved biomass. Here, not only is a predominance of isoprenoids observed but also signi®cant quantities of novel high-molecular-weight components. In some instances, it has been shown that
Fig. 7. Structures of thiophene, thiane, and thiolane isoprenoids present in the A2 and A4 fractions of the KCFhudlestoni``oil shale'' facies and their products formed upon Raney nickel desulfurization.
Fig. 8. Gas chromatogram showing the desulfurized and hydrogenated polar fraction of the KCFhudlestoni``oil shale'' facies. Labels denote structures shown in the Appendix and Fig. 7 and squares denoten-alkanes.
the distribution of biomarkers in the extractable frac-tion of ancient rocks is not representative of the source contributions to the total preserved organic matter (Goth et al., 1988; Tegelaar et al., 1989; Hartgers et al., 1994; Sinninghe Damste and Schouten, 1997). However, both pyrolysis and o-line pyrolysis clearly reveal the predominance of isoprenoids in the kerogen of the KCF
hudlestoni``oil shale'' facies.
It seems likely that all isoprenoids in the KCF hudle-stoni``oil shale'' facies are related. The13C values of all
high- and low-molecular-weight isoprenoids in the free hydrocarbon and polar desulfurized fractions and gener-ated during o-line pyrolysis of the kerogen are similar and distinct from other hydrocarbons. However, it is unclear whether both the low- and high-molecular-weight isoprenoids were biosynthesized by the precursor organ-ism or if only the high-molecular-weight compounds were biosynthesized and low-molecular-weight components were generated upon thermal cracking. It seems likely that the latter explanation is largely true. The subordinate series of low-molecular-weight acyclic isoprenoids with extended n-alkyl tails are likely products of thermal cracking of the high-molecular-weight compounds.
The location of the sulfur atoms in the high-mole-cular-weight isoprenoid sulfur compounds provides fur-ther clues to the structure of the original biological compounds. Sulfur incorporation into sedimentary organic matter is known to occur relatively rapidly (Kok et al., 2000; Werne et al., 2000), and thus, is expected to react with pre-existing (i.e. biosynthesized) functionalities. Thus, the location of the thiophene, thiane, and thiolane moieties suggests that the novel high-molecular-weight isoprenoids were biosynthesized
as such and with interior functional moieties, such as double bonds or keto groups (Schouten et al., 1993; de Graaf et al., 1992).
As stated previously, the majority of the compounds reported here are novel; however, there are some simila-rities to previously reported regular and irregular iso-prenoids and linearly extended phytane skeletons containing thiophene moieties (Peakman et al., 1989a). Speci®cally noteworthy similarities are the extended n -alkyl tails and thiophene moieties in these structures. In contrast to the isoprenoids reported here these compounds are less abundant than co-occurringn-alkanes, but they have been observed in a variety of bitumens and oils (Peakman et al., 1989b) and could have a related source.
4.3. Sources of isoprenoids
In the saturate fractions of both the KCFhudlestoni
``oil shale'' facies and the 2/2-5 oil, the predominant compounds are 13C-enriched isoprenoids. This is in
marked contrast to typical marine oils and sediments, in which a mixture of components derived from diverse phytoplankton, bacteria, and possibly terrigenous inputs are present, and it seems likely that the primary source of organic matter to the KCF facies and 2/2-5 oil is a single isoprenoid-rich organism. However, the nat-ure of this organism and reason for its imprint on both the 2/2-5 oil and the KCF is dicult to ascertain.
