Bacterial and algal markers in sedimentary organic matter deposited under natural sulphurization...

Pergamon Org. Geochem. Vol. 26, No. 9/10, pp. 605-625, 1997

© 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain

Plh S0146-6380(97)00034-X o 146-6380/97 $17.00 + o.oo

Bacterial and algal markers in sedimentary organic matter deposited under natural sulphurization conditions (Lorca Basin, Murcia, Spain)

MARIE RUSSELL'*, JOAN O. GRIMALT', WALTER A. HARTGERS't , CONXITA TABERNER-' and JEAN MARIE ROUCHY 3

'Department of Environmental Chemistry (C.I.D.-C.S.I.C.), Jordi Girona 18, 08034 Barcelona, Catalonia, Spain, -'Institute of Earth Sciences (C.S.I.C.), Lluis So16 Sabaris s/n, 08028 Barcelona,

Catalonia, Spain and ~Laboratory of Geology (C.N.R.S.U.R.A. 723), National Museum of Natural History, 43, rue Buffon, 75005 Paris, France

Abstract--Free lipids, sulphur-bound lipids present in macromolecular fractions and kerogen pyrolysis products have been studied in shales and early diagenetic carbonates replacing gypsum from a Messi- nian sequence of Lorca Basin. The high abundance of phytane, 2,3-dimethyl-5-(2,6,10-trimethylundecyl)thiophene, mid-chain C20 isoprenoid thiophenes and bithiophenes and bis-O-phytanyl and O- phytanyl-O-sesterterpanyl glycerol ethers indicates that the organic matter in all these samples was deposited under hypersaline conditions. The isopranyl glycerol ethers are essentially found in the sulphur-bound macromolecular matter which contrasts with the low concentrations of these compounds as free lipids. However, the distributions of these isopranyl glycerols is paralleled by the occurrence of free phytanic acid (shales and laminated carbonate) and 3,7,11,15,19-pentamethyleicosanoic acid (only in the laminated carbonate). The bacterial inputs are represented by 2-hydroxytetracosanoic acid, n- octadec-1 l-enoic acid, hopanoic acids and the distributions of iso- and anteiso-C~5 and C17 homologues and minor amounts of iso-Ci6 and anteiso-Cl4. These branched fatty acids are characteristic of sulphate-reducing bacteria. The relative proportions of the iso- and anteiso-compounds in the total fatty acid distributions are correlated with the proportion of reduced sulphur in the sediments. © 1997 Else- vier Science Ltd

Key words--algal organic matter, bacteria, fatty acids, alkylthiophenes, alkylthiolanes, alkylbenzo[b]thiophenes, sulphurization of organic matter, Messinian evaporites, diagenetic carbonates, organic- rich shales

INTRODUCTION

The recognition of algal and bacterial inputs in the lipid sedimentary record is usually obscured by post-depositional diagenetic transformations that strongly modify the original composition. However, in anoxic environments with abundant sulphate, preservation of the lipid mixtures is apparently enhanced by the formation of sulphur-bound compounds. At low iron concentration (Gransch and Posthuma, 1974), hydrogen sulphide generated by sulphate-reducing bacteria binds to the organic molecules, increasing their potential preservation (see Sinninghe Damst6 and de Leeuw (1990) for a review). The composition of these organo-sulphur molecules has been studied intensively and the sulphurization process has been reported to involve the selective preservation of the algal inputs orig- inally present at deposition (Kohnen et al., 1991).

*Present address: Department of Organic and Environmental Geochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK

tPresent address: Petroleum and Environmental Geochemistry Group, Department of Environmental Sciences, University of Plymouth, Plymouth PL4 8AA, UK

A substantial number of the studies dealing with this sulphurization process have been devoted to structural elucidation, either the GC amenable organo-sulphur molecules or the compounds sulphur-bound to macromolecules. However, knowl- edge of the mechanisms of sulphur incorporation to the lipid molecules must be improved. More information, particularly on the biases between orig- inally sedimented and the fraction consisting of sulphurized "preserved" components is needed if the sulphur-bound molecular distributions are to be considered for palaeoenvironmental assessment.

The comparison of distributions of organo-sulphur molecules and functionalized lipids may contribute to a better understanding of the origin of the sulphur-bound material. In this respect, the investigation of the lipids characteristic of sulphate- reducing bacteria, namely the fatty acid fraction, is important. These objectives can be addressed by the study of sedimentary sequences in which, in addition to organo-sulphur compounds produced by natural sulphurization, a substantial amount of functionalized lipids can still be encountered.

The Lorca Basin (Fig. 1) is one of a number of Neogene basins in southeast Spain that contains

605

606 M. Russell et al.

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f ~ 0 50 10~1 km

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7277~ diatomites

Marls and thin-bedded gypsum Gypsum (9errata Fro.) Marls and sandstones Marls and diatomites Sulphur-bearing carbonates Marls with carbonate intercalations Marls Sequence of studied organic-rich shales and sulphur-bearing carbonates

Pliocene Tortonian

Fig. 1. Location map of the Lorca Basin (a). Stratigraphic section of the uppermost (Messinian) part of the basin representing the outcrop of La Serrata close to the El Volcfin gypsum quarry (b). Schematic lilhological column of the studied sequence (c). Only ['our of the six carbonate levels with sulphur crop

out in this part of the basin.

Messinian sediments. This basin encompasses a wide range of environments from normal marine to hypersaline, including sequences of sulphur-rich carbonates associated with organic rich shales that contain large amounts of organo-sulphur compounds and functionalized lipids. Some features of the carbonate beds, such as the presence of elemental sulphur and the replacement of sulphate by carbonate with preservation of the original gypsum textures suggest that the origin of part of these carbonates could be related to the action of bacterial sulphate reduction processes, as in the examples studied by Pierre and Rouchy (1988) and Anaddn et

al. (1992). Preliminary isotopic data (bl3C of carbonates and 634S and cgSO of sulphates and native sulphur) are in agreement with such a process. Thus, these organic-rich shales and associated carbonates are a priori good environments for the assessment of the relative importance of algal and bacterial inputs in the distributions of fi'ee and sulphur-bound molecules.

GEOLOGICAL SETTING

The Lorca basin (Fig. la) displays a well-exposed Messinian sedimentary series and records the tran- sition from deposition under normal salinity (marls) to restricted (diatomite-bearing deposits) and hypersaline conditions (massive gypsum). Previous geo-

logical studies in the area have focused on the structural and stratigraphic aspects (Geel, 1976; Montenat et al., 1987). Information on the age of the sedimentary units infilling the basin is available from the studies of Geel (1976) and Montenat et al.

(1987). A more recent study by Dinares-Turrell et

al. (1997) gives new insights on the age of the marine infill of the basin and provides the time con- straints used for Fig. 1.

The Messinian deposits commence with marine marls and grade upwards to thick levels of thinly laminated diatomites, that are interbedded with silty claystones or marlstones and carbonate beds (Fig. lb). The uppermost part of the section, just below the massive gypsum of La Serrata Fm, consists of marls with intercalations of sandstones and thin diatomitic laminae (Fig. l b).

At least six main levels of sulphur-bearing dolo- mitic limestones, each less than l m thick, have been recognized within the diatomitic intervals in the basin (Fig. lb). As indicated by the petro- graphic features, these carbonate levels were formed after replacement of gypsum beds. Organic-rich shales are related to these sulphur-bearing carbonates (Benali et al., 1995). High organic contents in some beds suggest that those sediments were deposited in a restricted marine environment with both high productivity and stagnant bot tom conditions (Geel, 1976: Rouchy, 1982: Permanyer et al., 1994;

Bacterial and algal markers 607

Benali et al., 1995). Mining of the sulphur-rich levels continued from Roman times until approxi- mately 1939 and relatively unweathered material was exposed by this mining activity. Organic-rich shales have also been detected in boreholes (IGME, 1982).

