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Microbial degradation of marine evaporitic crude oils
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Geochimrca d Cosmochimrca Ada Vol. 55, pp. 1903-1913 Copyright 0 1991 Pergamon Press pk. Printed in U.S.A.
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Microbial degradation of marine evaporitic crude oils J. 0. GRIMALT,' M. GRIFOLL,'A. M. SOLANAS,’and J. ALBAIG~S’ ‘Department of Environmental Chemistry (CID-CSIC), Jordi Girona, 18,08034-Barcelona, Spain *Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain (Received February 1, 1990; accepted in revised form April 9, 199 I)
Abstract-The composition of alkylbenzenes and organosulphur compounds in crude oils is strongly modified by biodegradation, even at low levels of transformation where the composition of steranes, hopanes, alkylnaphthalenes, and alkylphenanthrenes has not been altered. These conclusions emerge from the comparison of preserved and biodegraded crude oils of marine evaporitic origin (Amposta and Varadero oils) and controlled experiments of microbial transformation of oils containing high amounts of aryl hydrocarbons and organic sulphur species. The compositions of straight chain compounds, such as n-alkanes, n-alkylbenzenes, 2,5-di-n-alkylthiolanes, 2,6,-di-n-alkylthianes and 2,4dialkylbenzo[blthiophenes are more easily modified than those of compounds with isoprenoid side-chains. The experiments also show that benzo[b]thiophenes are markedly more resistent to microbial attack than thiolanes and thianes. INTRODUCTION
ecules and the stability of the organosulphur compounds upon
AN UNDERSTANDINGOF THE COMPOSITIONAL changes produced during the microbial degradation of crude oils is important to petroleum geochemistry. This topic has been addressed in terms of biomarkers (CONNAN, 1984; SEIFERTand MOLDOWAN, 1979; MCKIRDY et al., 1983; RUBINSTEINet al., 1977; SEIFERTet al., 1984; SOFERet al., 1986; VOLKMAN et al., 1983, 1984; WEHNER et al., 1986; WILLIAMSet al., 1986), aflording valuable information on the transformations of acyclic isoprenoids, hopanes and steranes, used for oil-oil and oil-source rock correlations (SEIFERTand MOLDOWAN, 1979; MCKIRDY et al., 1983; VOLKMAN~~al., 1983; SEIFERT et al., 1984). Stable isotope ratios (SOFER et al., 1986; WEHNER et al., 1986) and other techniques such as 13CCPJMAS nuclear magnetic resonance (LANDAISet al., 1988), fluorescence spectroscopy (DUMKE and TESCHNER,1988), and hydrous pyrolysis (JONES et al., 1988) have also been used in the study of biodegraded oils. Nevertheless, some questions remain to be addressed. One of them concerns marine evaporitic crude oils, which have received limited attention in biodegradation studies. This gap is important in terms of molecular marker geochemistry because most of the oils reported to contain significant amounts of organosulphur compounds were generated from marine evaporitic source rocks (SINNINGHEDAMSTB et al., 1989). Marine evaporitic environments (also quoted as hypersaline environments) are anoxic with the amount of hydrogen sulphide far exceeding the amounts of available iron and other heavy metals; they therefore provide good conditions for the formation of organosulphur molecules (DE LEEUW and SINNINGHEDAMST& 1990). Accordingly, studies on sedimentary sequences including marine evaporite deposits have shown an enrichment of organosulphur compounds in this type of facies (SINNINGHEDAMSTBet al., 1986; TEN HAVEN et al., 1987). However, many aspects related to the occurrence of these compounds still need to be clarified (e.g., the geochemical pathways of sulphur incorporation into many organic mol1903
diagenesis and catagenesis). In this respect, we have found several crude oils of marine evaporitic origin with high sulphur content (4-6%) in which no low molecular weight organic sulphur compounds (LMOSC), those amenable to gas chromatographic analysis, can be observed. Biodegradation was suspected to account for the absence of these molecular species. Another common feature of many marine evaporitic crude oils is their high content of long-chain alkylbenzenes (ALBAIGBSet al., 1986), especially those with an isoprenoid side chain (SUMMONSand POWELL, 1987; SINNINGHEDAMST~ et al., 1988). However, we have observed that these hydrocarbons are absent (or present at only trace levels) in partially biodegraded evaporitic crude oils. To date, no data are available to relate the lack of these compounds to microbial decomposition processes. This paper concerns the comparison of crude oils of marine evaporitic origin from Amposta (Tarragona Basin, Catalonia, NE