One explanation for the predominance of acyclic iso-prenoids is enhanced preservation of precursor com-pounds via sulfur incorporation. In the KCF, the release of high-molecular-weight irregular isoprenoids from the thiane/thiolane, thiophene, and polar fractions provides
Fig. 9. TIC trace of the ¯ash pyrolysates of the kerogen of the KCFhudlestoni``oil shale'' facies. Filled and open circles denote the pseudo-homologous series of isoprenoid (i-) alkanes andi-alk-1-enes, respectively, and rectangles denote the homologous series of
direct evidence that such processes did occur. Indeed, unsaturated analogs to lycopane (h±h) have been shown to react with sulfur and aromatize, resulting in com-pounds similar (VIII) or identical (IX) to those observed here (Grice et al., 1998a). The presence of iso-renieratane, a biomarker for green sulfur bacteria, which require both light and sul®de, throughout the KCF (van Kaam et al., 1997, 1998), clearly indicates that the availability of reduced sulfur was not restricted to the ``oil shale'' facies. Moreover, Sinninghe Damste et al. (1998) showed that the abundance of low-molecular-weight thiophenes released upon pyrolysis is high throughout the KCF and correlated to the %TOC. These observations reveal that sulfurization of organic matter was not restricted to the ``oil shale'' facies and is apparently unrelated to the presence or absence of the
13C-enriched isoprenoids. Thus, the occurrence of these
compounds in restricted intervals of the KCF is due not to changes in conditions favoring preservation but to the emergence of an isoprenoid-rich organism into a setting ideal for preserving its biomarkers.
The nature of such an organism is unclear. Lycopene is a carotenoid common in diverse microorganisms and higher plants (Schmidt, 1978 and references therein); however, because it is unsaturated at thirteen positions, sulfurization of lycopene would be expected to generate compounds in which sulfur moieties are not restricted to the interior of the molecule. The structures we observe are consistent with an origin from lycopadienes. Lyco-padienes have only been observed in the L strain ofB. braunii (Metzger et al., 1991) and certain thermophilic archaea (Lattuati et al., 1998). Although neither a freshwater alga nor a thermophilic archaea can be the direct source of the isoprenoids in these samples, it is tempting to suggest that a marine, mesophilic analog to one of the above organisms was present in the Jurassic epicontinental seas. Indeed, recent work (DeLong et al., 1998; Hoefs et al., 1997; Schouten et al., 1998) has high-lighted the ubiquity of archaea-derived compounds in marine waters and sediments. Moreover, the isoprenoid chains of such pelagic crenarchaeota are typically enri-ched in 13C relative to compounds derived from other
organisms (Hoefs et al., 1997). In addition, C21ÿ25
iso-prenoidal biomarkers isolated from Miocene/Pliocene deposits of the Dead Sea basin (Sdom formation) and inferred to be derived from halophilic archaea also exhibit a strong enrichment in13C (7%) relative to co-occurring
phytoplankton biomarkers (Grice, 1998b). Although unsaturated analogs of most of the C36ÿ40irregular
iso-prenoids observed here have never been reported, it is possible that a poorly studied or even non-extant archaeon could have thrived during this brief interval and served as a source for these compounds.
An alternative explanation is that the isoprenoids derive from a resistant isoprenoidal biopolymer (cf. HoÈld et al., 1998). Indeed, the presence of functional groups in
the interior of the isoprenoids is suggestive of the oxi-dative cross-linking that occurs in biological macro-molecules such as algaenan (Blokker et al., 1998). Such an isoprenoidal biopolymer Ð but not an isoprenoidal algaenan Ð has been isolated from B. braunii race L (BertheÂas et al., 1997). However, it is highly unlikely that this lacustrine alga contributed isoprenoids to the marine 2/2-5 oil and KCF sediments. Also, the iso-prenoidal biopolymer inB. brauniirace L would not be as resistant to degradation as the co-existing aliphatic algae-nan (Derenne et al., 1989), and such an organism is not expected to generate sedimentary organic matter domi-nated by isoprenoids. Other than the L race ofB. braunii, there are no other modern organisms known to biosynthe-size isoprenoidal biopolymers. Nonetheless, it remains possible that an organism living during the Jurassic could have synthesized such a biopolymer, which would have been preferentially preserved relative to other sources of organic matter as has been observed for aliphatic algaenans (Tegelaar et al., 1989; de Leeuw and Largeau, 1993).