EXPERIMENTAL

Inorganic composition

Mineralogy was obtained by X-ray diffraction (XRD) of powdered samples using a Siemens D-500 diffractometer fitted with a Cu tube (K = 1.5418) and a secondary graphite monochromator. 20 angle was from 4 to 60 °. The use of mineral phase-specific reflectivity factors and area integration of selected peaks provided wt% semi-quantitative data of the crystalline phases above detection limit (Chung, 1974). The non-quantified fraction should essentially represent opal and other amorphous material, such as organic matter. Total carbonate contents were determined by calcimetry. The proportion of each component carbonate mineral was calculated from their respective peak heights. These results were used as a control of the carbonate results from the XRD method.

Thermogravimetry was performed with a SETARAM TG92 thermoanalyzer by heating from 20 to 920 ° at a 10°/min rate in an Ar atmosphere. One hundred mg sample aliquots allowed the determination of the weight losses for S ° and gypsum. The gypsum determinations were compared with those obtained by XRD. The S ° measurements were compared with the elemental analysis data to calcu- late the amounts of sulphur-bound organic matter.

The organic matter-rich shales were observed under scanning electron microscopy (SEM) for tex- tural characterization and to better constrain the mineralogical composition. A Jeol 840 (JEOL(~) fitted with a solid XRD with a Si(Li) diode and an AN10000 LINK analyzer was used. Working conditions were 15 KeV, gun current 3 nA and working distance 39 ram.

Materials and reagents for organic matter analysis

Chromatographic-grade dichloromethane and n- hexane, methanol and iso-octane were used. Neutral silica gel and alumina were extracted with dichloromethane:methanol (2:1, v/v) in a Soxhlet apparatus for 24 h. After solvent evaporation, the silica and the alumina were heated for 12h at 120°C and 350°C, respectively. Five percent of Milli-Q grade water was added to these adsorbents for deactiva- tion. The ethereal solutions of diazomethane were prepared following standard methods (Vogel, 1978).

Bulk organic matter analyses

Sediment sample aliquots were treated for the removal of calcite, dolomite and sulphates prior to

elemental analysis as follows: 0.2 N HC1 was added slowly until no CO2 bubbling was observed. Then, 37 N HC1 was added and the sample was heated at 60°C for 2 h. After centrifugation, the supernatant was removed and the residue rinsed with distilled water until a neutral pH was obtained. An excess of distilled water was added and the samples were heated at 100°C for 30 min to ensure that any sulphate was removed. The samples were centrifuged again, the supernatant discarded and the residue was rinsed with methanol and dried overnight at 60°C. The samples were subsequently analyzed by XRD to ensure that all carbonate and sulphate had been removed. The samples were then analyzed for elemental carbon, hydrogen, nitrogen and sulphur using a Carlo Erba instrument, model EA1108.

Analysis o f fatty acids and neutral lipids

The samples were extracted with 250ml of dichloromethane:methanol (2:1, v/v) in a Soxhlet apparatus for 48 h and the extracts were concentrated by vacuum rotary evaporation (ca. 2 ml). The extracts were hydrolyzed overnight at room temperature with 35 ml of 6% KOH in methanol. The corresponding neutral and acidic fractions were recovered by successive extractions with n-hexane (3 x 30 ml), the latter after acidification (pH 2) with aq. 6 N HCI. Both extracts were vacuum evapor- ated and concentrated to ca. 0.5 ml. The acidic fractions were methylated with an ethereal solution of diazomethane for gas chromatographic analysis. The neutrals were fractionated by column chromatography in a 34 x 0.9 cm i.d. column filled with 7 g of 5% water deactivated alumina (70-230 mesh, Merck; top) and silica (70-230 mesh, Merck; bot- tom). Six 20 ml fractions of increasing polarity were collected: (1) n-hexane; (2) n-hexane-dichloromethane (9:1, v/v); (3) n-hexane-dichloromethane (4:1); (4) n-hexane-dichloromethane (1:4); (5) dichloromethane-methanol (9.5:0.5); and (6) dichloromethane-methanol (9:1). All fractions were analyzed by gas chromatography (GC) and mass spectrometry coupled to gas chromatography (GC- MS). Prior to instrumental analysis they were evap- orated to dryness and redissolved in iso-octane. Fractions 5-6 were silylated with bis(trimethylsilyl)- trifluoroacetamide (BSTFA, 100 #1, 80°C, 30 min).

GC was performed with a Carlo Erba Mega Model HRGC 5300 equipped with a flame ioniz- ation detector and a splitless injector. A capillary column coated with DB-5 (30 m x 0.25 mm i.d.; film thickness 0.2 #m) was used. Hydrogen was the carrier gas (50 cm/s). The oven temperature program was 70 to 310°C at 6°C/min with a final holding time of 20 min. Injector and detector temperatures were 300 and 330°C, respectively. The injection was performed in splitless mode (iso-oetane, hot needle technique) keeping the split valve closed for 35 s. Nitrogen was used as make up gas (30 ml/min).


deca

Curie point py-GC-MS

~acaon ~MeOH (2:0

GC,GC-MS

~saponification 1N KOH/MeOH

GC,C,C-MS ~ c o l u m n chromatography

, , ? , ;,y,aoo. ! I .ol~rs for

q¢ also analysed by GC and GC-MS ~ [ desulphurization

column NizB desulphurization (l) chromatography (2)

desulphurized apolars

~ PtQ/H2 silylation GC,GC-MS

GC,GC-MS

Fig. 2. Analytical flow diagram of the sample fractionation performed in this study. The shaded boxes correspond to the fractions discussed in text.

Detector gas flows were hydrogen (30 ml/min) and air (300 ml/min).

GC-MS was performed with a Fisons MD-800 quadrupole instrument. The gas chromatograph was equipped with a fused silica capillary column (30m x0 .25mm i.d.) coated with HP-5MS (film thickness 0.25 pm). Helium was used as carrier gas. The oven temperature program was 60°C (solvent delay 4min) to 310°C at 4°C/min (holding time 15 rain). Injector, transfer line and ion source temperatures were 300, 280 and 200°C, respectively. The injection was in the splitless mode (iso-octane, hot needle technique) keeping the split valve closed for 48 s. Spectra were obtained in the electron impact mode (70 eV) through scanning from masses 50 to 700 every second.

Quantitation was performed from the GC traces using n-dodecylbenzene as internal standard. This standard was added to both the acidic and neutral fractions prior to GC analysis.

Nickel boride desulphurization/hydrogenation

The polar fractions 4 6 (Fig. 2) were combined and treated with Ni2B for desulphurization using the method of Schouten et al. (1993). 5 30 mg of sample were mixed with 500 mg of anhydrous NiC12 and 500 mg of NaBH4 for reduction. The amounts of these reagents are around five times higher than

those previously described (Schouten et al., 1993) to improve the yields (Hartgers et al., 1996). The reac- tion products were subsequently separated on a short column of alumina (70-230 mesh, Merck, activated at 150°C for 2 h) into apolar (four column volumes of hexane:dichloromethane, 9:1, v/v) and polar (four column volumes of MeOH: dichloromethane, 1:1, v/v) fractions. Prior to chromatographic analysis the polar fractions were silylated with BSTFA (70°C, 30 min). Incomplete hydrogenation led to the presence of unsaturated hydrocarbons in the apolar fractions. Since these provide no information as to the number and/or position of the C S linkages, catalytic hydrogenation with PtO2 under hydrogen was performed (12 h, room temperature, with a small amount of acetic acid added). Both alumina-separated apolar and polar fractions were analyzed by GC and GC-MS.