Spain) and Varadero (Varadero-Cardenas region, N Cuba). The major differences in their composition lie in the strong depletion of n-alkanes, aryl hydrocarbons, and LMOSC in Varadero oils. In principle, these differences were attributed to microbial degradation, a hypothesis tested by means of degradation experiments of Amposta oil using microbial strains isolated from freshwater and marine petroleum-rich environments. Hence, the major compositional features of Varadero oils and the original and artificially biodegraded Amposta oil are investigated, and their differences are discussed in terms of possible biodegradation processes. EXPERIMENTAL Organisms The isolation of strain F2 1 is described in SOLANASet al. ( 1984). In short, ballast water from an oil tanker was used as inoculum of sterilized artificial seawater (100 mL) supplemented with KN03 (0.0 1 M) and Na2HFQ (0.00035 M). Arabian light crude oil residues were used as the only source of carbon and energy. The incubation was
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performed at 30°C with continuous stirring (120rpm) for five days. The resulting mixture was transferred to another flask containing the same fresh medium and the operation was repeated. The samples were plated in the same medium with agar. After several successive incubations the isolated cultures were identified as Pseudomonasspp. This was based on morphological characterization and on a series of biochemical tests that used the culture media described by AUSTIN etal. ( 1977). The bacterial strain A 104 was isolated from a waste water treatment system of a petroleum refinery in Tarragona (Catalonia, NE Spain). Batch enrichment was carried out by inoculating sludge samples on the minimal salts medium described below supplemented with Arabian crude oil fO.l%, w/v). The cultures were incubated at 22°C with shaking. After several transfers different bacterial strains were isolated by making serial dilutions and plating on Tripticase Soy Agar (ADSA, Spain). Strain A 104 was selected for the quick growth in mineral medium containing Arabian light crude oil, and for the early production of crude oil emulsions. This isolate is a non-motile, gram negative coccoid rod, catalase positive, cytochrome oxidase negative and uses glucose only in aerobic conditions. Biodegradation Assays of the Crude Oil The biodegradation assays were perhormed with two different media depending on the bacterial strain. In tbe experiences with Al04 strain a minimal salts medium (a solution of Na2S04 ( 1 g), MgSO, *7H20 (0.2 g), K2 HPQ - 3HzO (0.65 g), NH&l (1 g), KNOr (2 g), and FeSO, (traces) in 1 liter of distilled water) was used. The assays with F21 were performed in artificial seawater (ADSA, Spain) supplemented with KNOs (0.01 M) and Na*HPO., (0.~35 M). Crude oil was added to both media aseptically to 0.1% of final concentration. The starter cultures were obtained by growing the strains in 250 mL Erlenmeyer flasks containing 50 mL of the respective mineral media and hexadecane (O.l%, w/v) as source of carbon. AAer 72 hours of incubation the cultures were centrifuged, washed twice, and resuspended in the same volume of mineral media. 5 mL of these suspensions were used to inoculate 250 mL Erlenmeyer flasks contaming 50 mL of the same medium and 0.1% of Amposta oil. All flasks were incubated at 22°C on an orbital shaker (150 mm) durine 15 or 40 days. Duplicate cultures sterilized before the addition of the crude oil and incubated under the same conditions were used as blanks. Crude Oil Analyses The flask contents from each incubation were blended and the homogenate extracted with n-hexane (3 X 30 mL). The extracts were vacuum evaporated to about 0.5 mL. The crude oils were also extracted with n-hexane using a mechanical stirrer; the n-hexane soluble fraction was decanted and vacuum evaporated to 0.5 mL. All concentrated extracts were fractionated follo~ng previously established methods (ACEVES et al., 1988). About 0.5 g were introduced into a column filled with 8 g each of 5% water-deactivated alumina (70230 mesh, Merck) (top) and silica (70-230 mesh, Merck) (bottom). The following fractions were collected: (Fl), 20 mL of n-hexane (aliphatic hydrocarbons); (FZ), 20 mL of 10% dichloromethane in nhexane (monocyclic aromatic hydrocarbons); and (F3), 40 mL of 20% dichloromethane in n-hexane (polycyclic aromatic hydrocarbons). With this method the organosulphur compounds are separated into thiophenes (Fl), ~n~~ophen~ (F2) and thiolanes and thianes (F3). These fractions were concentrated to small volumes by vacuum rotary evaporation and submitted to instrumental analysis.