4.4. Causes of13C-enrichments
The high 13C values are a striking characteristic of
the isoprenoids in both the 2/2-5 oil and the KCF ``oil shale'' facies. Low CO2or high algal growth rates
typi-cally result in low carbon isotope fractionation ("p) by
marine phytoplankton (Goericke et al., 1994; Laws et al., 1995; Popp et al., 1998) and, thus, 13C-enriched
biomass. In such a situation, most phytoplankton are expected to respond to the environmental conditions, and compounds derived from all organisms should exhibit similar enrichment. However, in the KCF oil shale faciesn-alkanes show no enrichment in13C
rela-tive to those in adjacent units. Based on this as well as the carbon isotopic compositions of other biomarkers and carbonate, it seems unlikely that either CO2(aq)
con-centrations or DIC13C values varied (van Kaam-Peters
et al., 1998). Thus, the13C-enrichment of the acyclic
iso-prenoids is speci®c to an organism rather than solely the result of surface-water nutrient concentrations.
The observed 13C-enrichment of acyclic isoprenoids
relative to n-alkanes could indicate that the source of the isoprenoids grew during episodic blooms. In this situation, the isoprenoid-rich organism would have grown under environmental conditions decoupled from ``normal'' conditions during which other organisms grow. Moreover, bloom conditions Ð high growth rates and lowered CO2(aq)concentrations Ð are expected to cause 13C-enrichment in algal biomass. Such blooms, because
they represent episodic high productivity events, also might be preferentially preserved relative to non-bloom-ing organisms and account for the high abundance of isoprenoids in our samples.
An alternative possibility is that the isoprenoid-rich organism is physiologically distinct from the algae that
predominate during most of the time of KCF deposi-tion. For example, CO2 diusion into the cell of the
isoprenoid-rich organism could have been limited. The-oretical considerations indicate that physiological lim-itations to CO2 diusion can exert a large control on algal13C values (Rau et al., 1996). Such an explanation
has been invoked to explain the observed 13
C-enrich-ment of algae such as B. braunii that biosynthesize algaenan and are characterized by a thick cell wall (Boreham et al., 1994). Another physiological dier-ence that could aect the carbon isotopic composition of an organism is the use of a carbon ®xation pathway other than the Calvin cycle. Fractionation during car-bon assimilation via either the reverse TCA cycle (Sir-evaÊg et al., 1977) or the 3-hydroxypropionate pathway (van der Meer et al., submitted) can be signi®cantly lower than that during the Calvin cycle. However, all known algae utilize the Calvin cycle during photo-synthesis, and thus, this explanation implies that the source of the isoprenoids was either an archaeon or a bacterium.
5. Conclusions
The 2/2-5 oil and the bitumen of the hudlestoni ``oil shale'' facies of the KCF share profound and diagnostic characteristics that clearly suggest a relationship. Both the oil and the bitumen contain novel high-molecular-weight isoprenoids and the saturate hydrocarbon frac-tions of both are dominated by 13C-enriched
iso-prenoids. Although the latter cannot be a source rock for the 2/2-5 oil, the presence of an isotopically enriched
source rock facies in the Central Graben (Burkwood et al., 1990) suggests that an equivalent facies is.
There are multiple explanations for the unique char-acteristics of the 2/2-5 oil and the bitumen of the KCF ``oil shale'' facies. The high abundances of regular iso-prenoids and irregular isoiso-prenoids with unprecedented structures, both of which are characterized by elevated
13C values indicate that the source organism is
uncom-mon and if extant, has not been studied. Consequently, we cannot state with certainty the ecology or physiology of this organism. The sum of the evidence is consistent with either an algae that grew in strong and episodic blooms or biosynthesized a resistant cell wall or an archaeon rich in lycopadienes and related unsaturated irregular isoprenoids. Both explanations explain the predominance of isoprenoids in the KCF ``oil shale'' facies and the 2/2-5 oil and the fact that those iso-prenoids are enriched in13C. Regardless of source, the
organism is clearly uncommon in both ancient and modern settings. Nonetheless, in these samples it is the predominant source of organic carbon, is largely responsible for high TOC contents in the KCF facies, and governs the unique carbon isotopic characteristics of the saturate hydrocarbon fraction of the 2/2-5 oil.
Acknowledgements
We gratefully acknowledge analytical support provided by M. Baas and M. Dekker. NIOZ Contribution No. 3522.