Pyrolysis gas chromatography-mass spectrometry (py G ~ M S )

Pyrolyses were carried out using a Pi-rho Curie- point pyrolyser and control unit (Horizon Instruments Ltd) at 610°C. Samples were pressed onto the wire in a pre-cleaned stainless steel press (15 tonnes; Venema and Veurink, 1984). The maxi- mum pyrolysis time was 2 s. The pyrolysis unit was interfaced to the split/splitless injector of a Hewlett


Table I. Semi-quant i ta t ive determinat ion of the composi t ion of the studied samples (%). The last column represents the errors of the technique plus the contr ibut ions of amorphous inorganic phases. These amorphous phases are shown to be significant for shale 1 and

shale 2 (see text for details)

Calcite Dolomite Total Quar tz Clays (illite G y p s u m S ° Total O M Non- Sample carbonates and chlorite) quantified

Brecciated 382 50 ~ 88 ~ 0.9" N D a,b 2.42 0 . 8 1 c - - 0.013 d 11 carbonate Laminated 82" 6.32 88 ~' 94 e 1.92 N D a.b 0 . 8 1 c 1.1 e 2.6 b 0.27 d 62 carbonate Shale I 8.3 ~' N D 2.b 8.3 a 9.0" 132 10 a 1 1 c ~2.9 b 31 d 25 Shale 2 112 3.3 ~' 1 5 ~ I 1 ~ 8.52 122 6.1 a 6.0 ~ 5.1 e ~3.2 b 30 d 27

" As determined by X-ray diffraction. hND, not detected. CAs determined by thermogravimetry . dDetermined f rom elemental analysis after HCI treatment. ~Calculated f rom weight loss after HCI treatment. rWhere there is more than one value, the value in italics corresponds to the most reliable measurement .

Packard HP8590 GC, which was fitted with a cryo- genic cooling unit (liquid CO2). The pyrolysis products were swept onto a GC column (DB-1; 3 0 m x 0 . 2 m m i.d.; film thickness 0.33 #m) in a stream of helium carrier gas (0.5 ml/min), which was fed directly into the source of a VG TS-250 mass spectrometer. The oven temperature program was -40 to 300°C at 6°C/min and held isothermally at 300°C for 15 min. The MS conditions were: electron voltage 70 eV, trap current 200/~A, magnet scanned from 40 to 500 Da every second at a resol- ution of 500.

Pyrolysis products were identified by their mass spectra, relative retention indices and by comparison of spectra reported in the literature. Compound concentrations were calculated by comparison of their peak areas to that of t-butylstyrerene, which is produced on pyrolysis of the polymeric internal standard (poly-(p-4-butyl)-styrene, Polysciences Inc., Warrington, U.K.). Data were semi-quantitative since relative response factors were taken to be 1.

R E S U L T S A N D D I S C U S S I O N

Sedimen tology

The sequence selected for study corresponds to the third carbonate level and associated organic matter rich shales shown in Fig. l(c). The thickness of the sequence ranges between 70 and 100 cm (50-70 cm for the carbonate bed). Laterally to the east, in relatively marginal areas, this carbonate bed passes to gypsum as do most of the similar carbonates in this basin (Rouchy, 1982; Dinares-Turrell et al., 1997). The four samples chosen for organic geochemical analysis have been selected for their position in the studied sequence and for their features observed in the field. The textures and composition of these samples observed under transmitted light microscopy and SEM suggest that they have been formed under different depositionai environments and have been affected by diverse diagenetic processes.

Table 1 describes the bulk composition of the samples selected for study. The XRD values provide

semi-quantitative data. The accumulated error in the quantitative determinations can be estimated by the percentage of undescribed material which, in addition to errors, also encompasses the relative content of amorphous inorganic phases. The expected content of amorphous inorganic phases in the carbonates is very low. Thus, the values of 6-11% in these two samples probably give an estimate of the relative errors of quantitation. In contrast, the shales have 25-27% of undescribed material, which is too high to correspond to errors in the technique. In this respect, XRF analyses show that there is an excess of SiO2 (ca. 7%) when compared with the content of quartz and clay minerals (illite and chlorite). Part of the non-quantified phases could correspond to opal (difficult to detect with XRD when present in small amounts).

The brecciated carbonate displays the most typical features of the carbonate bed from the studied sequence and was formed by partial replacement of former gypsum crystals which were disseminated in a dolomite matrix. This carbonate consists at present of dolomicrite fragments outlined by fractures and gypsum crystal moulds (average l cm in size), both infilled by sparry calcite. The calcite filled fractures are seen only to cross-cut this carbonate deposit and not the organic-rich shales or diatomites, which suggests a local source of CO2 for later carbonate precipitation. The shapes of the gypsum moulds and their relation to the surround- ing dolomicrite show that gypsum grew mostly interstitially in a soft matrix. In some cases, the disposition of the gypsum pseudomorphs suggests replacement after precursor selenite crystals which were formed by sub-aqueous growth. Thus, the salinities required for gypsum precipitation were mainly reached interstitially, although in some parts they were achieved at the sediment-water interface, as evidenced by the precursor selenite crystals. The original textures of the gypsum before replacement by carbonates are suggestive of those typical of solar saltern-like shallow water evaporite deposits. The presence of interstitial gypsum suggests occasional dessication. Reduced sulphur and total or-


Table 2. Organic matter elemental analysis after removal of carbonates and sulphates

Sample %C ~ %H %N %S b Tm~,x(°C)

Brecciated carbonate 0.01 c c 0.02 ND d Laminated carbonate 0.20 0.02 0.01 2.75 ND d Shale 1 23 2.9 0.42 6.9 406 Shale 2 22 2.6 0.45 7.5 405

"All percentages are in weight. bRepresents organic sulphur, elemental sulphur and, in very minor amounts, pyrite. ~Organic content too low for determination. 'IND~ not determined.

ganic carbon contents are very low (Table 2). Thermogravimetry data proves that there is no gypsum left after replacement by carbonate.

The laminated carbonate displays textures which are only locally present in the studied carbonate bed and apparently has most of the original sedimentary features preserved. It consists mainly of calcite with lesser amounts of dolomite (Table 1). The sample corresponds to a laminated micrite with gypsum pseudomorphs (a few millimetres in length) preferentially aligned along some laminae. This texture also suggests sporadic dessication. Cavities resulting from dissolution of gypsum crystals are infilled by sparry calcite and elemental sulphur crystals. The laminated texture of this sample and the disposition of the original gypsum crystals along laminae resembles that of cyanobacterial mats found in recent hypersaline environments, where gypsum crystals grow interstitially displacing the original sediment. The reduced sulphur and total organic carbon contents are more than one order of magnitude higher than those of the brecciated carbonate sample (Table 2).

The two organic-rich shales have been collected in old sulphur mines, where the best preserved samples can be obtained. Their position with respect to the carbonate bed can be elucidated by investigation of the sections in the old sulphur mines and the relatively unweathered outcrops exposed in the new rock-cuts of the concrete factory on the eastern side of La Serrata. In both samples the main mineral components are calcite, clay minerals (illite and chlorite) and quartz with minor amounts of gypsum and dolomite (Table 1). Sample 2 is richer in calcite than sample 1. Elemental sulphur aggregates between 5 and 10/tm in size, as well as nodules and thin laminae of sulphur (up to 10 ~tm in size) are observed under SEM. This disposition suggests interstitial growth during the early stages of diagenesis. Small fragments of feldspars and framboidal pyrite have also been detected by SEM observations. These two samples have high contents of reduced sulphur and total organic carbon (Table 2).

Curie-point p y r o l y s i s - G C - M S

Curie-point pyrolys is-GC-MS was carried out on the Soxhlet-extracted decarbonated sediments, which were re-extracted by ultrasonication to remove traces of soluble organic material. Pyrolysis of the brec-

ciated carbonate (Fig. 3a) generated a simple distribution of products, the most significant compounds being n-C6 alkene/alkane, benzene, toluene and phe- nol. Organo-sulphur compounds were not detected among the pyrolysis products in this sample.

The most significant compounds generated on pyrolysis of the laminated carbonate kerogen (Fig. 3b) are benzene, toluene and styrene, with subordinate amounts of C5 and C6 alkylthiophenes. There are also minor amounts of n-alkenes/alkanes in the C6 C23 range.