Associate EditorÐS.J. Rowland
References
Albaiges, J., 1980. Identi®cation and geochemical signi®cance of long chain acyclic isoprenoid hydrocarbons in crude oils. In: Douglas, A.G., Maxwell, J.R. (Eds.), Advances in Organic Geochemistry, 1979. Pergamon, Oxford, pp. 19±28. Aquino-Neto, F.R., Triguis, J., Azevedo, D.A., Rodrigues, R.,
Simoneit, B.R.T., 1992. Organic geochemistry of geo-graphically unrelatedTasmanites. Organic Geochemistry 18, 791.
Bailey, N.J.L., Burwood, R., Harriman, G.E., 1990. Applica-tion of pyrolysate carbon isotope and biomarker technology to organofacies de®nition and oil correlation problems in North Sea basin. Organic Geochemistry 16, 1157±1172. BertheÂas, O., Delahais, V., Metzger, P., Largeau, C., 1997.
Acetal-containing macromolecular lipids in Botryococcus braunii(B and L races). Chemical structure and relationship with algaenans. In: Hors®eld, B. (Ed.), Abstracts from the 18th International Meeting of Organic Geochemists, Maas-tricht, The Netherlands, Vol. II. Forschungszentrum JuÈlich GmbH, pp. 855±856.
Bjorùy, M., Hall, P.B., Moe, R.P., 1994. Stable carbon isotope variation ofn-alkanes in Central Graben oils. In: Telnñs, N., van Graas, G., éygard, K. (Eds.), Advances in Organic Geochemistry 1993. Pergamon, Oxford, Organic Geochem-istry, pp. 355±381.
Blokker, P., Schouten, S., van den Ende, H., de Leeuw, J.W., Hatcher, P.G., Sinninghe DamsteÂ, J.S., 1998. Chemical structure of algaenans from the fresh water algaeTetraedron minimum,Scenedesmus communis, andPediastrum boyanum. Organic Geochemistry 29, 1453±1468.
Boreham, C.J., Summons, R.E., Roksandic, Z., Dowling, L.M., Hutton, A.C., 1994. Chemical, molecular and isotopic dierentiation of organic facies in the Tertiary lacustrine Duaringa oil shale deposit, Queensland Australia. Organic Geochemistry 21, 685±712.
Chappe, B., Michaelis, W., Albrecht, P., 1980. Molecular fos-sils of archaebacteria as selective degradation products of kerogen. In: Douglas, A.G., Maxwell, J.R. (Eds.), Advances in Organic Geochemistry 1979. Pergamon, Oxford, pp. 265± 274.
Collister, J.W., Summons, R.E., Lichtfouse, E., Hayes, J.M., 1992. An isotopic biogeochemical study of the Green River oil shale. In: Eckhardt, C.B., Maxwell, J.R., Larter, S.R., Manning, D.A.C. (Eds.), Advances in Organic Geochemistry 1991. Pergamon, Oxford, Organic Geochemistry 19, pp. 265± 276.
Cox, B.M., Gallois, R.W., 1981. The stratigraphy of the Kim-meridge Clay of the Dorset type area and its correlation with some other Kimmeridgian sequences. Report Institute Geo-logical Science 80, 1±44.
DeLong, E.F., King, L.L., Massana, R., Cittone, H., Murray, A., Schleper, C., Wakeham, S.G., 1998. Dibiphytanyl ether lipids in nonthermophilic crenarchaeotes. Applied and Environmental Microbiology 64, 1133±1138.
Derenne, S., Largeau, C., Casadevall, E., Berkalo, C., 1989. Occurrence of a resistant biopolymer in the L race of
Botryococcus Braunii. Phytochemistry 28, 1137±1142. Derenne, S., Largeau, C., Casadevall, E., Berkalo, C.,
Rous-seau, B., 1992. Chemical evidence of kerogen formation in source rocks and oil shales via selective preservation of thin
resistant outer walls of microalgae: origin of ultralaminae. Geochimica et Cosmochimica Acta 55, 1041±1050.
Derenne, S., Metzger, P., Largeau, C., Van Bergen, P.F., Gatellier, J.P., Sinninghe DamsteÂ, J.S., Tegelaar, E.W., de Leeuw, J.W., Berkalo, C., 1992. Similar morphological and chemical variations ofGloeocapsomorpha priscain Ordovi-cian sediments and cultured Botryococcus braunii as a response to changes in salinity. Organic Geochemistry 19, 299±313.