In contrast, the pyrolysates of the two shales are dominated by organo-sulphur compounds, namely C4 C20 alkylthiophenes, with maxima at the C5 C8 homologues. These compounds were identified by comparison with previously reported GC retention time and mass spectral data (Eglinton et al., 1992). C8-CI2 benzothiophenes are present in minor amounts. Minor alkylthiolanes in the range C2-C20 are also present. The dominance of organo-sulphur compounds in the pyrolysates reflects the incorporation of abiotic sulphur species into macromolecular organic matter leading to the formation of sulphur-rich kerogens (Sinninghe Damst~ et al., 1989a). The internal distribution of alkylthiophenes shows relative high abundances of 2,3- and 2,3,5- di- and tri-substituted thiophenes (Fig. 3c,d), similar to the distribution encountered in pyrolysates of Monterey kerogens (Eglinton et al., 1992). Their presence is attributed to an isoprenoid and steroid origin. Furthermore, C6-C30 n-alkene/alkane doub- lets are important pyrolysis products of the two shales. The abundance of these aliphatic hydrocarbons and the characteristic alkylthiophene distribution indicate that the kerogens of the two shales can be classified as marine-derived, sulphur-rich kerogens (Type II-S; Eglinton et al., 1992).

A useful indicator of the degree of sulphurization is the ratio of 2-methylthiophene to toluene (Eglinton et al., 1990; Sinninghe Damst~ et al., 1993). This ratio has values of 6 for both shales and 0.1 and 0 for the laminated carbonate and the brecciated carbonate, respectively. The changes in this ratio parallel the abundance of reduced sulphur in the samples (Table 2). Furthermore, the solvent-extractable sulphur-containing compounds follow a parallel trend. These compounds are dominant in the two shales, occur as minor products in the laminated carbonate and are absent in the brecciated carbonate (Fig. 4).


Prist-l-ene is present in significant amounts in both shale pyrolysates. Its origin has been attributed to cleavage of the phytenyl side chain of macro- molecularly-bound chlorophyll a and b, although bound archaebacterial lipids (phytenyl glycerol ethers) or ~-tocopherol (Goossens et al., 1984), which is present in small amounts in methanogenic bacteria (Hughes and Toney, 1982) can contribute as well.

H y d r o c a r b o n s

In the brecciated carbonate the dominant compounds are C17 C34 n-alkanes, pristane and phytane (Fig. 4). Phytane predominates over pristane (Pr/ Ph = 0.2) which, in the context of these samples, is indicative of deposition under hypersaline conditions (ten Haven e t al. , 1985). The C29-C33 odd carbon numbered n-alkanes reflect contributions from higher plants (Eglinton and Hamilton, 1967). Phytane is the major alkane in the shales where other saturated hydrocarbons are only found at trace levels. No significant GC resolved n-alkanes have been found in the laminated carbonate. Further separation by Ag + thin-layer chromatography (Kohnen et al. , 1990) confirmed the lack of significant GC resolved n-alkanes in this sample.

The apolar compounds in the shales and laminated carbonate are essentially constituted of organo-sulphur compounds, namely benzo[b]thiophenes, thiophenes, bithiophenes and thiolanes. The relative proportions of these compounds are represented in Fig. 4 by means of the corresponding gas chromatographic traces. The identifications of these compounds were performed by mass spectral interpretation, comparison with samples of known composition (Sinninghe Damst6 et al. , 1989b; Grimalt et al. , 1991a) and with published mass spectra (Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst~ et al.~ 1986, 1987a,b).

Short-chain alkylbenzo[b]thiophenes are the major apolar compounds, ranging between C9 and CI4 in the shales and between Ci0 and C15 in the laminated carbonate. These compounds encompass complex mixtures of isomers with several alkyl-sub- stitution patterns dominated by the C3 and the C4 substituted homologues (Fig. 4) in the laminated carbonate and the C~-C4 substituted homologues in the shales. In the laminated carbonate these compounds constitute almost the only identified organo-sulphur compounds in the hydrocarbon fraction. Only traces of some alkylthiophenes (e.g. 2,3-dimethyl-5-(2,6,10-trimethylundecyl)thiophene, I), have been identified (Fig. 5).

Conversely, significant relative abundances of alkylthiophenes and, to a lesser extent, alkylthiolanes are present in the shales. The most abundant alkylthiophenes constitute complex mixtures ranging between C9 and C~4. In addition to these, at higher carbon numbers, Ca5-C26, the alkylthio-

phenes exhibit distributions of 2,5-dialkyl substituted isomers encompassing methyl/C,_ 5, ethyl/ C,, _ 6, propyl/C, _ 7, butyl/Cn _ s and pentyl/C, _ 9 groups (n = total carbon length). These compounds have been identified by interpretation of the G C - MS data and comparison with previously reported spectra (Sinninghe Damst6 e t al., 1986, 1989c).

Another major group of alkylthiophenes is constituted by C20 isoprenoid thiophenes (Fig. 5), including mid-chain thienyl compounds, 4- (4,8-dimethylnonyl)-2-(2-methylbutyl)thiophene (II), 5 - (2,6 - dimethylheptyl) - 3 - methyl - 2 - (3 - methylpentyl)thiophene (III), 2-(3,7-dimethyloctyl)-3-methyl-5- (2-methylbutyl)thiophene (IV), 5-(3,7-dimethylnonyl)-4-methyl-2-(2-methylpropyl)thiophene (V), and compounds having the thiophene ring at one end of the side chain, 2,3-dimethyl-5-(2,6,10-trimethylundecyl)thiophene (I), 4-methyl-2-(2,6,10-trimethyldode- cyl)thiophene (VI), 4-ethyl-2-(2,6,10-trimethylundecyl)thiophene (VII) and 3-methyl-2-(3,7,11-tri- methyldodecyl)thiophene (VIII). All these compounds have been identified by comparison with published mass spectra (Brassell et al. , 1986b; Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst~ et al. , 1989b; Rullk6tter et al. , 1988) and interpretation of the G C - M S data.

In both shales, the C20 isoprenoid thiophenes are more abundant than the C~5 C26 linear alkylthiophenes. The dominance of these compounds is consistent with the high concentrations of phytanic acid and the predominance of phytane among the aliphatic hydrocarbons of these samples, as an origin from the phytyl side chain of chlorophyll a or from archaebacterial lipids is proposed for the C20 isoprenoid thiophenes (Brassell et al. , 1986b; Sinninghe Damst6 et al., 1989b; Rullk6tter et al. , 1988). No organo-sulphur compounds corresponding to the sulphurization of regular isoprenoids with 15, 25, 30 carbon atoms have been identified. The occurrence of the C20 isoprenoid thiophenes evidences the early sulphurization of the sedimentary lipids as a consequence of sulphate-reducing processes. On the other hand, the dominance of compound I and the high relative proportion of mid-chain thiophenes in the distribution of these isoprenoid compounds (Fig. 5) is characteristic of hypersaline depositional environments (Sinninghe Damst6 et al. , 1987a).

The alkylthiophenes are not detected in the brecciated carbonate and only found at trace levels in the laminated carbonate. In this sample compound I is also the major species of this group (Fig. 5), which again points to hypersaline conditions of deposition for the organic matter found in this section.

In the shales, the sulphurization of the Cz0 isoprenoids is also reflected in a distribution of bithiophenes (Fig. 5), among which 5'-(3-methylpentyl)-5- (2 - methylpropyl) - 3,4' - dimethyl - 2,2' -bithiophene

l ?.

h.

(a)

brec

ciat

ed

is.

carb

onat

e

~ I.S

5 6

(b)

lam

inat

ed

carb

onat

e

,, 6

[I

7 '~

sh

ale

1 1

8 YY

:

~*

s IS

i

,, IT

I

'° V

~ i'

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T

i so

~

I ,

,to

u

• [, 6

7 6S

60

5S

so

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2S

10

IS

~O

(d)

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e 2

v

r,

I'O

¢..

rete

ntio

n ti

me

~l

~

Fig.