Dubreuil, C., Derenne, S., Largeau, C., Berkalo, C., Rous-seau, B., 1989. Mechanism of formation and chemical struc-ture of Coorongite-I. Role of the resistant biopolymer and of the hydrocarbons of Botryococcus braunii. Organic Geo-chemistry 14, 543±553.
Foster, C.B., Wicander, R., Reed, J.D., 1989. Gloeocapsomor-pha prisca Zalessky, 1917: a new study, Part I taxonomy, geochemistry, and paleoecology. Geobios 22, 735±759. Freeman, K.H., Hayes, J.M., 1992. Fractionation of carbon
isotopes by phytoplankton and estimates of ancient CO2 levels. Global Biogeochemical Cycles 6, 185±198.
Gelin, F., Boogers, I., Noordeloos, A.A.M., Sinninghe DamsteÂ, J.S., Hatcher, P.G., de Leeuw, J.W., 1996. Novel, resistant microalgal polyethers: an important sink of organic carbon in the marine environment? Geochimica et Cosmochimica Acta 60, 1275±1280.
Goericke, R., Montoya, J.P., Fry, B., 1994. Physiology of iso-topic fractionation in algae and cyanobacteria. In: Lajtha, K., Michener, R.H. (Eds.), Stable Isotopes in Ecology and Environmental Science. Blackwell Scienti®c Publications, Oxford, pp. 187±221.
Goth, K., de Leeuw, J.W., PuÈttmann, W., Tegelaar, E.W., 1988. Origin of Messel oil shale kerogen. Nature 336, 759± 761.
de Graaf, W., Sinninghe DamsteÂ, J.S., de Leeuw, J.W., 1992. Laboratory simulation of natural sulphurization: I. Forma-tion of monomeric and oligomeric isoprenoid polysulphides by low-temperature reaction of inorganic polysulphides with phytol and phytadienes. Geochimica et Cosmochimica Acta 56, 4321±4328.
Grice, K., Schouten, S., Nissenbaum, A., Charrach, J., Sin-ninghe DamsteÂ, J.S., 1998a. A remarkable paradox: sul-furised freshwater algal (Botryococcus braunii) lipids in an ancient hypersaline euxinic ecosystem. Organic Geochem-istry 28, 195±216.
Grice, K., Schouten, S., Nissenbaum, A., Charrach, J., Sin-ninghe DamsteÂ, J.S., 1998b. Isotopically heavy carbon in the C21to C25regular isoprenoids in halite-rich deposits from the Sdom Formation, Dead Sea Basin, Israel. Organic Geo-chemistry 28, 349±359.
Hartgers, W.A., Sinninghe DamsteÂ, J.S., Requejo, A.G., Allan, J., Hayes, J.M., de Leeuw, J.W., 1994. Evidence for only minor contributions from bacteria to sedimentary organic matter. Nature 369, 224±227.
Hayes, J.M., 1993. Factors controlling13C contents of sedi-mentary organic compounds: principles and evidence. Mar-ine Geology 113, 111±125.
Hayes, J.M., Freeman, K.H., Popp, B.N., Hoham, C.H., 1990. Compound-speci®c isotopic analyses: a novel tool for recon-struction of ancient biogeochemical processes. In: Durand, B., Behar, F. (Eds.), Advances in Organic Geochemistry. Pergamon, Oxford, Organic Geochemistry 16, pp. 1115±1128.
Hayes, J.M., Strauss, H., Kaufmann, A.J., 1999. The abun-dance of13C in marine organic matter and isotopic fractio-nation in the global biogeochemical cycle of carbon during the past 800 Ma. Chemical Geology 161, 103±125.
Hoefs, M.J.L., Schouten, S., de Leeuw, J.W., King, L.L., Wakeham, S.G., Sinninghe DamsteÂ, J.S., 1997. Ether lipids of planktonic archaea in the marine water column. Applied and Environmental Microbiology 61, 3090±3095.
HoÈld, I.M., Schouten, S., van Kaam-Peters, H.M.E., Sinninghe DamsteÈ, J.S., 1998. Recognition of n-alkyl and isoprenoid algaenans in marine sediments by stable carbon isotopic analysis of pyrolysis products of kerogens. Organic Geo-chemistry 28, 179±194.
Huang, Y., Peakman, T.M., Murray, M., 1997. 8b,9a,10b -rimuane, a novel, optically active tricyclic hydrocarbon of algal origin. Tetrahedron Letters 38, 5363±5366.
van Kaam-Peters, H.M.E., Schouten, S., KoÈster, J., de Leeuw, J.W., Sinninghe DamsteÂ, J.S., 1997. A molecular and carbon isotope biogeochemical study of biomarkers and kerogen pyrolysate of the Kimmeridge Clay formation: paleo-environmental implications. Organic Geochemistry 27, 399± 422.
van Kaam-Peters, H.M.E., Schouten, S., KoÈster, J., Sinninghe DamsteÂ, J.S., 1998. Controls on the molecular and and car-bon isotopic composition of organic matter deposited in a Kimmeridgian euxinic shelf sea: evidence for preservation of carbohydrates through sulfurisation. Geochimica et Cosmo-chimica Acta 62, 3259±3283.
Kissin, Y.V., Feulmer, G.P., Wayne, W.B., 1986. Gas Chro-matographic analysis of polymethyl-substituted alkanes. Journal of Chromatographic Science 24, 164±169.
Kohnen, M.E.L., Sinninghe DamsteÂ, J.S., Rijpstra, W.I.C., de Leeuw, J.W., Alkylthiophenes as sensitive indicators of palaeoenvironmental changes: A study of a Cretaceous oil shale from Jordan. In: Orr, W.L., White, C.M. (Eds.), Geo-chemistry of Sulfur in Fossil Fuels. ACS Symposium Series 429, American Chemical Society, pp. 444±485.
Kok, M.D., Rijpstra, W.I.C., Robertson, L., Volkman, J.K., Sinninghe DamsteÂ, J.S., 2000. Early steroid sulfurisation in surface sediments of a permanently strati®ed lake (Ace Lake, Antarctica). Geochimica et Cosmochimica Acta 64, 1425± 1436.
Koopmans, M.P., KoÈster, J., van Kaam-Peters, H.M.E., Kenig, F., Schouten, S., Hartgers, W.A., deLeeuw, J.W., Sinninghe DamsteÂ, J.S., 1996a. Dia- and catagenetic pro-ducts of isorenieratene: molecular indicators for photic zone anoxia. Geochimica et Cosmochimica Acta 60, 4467±4496. Koopmans, M.P., de Leeuw, J.W., Lewan, M.D., Sinninghe
DamsteÂ, J.S., 1996b. Impact of dia- and catagenesis on sul-phur and oxygen sequestration of biomarkers as revealed by arti®cial maturation of an immature sedimentary rock. Organic Geochemistry 25, 391±426.
Koopmans, M.P., Rijpstra, W.I.C., de Leeuw, J.W., Lewan, M.D., Sinninghe DamsteÂ, J.S., 1998. Arti®cial maturation of an immature sulphur- and organic matter-rich limestone from the Ghareb Formation, Jordan. Organic Geochemistry 28, 503±521.
Largeau, C., Casadevall, E., Kadouri, A., Metzger, P., 1984. Formation of Botryococcus-derived kerogens-Comparitive study of immature torbanites and of the extant alga Botryo-coccus braunii. Organic Geochemistry 6, 327±332.
Largeau, C., Derenne, S., Casedevall, E., Kadouri, A., Sellier, N., 1986. Pyrolysis of immature Torbanite and of the resistant biopolymer (PRB A) isolated from extant algaBotryococcus braunii. Mechanism of formation and structure of Torbanite. Organic Geochemistry 10, 1023±1032.