3.

Pyro

lysa

tes

of t

he s

olve

nt-e

xtra

cted

an

d de

carb

onat

ed

sam

ples

. 5

= 2-

met

hylt

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hene

, 6

= 2,

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met

hylt

hiop

hene

, 7

= 2-

ethy

l-5-

met

hylt

hiop

hene

, 8

= 2,

3,5-

tri-

m

ethy

lthi

ophe

ne.

V:

n-al

kene

/alk

ane

doub

lets

: -~

: co

nlam

inan

t;

I.S.

: in

tern

al s

tand

ard.

I.S.

(a)

bre

ccia

ted

carb

onat

e

LS.

= O

.087

pg/g

sed

imen

t

31

phyt

ane

29

I pn

stane

27

I

i 19

21

25

17

al

l_

I.S.

alkyl

benz

o-lb

l-thi

ophe

ncs *

12

11 12

(b)

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inat

ed

carb

onat

e

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= O

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1~g/

g se

dim

ent

Cm, Is

opre

noid

th

ioph

enes

rete

ntio

n ti

me

C20 h

opre

noid

th

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encs

alkyl

bcnz

o-lb

l-thi

ophe

ncs *

11

(c)

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e 1

l.S.

= 14

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g/g

sedi

men

t

Fl

* ph

ytan

e 12

\

I.S.

• ~

\ C2

o I~p

reno

id

• tO

9

,-~

I I

I I 2

0 bi

thio

phen

es

9ATI

/

19

A

23A

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A

AT

alkyl

benz

o-lb

l-thi

ophe

ncs*

11

9AT

J I I *

10

8A

*

12

C m

Isop

reno

id

thio

phen

cs

2phy

tane

(d)

shal

e 2

l.S.

= 3.

71ag

/g s

edim

ent

C2o

Isopr

enoi

d ,

thio

lane

C~o l

sopr

enoi

d 24

'- bith

ioph

enes

Fig.

4.

Gas

chr

omat

ogra

ms

show

ing

the

hydr

ocar

bon

com

posi

tion

of

the

carb

onat

e an

d sh

ale

sam

ples

sel

ecte

d fo

r th

is s

tudy

. N

umbe

rs r

efer

to

tota

l nu

mbe

r of

car

bon

atom

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T:

alky

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ogue

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rnal

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ndar

d.

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m

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100-

0

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m/z

308

C

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nes

VII

I+ u

nkno

wn

100

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._ ~

, j~

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..

,

, ,

.

,

m/z

312

C

20 is

oprc

noid

thio

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linea

r V

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iv

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100]

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X

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. v-

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7,

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IO

0 X

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m/z

334

C

20 is

opre

noid

bith

ioph

enes

XI XI

xI

.Ig

lam

inat

ed

carb

onat

e

shal

e 1

shal

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RT

(r

ains

) 31

.5

t~ 7:

Fig.

5. m

/z 3

08, 3

12 a

nd 3

34 m

ass

frag

men

togr

ams

show

ing

the

com

posi

tion

of t

he i

sopr

enoi

d th

ioph

enes

, th

iola

nes

and

bith

ioph

enes

in

the

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and

the

sh

ale

sam

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sel

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r th

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.


(IX), 5-(2-methylbutyl)-5'-(3-methylbutyl)-3,4'- dimethyl-2,2'-bithiophene (X) and 5-(2,6-dimethylheptyl)-3,4',5'-trimethyl-2,2'-bithiophene (XI) have been identified. The mass spectra of compounds IX-XI have already been described (Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst6 et al., 1989a). These compounds have been found in the hypersaline-sourced Rozel Point oil (Sinninghe Damst6 and de Leeuw, 1987; Sinninghe Damst+ et al. , 1989a), which is consistent with the origin of the above described isoprenoid thiophenes.

Alkylthiolanes are absent in the carbonates and are generally found in low concentrations in the shales. They encompass distributions between C11 and C28. The highest relative abundances also correspond with a distribution of thiolanyl C20 regular isoprenoids (Fig. 5). In this group the cis- and t r a n s - i s o m e r s of 2-(3,7-dimethylnonyl)-3-methyl-5- (2-methylpropyl)thiolane (XII), 2-(2,6-dimethylheptyl)-5-(3-methylpentyl)-3-methylthiolane (XIII), 2- (2,6-dimethylheptyl)-4-(4-methylhexyl)thiolane (XIV), 4-(4,8-dimethyldecyl)-2-(2-methylpropyl)thiolane (XV) and 3-methyl-2-(3,7,11-trimethyldode- cyl)thiolane (XVI) have been identified based on comparison with previously reported mass spectra (Sinninghe Damst6 and de Leeuw, 1987). Some of these compounds, e.g. XVI, have also been found in other Messinian sediments (Sinninghe Damst+ et al. , 1986) and in crude oils of hypersaline origin, such as Rozel Point oil (Sinninghe Damst6 et al. , 1987b). The abundance of mid-chain thiolanes is in agreement with the above reported occurrence of mid-chain thiophenes in the shale samples. However, the parallelism does not involve the same isomers. In this respect, it has to be mentioned that despite the fact that compound I is the major isoprenoid thiophene not even a trace of the 2,3- dimethylthiolanyl counterpart has been found.

The proportion of alkylbenzo[b]thiophenes vs. alkylthiophenes and alkylthiolanes has been suggested to be maturity-related, showing a higher proportion of the former in more mature samples (Sinninghe Damst6 et al. , 1989a). Maturity differences between these Lorca shales and carbonates are unlikely due to the short stratigraphic range of the samples selected for study. Furthermore, the laminated carbonate, the sample containing the highest proportion of alkylbenzo[b]thiophenes vs. alkylthiophenes and alkylthiolanes, is the one with a higher relative abundance of unsaturated fatty acids (see next section), which is not compatible with a higher maturity level than the shales.

Conversely, sulphur may act as a low oxidizing agent, leading to partial aromatization of molecules with cyclic structures (Alexander et al. , 1987). In this respect, the sample exhibiting the highest S/C ratio is the laminated carbonate (S/C = 5.15; Table 2), whereas the shales have ratios of 0.11- 0.13 (Table 2). Likewise, the low sulphur content in

the brecciated carbonate (0.02%, Table 2) is reflected in the absence of organo-sulphur compounds, either aliphatic or aromatic.

F a t t y ac ids

The fatty acid distributions of the samples considered in this study are represented in Fig. 6. The concentrations of the major individual compounds are listed in Table 3 and total concentrations are given in Table 4. C18:1A9 is a predominant species in the carbonates and C16:0 dominates the fatty acid distributions in the shales. C~6:0 maximized distributions lacking unsaturated compounds, as in the shales, are generally found in ancient sediments (de las Heras, 1988). Fatty acid distributions containing large amounts of unsaturated species are also found in recent sediments from normal marine environments (Chuecas and Riley, 1969; Grimalt and Albaig6s, 1990; Volkman et al. , 1980) and hypersaline systems (Barb6 et al., 1990; Grimalt et al. , 1992). The unsaturated acids like Cis:J69 are nor- mally lost after deposition (Rhead et al. , 1971) and are not generally found in sediments as old as 6My. However, in some cases they can be preserved (Parker, 1969) and their occurrence in the Lorca carbonates constitutes one of these unusual cases.

The fatty acid concentrations in these carbonates are low, 0.3-7.9 #g/g sediment for the total concentrations, when compared with those in the shales (170 180/~g/g sediment). However, when these amounts are referred to the total organic carbon content, they correspond to 3 4 mg/g C in the carbonates, which is a higher amount than in the shales (0.74 0.82mg/g C). The fatty acid distributions from the carbonates, particularly their proportion of unsaturated compounds, is close to the distributions currently found in the algae or cyanobacteria (Chuecas and Riley, 1969). The survival of this algal/cyanobacterial signature is particularly significant in the brecciated carbonate, where even C~s:2 is found in a significant relative proportion. These distributions are unlikely to be related with diatom contributions due to the low predominance of C16 fatty acids (Volkman e t al., 1980).

Another distinct feature of the fatty acids in these samples is the high relative proportion of phytanic acid (Fig. 6; Tables 3 and 4). This compound could derive from the phytyl side chain of chlorophylls, therefore, is a potential marker of algae, cyanobacteria or purple sulphur bacteria. However, in cultures of algae (Chuecas and Riley, 1969), cyanobacteria (Rezanka et al., 1983; Piorreck et al. , 1984) or purple sulphur bacteria (Grimalt et al. , 1992) this compound is a minor component. Likewise, in recent environments (Grimalt and Albaig~s, 1990; Grimalt and Olive, 1993; Berdi6 et al., 1995), even those corresponding to highly productive sites (Grimalt et al., 1991b, 1992), phytanic

? Cl

o ID

al

m

i

l.S. 18

:1

I.S.

18:1

(a)

brec

ciat

ed

carb

onat

e l.

S. =

O.0

871~

g/g

sedi

men

t

~ 16

18

:!

i 16

I I

18:2

/~/

",,L

..

..

..

..

.

:::I

:±]L

__~L

~

..-..:

:

[

phyt

anic

18:

1

16

(b)

lam

inat

ed

carb

onat

e

l.S.

= 0

.925

1ag/

g se

dim

ent

C~

isop

reno

id

18

/ / 26

/

22

I

\ 24

28

bi

shom

ohop

anoi

c

acid

32

~

rete

nti

on t

ime

--ti

~

a-15

,

i'15/

i. ~

tl ,~

/j 15

2-hy

drox

y ac

ids"

(C

) 24

* sh

ale

1

I.S.

= 7

4~tg

/g s

edim

ent

26*

phyt

anic

I a

cid

l!ll

18

23

17

22~

r I bi

shom

ohop

anoi

c

i~ll

20

IbHl

,<i

d /'

/ 22

ii

;~

(d)

shal

e 2

l.S.

= 1

8.5p

.g/g

sed

imen

t

phyt

anic

ac

id

18

. 26

bi

shom

ohop

inoi

c 22

24

'11/2

8 30

acid

2

Fig.

6.

Gas

chr

omat

ogra

ms

show

ing

the

fatt

y ac

id c

ompo

siti

on (

as m

ethy

l es

ters

) of

the

car

bona

te a

nd s

hale

sam

ples

sel

ecte

d fo

r th

is s

tudy

. N

umbe

rs r

efer

to

carb

on

chai

n le

ngth

of

the

n-al

kano

ic/a

lken

oic

acid

s. S

hale

I t

race

was

obt

aine

d af

ter

sily

lati

on.

I.S.:

inte

rnal

sta

ndar

d; ,

: co

ntam

inan

t pe

aks.

Bacterial and algal markers

Table 3. Fatty acid concentrations in/ag/g of sediment (in brackets tlg/g TOC)

617

Compound Brecciated carbonate Laminated carbonate Shale 1 Shale 2

,v/°C 12:0 H°C 13:0 iso-C 14:0 ~lllteiso-Ci4:o I1-C14:0 iso-C~5:o anteiso-C 15:0 II°C~5:0 iso-Ci6:o • r/-C 16:0 iso-C 17:0 anteiso-ClT: 0 H-El7:0 t/-Cis:0 n-Ci8:2 //-Ci8:1 (Ag) n-CIs:l (All) Phytanic acid r/-Ci8:l //-Ci9:0 /'/-C2o:0 n-C21:0 isoprenoid-C~s I?'C22:0 n-C23:0 I1-C24:0 rt-C25:o 2-hydroxy-C24:o n-C26:o n-C27:o t/-C28:0 r/-C29:0 n-C30:o //-C31:0 n-C32:0 ~fl-C32 hopanoic tiff-C32 hopanoic

0.005(52)

0.059(590)

0.001(10) 0.016(155) 0.049(490) 0.17(1700) 0.013(130)

0.002(15)

0.086(43) 0.92(4) 2.0(9) 0.020(10) 1.8(8) 2.4(11) 0.010(5) 1.5(7) 0.004(2) 1.1(5) 0.16(81) 5.5(24) 5.7(26) 0.048(24) 3.7(16) 5.3(24) 0.038(19) 3.2(14) 4.6(21) 0.058(29) 3.2(14) 4.6(21) 0.058(29) 2.5(11) 4.2(19) 1.7(850) 28(120) 41(185)

0.096(48) 2.3(10) 5.1(23) 0.086(43) 1.8(8) 3.7(17) 0.14(57) 3.9(17) 3.7(17)

0.68(340) 5.1(22) 9.7(44)

0.88(440) 0.19(95)

0.94(470) 0.028(14) 0.066(33) 0.20(100) 0.12(62) 0.15(75)

0.50(250) 0.076(38) 0.31(160) O.lO(52)

0.52(260) 0.066(33) 0.36(180) o.o28(14) 0.16(81) o.o2o(1o) 0.076(38) 0.028(14)

8.3(36)

0.92(4) 2.1(9)

18(80)

2.9(13) 5.7(26) 2.4(11)

2.6(12) 1.8(8)

2.2(10)

92(400) 7.5(34) 4.6(21)

1.6(7) 2.0(9) 1.15(5) 4.0(19)

%,7,11,15,19-pentamethyleicosanoic acid.

acid is only a minor constituent of the sedimentary and water column fatty acid distributions. Post- depositional processes are required for the presence of this compound as a free species of the sedimentary solvent-soluble fraction. In this respect, it has to be emphasized that phytanic acid is absent in the brecciated carbonate, the sample containing the highest relative proportion of unsaturated fatty acids, the compounds which are more easily removed by post-depositional transformations. As will be commented upon in the section describing the desulphurized fractions, there is an interesting correspondence between this acid and the presence of isopranyl glycerol ethers.

Another major fatty acid group in shale 1 is a series of C24-C26 2-hydroxy-acids that is dominated by 2-hydroxytetracosanoic acid. These compounds are present in minor amounts in shale 2 (Fig. 6; Tables 3 and 4). Similar 2-hydroxy acid series have previously been identified in soils (Grimalt and Saiz-Jimenez, 1989), diatomaceous oozes (Boon et al., 1977a), lacustrine sediments (Cranwell, 1981), cyanobacteriai mats (Matsumoto et al., 1987) and eelgrass (de Leeuw et al., 1995). These 2-hydroxy acid distributions have also been invoked to be derived from higher plant organic matter which has undergone severe oxic degradation over a long

period of time during sedimentation (Cranwell, 1981). C20-C26 fatty acid distributions are effec- tively found in the Lorca shales (C20-C32 in the laminated carbonate) and to a certain extent the 2- hydroxy acid distribution parallels these terrigenous compounds.