Lattuati, A., Guezennec, J., Metzger, P., Largeau, C., 1998. Lipids ofThermococuus hydrothermalis, an Archaea isolated from a deep-sea hydrothermal vent. Lipids 33, 319±326. Laws, E.A., Popp, B.N., Bidigare, R.R., Kennicutt, M.C.,
Macko, S.A., 1995. Dependence of phytoplankton carbon isotopic composition on growth rate and [CO2]aq: theoretical considerations and experimental results. Geochimica et Cos-mochimica Acta 59, 1131±1138.
de Leeuw, J.W., van Bergen, P.F., van Aarssen, B.G.K., Gate-lier, J.-P.L.A., Sinninghe DamsteÂ, J.S., Collinson, M.E., 1991. Resistant biomacromolecules as major contributors to kerogen. Philosophical Transactions of the Royal Society of London B 333, 329±337.
de Leeuw, J.W., Largeau, C., 1993. A review of macro-molecular organic compounds that comprise living organisms and their role in kerogen, coal and petroleum formation. In: Engel, M.H., Macko, S.A. (Eds.), Organic Geochemistry. Principles and Applications. Plenum Press, New York, pp. 23±72.
Liaaen-Jensen, S., 1979. Marine carotenoids. In: Faulkner, D.J., Fenicall, W.H. (Eds.), Marine Natural Products. Aca-demic Press, London, pp. 233±247.
McCarthy, E.D., Calvin, M., 1967. The isolation and identi®ca-tion of the C17saturated isoprenoid hydrocarbon 2,6,10-tri-methyltetradecane from a Devonian shale. The role of squalane as a possible precursor. Tetrahedron 23, 2609±2619. Merrit, D.A., Brand, W.A., Hayes, J.M., 1994. Isotope-ratio-monitoring gas chromatography: methods for isotopic cali-bration. Organic Geochemistry 21, 573±584.
Metzger, P., Largeau, C., Casadevall, E., 1991. Lipids and macromolecular lipids of the hydrocarbon-rich microalga
Botryococcus braunii. Chemical structure and biosynthesis. Geochemical and biotechnological importance. In: Herz, W. et al. (Eds.), Progress in the Chemistry of Organic Natural Products, Vol 57. Springer-Verlag, Berlin, pp. 1±70. Moldowan, J.M., Seifert, W.K., 1979. Head-to-head linked
isoprenoid hydrocarbons in petroleum. Science 204, 169± 171.
Pancost, R.D., Freeman, K.H., Patzkowsky, M.E., 1999. Organic-matter source variation and the expression of a late Middle Ordovician carbon isotope excursion. Geology 27, 1015±1018.
Peakman, T.M., Sinninghe DamsteÂ, J.S., de Leeuw, J.W., 1989a. Organic geochemical evidence for a series of C25ÿ28 sulphur-containing lipids comprising regular and irregular isoprenoid and unusually linearly extended phytane skele-tons. Journal of the Chemical Society, Chemical Commu-nications 16, 1105±1107.
Peakman, T.M., Sinninghe DamsteÂ, J.S., de Leeuw, J.W., 1989b. The identi®cation and geochemical signi®cance of a second series of alkylthiophenes comprising a linearly exten-ded phytane skeleton in sediments and oils. Geochimica et Cosmochimica Acta 53, 3317±3322.
Philp. R.P., 1985. Fossil fuel biomarkers. Methods in Geo-chemistry and Geophysics, Vol. 23. Elsevier, New York, 294 p. Popp, B.N., Laws, E.A., Biigare, R.R., Dore, J.E., Hanson, K.L., Wakeham, S.G., 1998. Eect of phytoplankton cell geometry on carbon isotope fractionation. Geochimica et Cosmochimica Acta 62, 29±77.
Rau, G.H., Riebesell, U., Wolf-Gladrow, D., 1996. A model of fractionation by marine phytoplankton based on diusive molecular CO2uptake. Marine Ecology Progress Series 133, 275±285.
Quandt, I., Gottschalk, G., Ziegler, H., Stichler, W., 1977. Isotope discrimination by photosynthetic bacteria. FEMS Microbiology Letters 1, 125±128.
Rau, G.H., Takahashi, T., DesMarais, D.J., Repeta, D.J., Martin, J.H., 1992. The relationship between (13C of organic matter and [CO2(aq)] in ocean surface water: data from a JGOFS site in the northeast Atlantic Ocean and a model. Geochimica et Cosmochimica Acta 56, 1413±1419.