The iso- and anteiso-Ci5 and C17 homologues constitute another important fatty acid group which occurs together with minor amounts of iso-C~6 and anteiso-Cl4. These acids are present in the laminated carbonate and in the two shales. These fatty acids are generally indicative of bacterial contributions. Iso- and anteiso-Cls and Cl7 have been found in pure cultures of Desulfovibrio desulfuricans (Boon et al., 1977b), enrichment cultures of sulphate-reducing bacteria (Grimalt et al., 1992), cultures of Bacillus species (Kaneda, 1967) and sedimentary microbial populations (Perry et al., 1979; Volkman et al., 1980). Their occurrence in the Lorca shales and carbonates is, therefore, consistent with the high sulphate-reducing activity associated to the replacement of gypsum by carbonate and reduced sulphur (in the form of organo-sulphur, pyrite and S°). In this respect, the good correspondence between the concentrations (/~g/g sediment) or relative proportion (% with respect total fatty acids) of these iso- and anteiso-compounds and the reduced sul-


Table 4. Total vs. bacterial fatty acids in the sediments selected for study

Sample Total Phytanic 2-hydroxyC24 Bacterial fatty acids

i - + a- n-Ct~:l I a , , ) Hopanoic

Brecciatedcarbonate 0.3 ~' 0.013 100 h 4.3

3000" 130 Laminatedcarbonate 7.9 0.94 0.37 0.19 0.028

100 12 4.7 2.4 0.35 3950 470 185 95 14

Shale 1 170 8.3 92 13.5 2.75 100 4.9 54 8.0 1.6 740 36 400 59 12

Shale 2 180 18 7.5 25.5 6.0 100 l0 4.2 14 3.3 820 82 34 120 27

"Concentrations in l~g/g sediment. h% relative to total fatty acids. ~Concentration in pg/g organic carbon.

phur content of the Lorca samples (Tables 2 and 4) gives good support for the sulphate-reducing origin of these fatty acids in the Lorca sedimentary sequences.

Another marker of bacterial activity, C1~:1611, is found in the carbonates but not in the shales, where its absence may be due to the above described lack of survival of unsaturated fatty acids in this type of sample. This fatty acid is generally found in a wide variety of microorganisms (Oliver and Colwell, 1973; Boon et al., 1977b). It has also been found to be a predominant compound in cultures of several purple sulphur bacteria living in carbonate environments, such as those leading to microbial mat formation, that contain high amounts of H2S due to the active sulphate reduction (Barb6 et al., 1990; Grimalt et al. , 1992).

17fl,21/~(H)-, minor amounts of 17/L21~(H)- and 17~,21/~(H)-bishomohopanoic acids are found in the shales. The laminated carbonate only contains the 17~,21/~(H)-isomer. These acids originaUe from the transformation of polyhydroxybacteriohopanes (Ourisson et al. , 1979) and are used as cyanobacterial or heterotrophic prokaryotic markers. In this respect, they have been found in microbial mats generated in carbonate environments (Barb6 et al., 1990; Grimalt et al., 1992).

Desu lphur i zed solvent ex t rac tab le po lar compounds

Further insight into the algal and bacterial organic matter record preserved in the Lorca carbonates and shales can be obtained by examination of the sulphur-bound lipid compounds. Nickel boride reduction allows the selective cleavage of the C-S and S-S bonds (Schouten et al., 1993). The polar and apolar fractions (Fig. 2) resulting from the application of this method to the carbonates and shales considered in this study are shown in Figs 7 and 8, respectively.

Phytane is the dominant compound in all exam- ined apolar fractions. However, its occurrence is not specifically indicative of sulphur-binding, since

Ni2B reduction of phytol (Prahl et al., 1996; Hartgers et al., 1996) and chlorophyll a (Hartgers et

al., 1997) may also generate the saturated phytane.

Another compound present in all samples is 17~,21/~(H)-pentakishomohopane. This marker of prokaryotic organisms is currently found in desulphurized fractions (Sinninghe Damst6 et al., 1995) and it has been found in high abundances in sulphur-rich Tertiary Catalan lacustrine oil shales (Sinninghe Damst6 et al., 1993). Early sulphur in- corporat ion into bacteriohopanepolyol appears to be the most likely process for the occurrence of this compound.

C27 steranes, both the 5/~,14ct,17/~(H)- and 5c~,14c~,17/q(H)-isomers, are also found in all samples (Fig. 7). The high dominance of C27 homologues indicates that the distribution is representative of algal inputs (Volkman, 1986). These steranes are not produced by Ni2B sterol reduction (Hartgers et

al., 1996), but are due to the preservation of steroid molecules by sulphur-binding. Their occurrence contrasts with the lack of sterols in the solvent- extractable polar fractions. Only the brecciated carbonate, the sample with higher relative content of unsaturated fatty acids, contains solvent-extractable sterols in detectable amounts. 4,24-Dimethyl-5~(H)- cholestane and a C30 4-methylsterene are found in the shales. Again, their occurrence is indicative of algal steroid inputs preserved by sulphur-binding.

The major group of apolar sulphur-bound compounds in the shales constitutes a modal distribution of C13-C35 n-alkanes, without even odd carbon number preference, that is maximized at n- Cl8 (Fig. 7). This distribution parallels the mixture of linear alkylthiophenes found in these samples. These n-alkanes probably correspond to a group of straight chain lipids preserved either as sulphur- bound polymer or as a mixture of alkylthiophenes. In this respect, the absence of n-alkanes in the carbonates is consistent with the lack of these hydrocarbons in the solvent-extractable mixtures (Fig. 4).

~ytM

e

t.S.

(a)

bre

ccia

ted

carb

onat

e

l.S. -

0.0

87~t

g/g

sedi

men

t

choJ

esta

nes

L'

~,

~

~. ~ ~

phyt

ane I,S,

(c)

shal

e 1

l.S. =

1.4

8~tg

/g s

edim

ent

C~

4-m

ethy

lster

ene

I /

~,

~/

(3~2

33 !

22

sta

nes

/ ! 5

23

eta

s

2526

31

ho

pxne

32

~ 37

38

L .

..

.

_~

to

~'

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Another distinct feature of the apolar fraction of the desulphurized mixture in the shales is the high relative proportion of the n-C37 and rt-C38 homologues (Fig. 7). These compounds are probably related to the C37-C39 linear di-, tri- and tetra- unsaturated methyl and ethyl alkenones that are specific markers of some Haptophycea (Brassell et al. , 1986a). Sulphur-binding is a known diagenetic mechanism for the preservation of these ketones in ancient sediments (Sinninghe Damst6 and de Leeuw, 1990; Sinninghe Damst6 e t al. , 1990). These compounds appear as C37-C38 alkylthiophenes after Ni2B desulphurization and as the equivalent n- alkanes after PtO2 hydrogenation. These C37-C38 n- alkanes may reflect the preservation of these ketones in the Lorca shales since the distributions of these ketones are largely dominated by the C37 and C38 homologues.

The GC traces of the polar fraction obtained after desulphurization (Fig. 2) are represented in Fig. 8. Bis-O-phytanyl glycerol ether is the dominant compound in the laminated carbonate and shale 2. This compound is also present in the brecciated carbonate and in shale 1. Another glycerol ether, O-phytanyl-O-sesterterpanyl glycerol, is also found in the laminated carbonate. These compounds are characteristic moieties of the ether- linked isoprenoid lipids from archaebacterial cells (Kates, 1978; Langworthy e t al. , 1982). Studies in recent and ancient marine evaporitic environments have shown that the dominance of bis-O-phytanyl glycerol is characteristic of hypersaline environments with salinities in the range of 50-100 g/1 and that the mixture of bis-O-phytanyl glycerol and O- phytanyl-O-sesterterpanyl glycerol is characteristic of environments with salinities higher than 250 g/1 (Barb6 et al. , 1990; Teixidor et al. , 1993). The occurrence of these two distributions in these hypersaline environments is due to inputs from methanogenic and halophilic bacteria, respectively (Teixidor et al. , 1993). In particular, the presence of O-phytanyl-O-sesterterpanyl glycerol reflects inputs from H a l o c o c c u s spp. (Teixidor et al. , 1993).

The palaeodepositional environments corresponding to the distributions of these isopranyl glycerol ethers are in agreement with the sedimentology of the Lorca samples. Thus, the occasional dessication episodes evidenced in the morphology of the carbonates could correspond to salinity increases leading to the development of halobacteria. These episodes are not observed in the shales since these were deposited under lower salinity than the carbonates and do not record any dessication episode. As mentioned above, the laminated carbonate has interbedded gypsum pseudomorphs and the brecciated carbonate was formed by replacement of former gypsum crystals disseminated in a dolomite matrix. The lack of O-phytanyl-O-sesterterpanyl glycerol in this sample may be due to the low or-

ganic carbon content, which makes the detection of this minor isopranyl glycerol ether very difficult.

These isopranyl glycerols are only present at trace levels as free molecules in the solvent-extractable polar fractions of these samples (Fig. 2). However, when these extracts are desulphurized they appear to be dominant peaks in the GC traces of the resulting polar mixture (Fig. 8). Thus, a substantial proportion of these compounds is preserved by sulphur-binding. The occurrence of these compounds in these polar fractions contrasts with the lack of sterols or hopanols. The elucidation of the type of sulphur-binding of these isopranyl glycerols requires further chemical analysis and is beyond the aim of the present paper.

In contrast, the sterols are not found either in the free or in the desulphurized polar fractions. The group of compounds eluting before bis-O-phytanyl glycerol ether in shale 2 corresponds to a mixture of hopanoid compounds (Fig. 8). As mentioned above, only the brecciated carbonate contains a significant amount of sterols as free molecular species and this sample, like the others, does not have sulphur-bound sterols.

As mentioned before, phytanic acid is a major component in the laminated carbonate and in the two shales (Fig. 6). High abundances of phytanic acid have also been encountered in sediments from recent environments where bis-O-phytanylglycerol ether is a major compound (Barb6 et al. , 1990; Teixidor, 1996), but not in cultures of algae, cyanobacteria or purple sulphur bacteria or in recent evaporitic environments even those from highly productive sites. Thus, the large amounts of this glycerol ether in the Lorca samples suggests, in principle, that phytanic acid could also be a product resulting from the partial oxidation of this ether.

An aspect that reinforces the genetic relationship between isopranyl glycerol ethers and phytanic acid in these Lorca samples is the occurrence of the regular isoprenoid C25 acid, 3,7,11,15,19-pentamethyleicosanoic acid, in the laminated carbonate. Only this sample contains this compound in significant amounts and this carbonate is also the only sample having O-phytanyl-O-sesterterpanyl glycerol in significant proportion.

CONCLUSIONS

The organic matter contained in the Messinian carbonates and shales selected for study from the Lorca Basin was deposited under hypersaline conditions. In the solvent-soluble fractions this is reflected in the high abundance of phytane (brecciated carbonate and shales) and in the predominance of the C20 alkylthiophene (I) (laminated carbonate and shales). The presence of mid-chain C20 isoprenoid thiophenes and bithiophenes in the shales is also in agreement with this origin. In the


sulphur-bound macromolecular matter the hypersaline environment is reflected in the high abundances of bis-O-phytanyl glycerol ether (all samples) and O-phytanyl-O-sesterterpanyl glycerol ether (laminated carbonate).

The high abundance of C2o isoprenoid alkylthiophenes, alkylbithiophenes and alkylthiolanes among the free apolar compounds is consistent with the high relative proportion of C20 regular isoprenoid species both in the solvent-soluble fractions (phytanic acid and phytane) and in the sulphur-bound macromolecular matter (phytane). Furthermore, the occurrence of free phytanic acid is possibly related to the preservation of bis-O-phytanyl glycerol ether by sulphur-binding to the macromolecular matter. This correspondence is reinforced by the specific occurrence of 3,7,11,15,19-pentamethyleicosanoic acid in the laminated carbonate, the only sample containing O-phytanyl-O-sesterterpanyl glycerol in significant proportion. The presence of C20 isoprenoid species is, therefore, related to the formation of organo-sulphur compounds, either as individual molecules (thiophenes) or sulphur-bound to the macromolecular matter (as a single molecule or ether-linked to glycerol).

The high abundance of isopranyl glycerols in the sulphur-bound macromolecular matter contrasts with the absence of other hydroxyl-substituted molecules, such as the sterols. The sterols are found only as free compounds in the brecciated carbonate, the sample with a higher relative proportion of unsaturated fatty acids. They are absent in the other samples, either as free compounds or as sulphur-bound molecules. The small amounts of 5fl(H),I4~(H),17/3(H)- and 5~(H),14~(H),17I~(H)- steranes, 4,24-dimethyl-5~(H)-cholestane and a C30 4-methylsterene in this macromolecular fraction are related to sulphur-bound steroid precursors having functionalities other than hydroxy groups.

The major group of apolar sulphur-bound compounds in the shales constitutes a modal distribution of Cl3 C35 n-alkanes without even-odd carbon number preference that roughly parallels the mixture of linear alkylthiophenes. These n-alkanes probably correspond to a group of straight chain lipids preserved either as sulphur-bound polymers or as a mixture of alkylthiophenes. Two staturated hydrocarbons, ,v/-C37 and n-C38, that are in the form of two C37-C38 alkylthiophenes prior to PtO2 hydrogenation, reflect sulphur-binding of the di-, tri- and tetra-unsaturated methyl and ethyl alkenones that are specific markers of some Haptophycea. The absence of n-alkanes in the carbonates is consistent with the lack of these hydrocarbons in the solvent-extractable mixtures.

Likewise, evidence of fatty acid sulphur-binding to the macromolecular matter has not been found and contrasts with the presence of free fatty acid

distributions in all samples, including unsaturated acids in the carbonates.

The markers of heterotrophic prokaryota in these sediments are represented by 2-hydroxytetracosanoic acid, n-octadec-ll-enoic acid, hopanoic acids and the distributions of iso- and anteiso-Ci5 and Cj7 homologues and minor amounts of iso-Ci6 and anteiso-Ci4. These branched fatty acids are characteristic of sulphate-reducing bacteria. The proportions of these iso- and anteiso-compounds relative to the total fatty acid distributions (Table 4) are correlated with the concentrations of sedimentary reduced sulphur (Table 2).

In these samples, the differences in relative content of alkylbenzo[b]thiophenes and alkylthiophenes cannot be maturity related. Thus, the laminated carbonate, the sample with the highest alkylbenzo[b]thiophene/alkylthiophene ratio, contains a higher proportion of unsaturated acids than the shales, which is not consistent with maturation. This sample is the one with highest S/C ratio, which leads to the question of whether the high abundance of sulphur could be responsible for the partial aromatization of some cyclic molecules. This transformation is consistent with the well-known properties of sulphur in laboratory experiments and has also been proposed to explain the aromatization of diterpenoid molecules in other environments (Alexander et al., 1987).

Acknowledgements--This work has been financed by the EC Human Capital and Mobility Program, Contract CHRX-CT93-0309. Thanks are given to Dr George Wolff for his contribution to the pyrolysis GC-MS data; Dr J. J. Pueyo (Faculty of Geology, University of Barcelona) and Mr J. Lopez (C.I.D.-C.S.I.C.) for helpful discussions; Ms R. Chaler (C.I.D.-C.S.I.C.) and Dr P. Teixidor (Faculty of Geology, University of Barcelona) for their assistance in the GC MS analyses; Mr G. Carrera (C.I.D.-C.S.I.C.), Mr A. Samper, Ms B. Ilia and Mr J. Ilia (Faculty of Geology, University of Barcelona) for their help in sample preparation for XRD and bulk organic matter analyses; and Dr F. Plana and Mr P. Elvira (IJA-CSIC) for XRD analyses. SEM observations were performed in the Serveis Cientifico T~cnics de la Universitat de Barcelona, with special thanks to Dr R. Fontarnau and Ms A. Dominguez for their invaluable help. Dr C. de las Cuevas performed the thermogravimetry analyses. Dr L. Schwark and an anonymous reviewer are thanked for their useful com- ments. Thanks are also due to Mr. Antonio Miguel Ruiz- Hernandez (Minas Volcan, S.A.) for permission to sample in the quarry

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Bacterial and algal markers

Appendix

( I f )

Oil)

(vl)

(viii)

(IX)

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Bacterial and algal markers in sedimentary organic matter deposited under natural sulphurization...

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Transcript of Bacterial and algal markers in sedimentary organic matter deposited under natural sulphurization...