Revill, A.T., Volkman, J.K., O'Leary, T., Summons, R.E., Boreham, C.J., Banks, M.R., Denwer, K., 1994. Hydro-carbon biomarkers, thermal maturity and depositional set-ting of Tasmanite oil shales from Tasmania, Australia. Geochimica et Cosmochimica Acta 58, 3803±3822.
Schmidt, K., 1978. Biosynthesis of carotenoids. In: Clayton, R.K., Sistrom, W.R. (Eds.), Photosynthetic Bacteria. Plenum Press, New York, pp. 729±750.
Schouten, S., van Driel, G.B., Sinninghe DamsteÂ, J.S., de Leeuw, J.W., 1993. Natural sulphurization of ketones and aldehydes: a key reaction in the formation of organic sulphur com-pounds. Geochimica et Cosmochimica Acta 57, 5111±5116. Schouten, S., Hoefs, M.J.L., Koopmans, M.P., Bosch, H.-J.,
Sinninghe DamsteÂ, J.S., 1998. Structural characterization, occurrence, and fate of archaeal ether-bound acyclic and cyclic biphytanes and corresponding diols in sediments. Organic Geochemistry 29, 1305±1319.
Sinninghe DamsteÂ, J.S., de Leeuw, J.W., 1990. Analysis, struc-ture and geochemical signi®cance of organically-bound sul-phur in the geosphere: state of the art and future research. Organic Geochemistry 16, 1077±1101.
Sinninghe DamsteÂ, J.S., Kok, M.D., Koster, J., Schouten, S., 1998. Sulfurized carbohydrates: an important sedimentary
sink for organic carbon? Earth and Planetary Science Letters 164, 7±13.
Sinninghe DamsteÂ, J.S., Rijpstra, W.I.C., de Leeuw, J.W., Schenck, P.A., 1988. Origin of organic sulphur compounds and sulphur containing high molecular weight substances in sedimantes and immature crude oils. In: Matevelli, L., Novelli, L. (Eds.), Advances in Organic Geochemistry. Per-gamon, Oxford, Organic Geochemistry 13, pp. 593±606. Sinninghe DamsteÂ, J.S., Schouten, S., 1997. Is there evidence
for a substantial contribution of prokaryotic biomass to organic carbon in Phanerozoic carbonaceous sediments? Organic Geochemistry 26, 517±531.
Sinninghe DamsteÂ, J.S., Wakeham, S.G., Kohnen, M.E.L., Hayes, J.M., de Leeuw, J.W., 1993. A 6,000-year sedimen-tary molecular record of chemocline excursions in the Black Sea. Nature 362, 827±829.
SirevaÊg, R., Ormerod, J.G., 1970. Carbon dioxide ®xation in green sulfur bacteria. Biochemistry Journal 120, 399±408. SirevaÊg, R., Buchanan, B.B., Berry, J.A., Troughton, J.H.,
1977. Mechanisms of CO2®xation in bacterial photosynth-esis studied by the carbon isotope fractionation technique. Archives of Microbiology 112, 35±38.
Tegelaar, E.W., de Leeuw, J.W., Derenne, S., Largeau, C., 1989. A reappraisal of kerogen. Geochimica et Cosmochi-mica Acta 53, 3103±3106.
Thiel, V., Peckmann, J., Seifert, R., Wehrung, P., Reitner, J., Michaelis, W., 1999. Highly isotopically depleted iso-prenoids: molecular markers for ancient methane venting. Geochimica et Cosmochimica Acta 63, 3959±3966.
Van der Meer, M.T.J., Schouten, S., van Dongen, B.E., Rijp-stra, W.I.C., Fuchs, G., Sinninghe DamsteÂ, J.S., de Leeuw, J.W., Ward, D.M. Biosynthetic controls on the13C-contents of organic components in the photoantotrophic bacterium
Chloro¯exus aurantiacus. Journal of Biological Chemistry, submitted.
Werne, J.P., Hollander, D.J., Behrens, A., Schaeer, P., Albrecht, P., Sinninghe DamsteÂ, J.S., 2000. Timing of the diagenetic sulfurization of organic matter: a precursor-pro-duct relationship in Holocene sediments of the anoxic Car-iaco Basin, Venezuela. Geochimica et Cosmochimica Acta 64, 1741±1751.