Gas chromatography (GC) was performed with a Cario Erba FTV 4 160 GC instmment equipped with a flame ionization detector and a splitless injector. A column of 25 m X 0.25 mm id. coated with CP-Sil CB (Chrompack) was used (film thickness 0.13 pm). Hydrogen was the carrier gas (50 cm-s-‘). The oven temperature was programmed from 60 to 300°C at 6°C * min-‘. Injector and detector temperatures were 300 and 330-C, respectively. The injection was in the splitlessmode (solvent &octane, hot needle technique) keeping the split valve closed for 35 s. GC coupled to mass spectrometry (GC-MS) was performed with a Hewie~-icky 5995 inst~ment equipped with an HP 300 data
system. A column of 2.5 m X 0.2 mm i.d. coated with HP-S (Hewlett Packard) was used (film thickness 0.11 pm). Helium was used as carrier gas. The oven temperature was programmed from 60 to 3OO’C at 4°C - min-‘. The injection conditions were the same as described above, MS temperatures were transfer line 3OO”C,ion source 2OO”C, and analyzer 230°C. Data were acquired in the electron impact mode (70 eV), scanning range m/z 50-650 at 1 s per decade. Carbon and sulphur determinations were performed with a Carlo Erba elemental analyzer Model 1500. RESULTS AND DISCUSSION
The Biomarker Composition of Amposta Oit Amposta oil occurs in Miocene reservoirs of the Tarragona Basin. Most of the crudes of this basin originate from the Casablanca Fo~ation (Miocene carbonate; DEMAISONand BOURGEOIS, 1985). However, Amposta is the most representative example of a group of sulphur-rich oils from the basin (Table 1) which does not originate from these deposits (ALBAIGES et al., 1986). The source rock of this group of oiis remains to be identified, but their molecular markers are characteristic of oils generated from marine evaporitic sediments. Diverse authors have contributed to the study of the composition of Amposta oil (ALBAIGBSand TORRADAS,1974, 1977; ALBAIG& et al., 1986; SEIFERTet al., 1983; SINNINGHE DAMSTE:et al., 1989), and several new molecular markers have been described as a consequence of these studies; namely, long chain acyclic isoprenoid hydrocarbons (ALBAIG~ZS, 1980; ALBAIGB et al., 1985), isoprenoid cyclohexanes (BARBE et al., 1988), and isoprenoid alkyl~nzenes (SINNINGHEDAMSTBet al., 1988). The gas chromatograms corresponding to the aliphatic and aromatic fractions of Amposta oil are presented in Fig. 1. The tr-alkanes range between Cr.&& showing the characteristic C22-C32 even-to-odd carbon number predominance (ALBAIG&and TORRADAS,1974). This feature, together with the pr~ominan~ of phytane over pristane, is indicative of marine evaporitic deposits (CONNAN et al., 1986; Fu JIAMO et al., 1986; MELLO et al., 1988; TEN HAVEN et al., 1985, 1987, 1988). The distributions of hopanes and steranes are shown in Fig. 2. Peak identifications are given in Table 2. These compounds were identified by comparison of the m/z 19 1,205, 2 17, and 23 1 mass ~~mento~arns with previously reported mass fragmentographic data (SEIFERT and MOUXIWAN, 1979) and with those of crude oils of known sterane and hopane composition (ALBAIGI?Set al., 1986). The position of the A-ring methyl substituent in the hopane compound no. 11 was assigned according to the mass spectral and retention time data reported by SUMMONSand JAHNKE(1990). The position and stereochemistry of the A-ring methyi substituents in the sterane compounds nos. 12, 13, 19, 23, 24, 25, and 26 were determined by comparison of selected mass fmgmentograms(m/z 217,231,372,286,400, and414)with those reported by SUMMONSet al. (1987,1988) and SUMMONS and CAPON ( 1988). Absence of 24-n-propylcholestanes was deduced by mass spectral examination of the m/z 414 molecular weight steranes and from the lack of a response in the m/z 304 mass fragmentographic trace (MOLDOWANet al., 1990). Examination ofthe m/z 358 mass fmgmentogram showed that only two Ga steranes are present. They represent two minor ~rn~un~, one coeluting with the C2, sterane
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Microbial degradation of crude oils
WPOSTA
5KxEGRADu) AIWOSTA
F2
F3
F3
I
FIG. 1. Gas chromatographic profiles of the aliphatic (FL) and aromatic (F2 and F3) hydrocarbons fractions of the origina Amposta oil and Amposta oil biodegraded in controlled experiments. Numbers refer to chain length of nafkanes: a, nor&Sane; b, pristane; c, phytane. M and F3 are mainly composed of afkyfbenzenes and poiycyclic aromatic hydrocarbons, respectively.
IARADERO-S
8 lx1
FIG. 2. Hopane (m/z 19 1)and sterane (m/z 217) distributions of the original Amposta oit, Amposta oil biodegaded in co~troi~~ experiments, Varadero-Y, and Varadero-S oiis. Peak identiftcations are given in Tabie 2.
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Tablel: SuIphur content in Amposta and Varadero oils, and in Amposta oil transformed by A104 and F21 strains. The weight losses observed tier laboratory biodegradation are also given.
Amposta Varadero Varadero Amposta* Amposta* Amposta*
Weight losses (%) after biodegradation F2 F3 Total Fl
%S
Sample
Y S A104 (15d) A104 (40d) F21(15d)
n.a. n.a. n.a. 18 al 19
5.8 4.4 5.6 6.0 5.3 5.6
n.a. n.8. n.a. 78 80 78
n.a. n.a. n.a. : 6
n.a. n.a. n.a. 1 1 1
n.a. not applicable. * Mixtures obtained after degradation of Amposta oil with F21 (Pseudomona spp.) and A104 strains.
compound no. 3 and the other corresponding to the unlabelled m/z 2 17 peak that elutes before this compound. The high abundance of gammacerane (I) and Cj4 and Cjs 17a(H),2 1/3(H) extended hopanes (Cx., > Cs3 and C35> &) represents a distinct feature of Amposta oil (ALBAIGBSet al., 1986) which is commonly observed in many evaporitic crude oils (CONNAN et al., 1986; Fu JIAMOet al., 1986; TEN HAVEN et al., 1985, 1987), although it may also be encountered in oils sourced from normal marine salinity environments (MELLO et al., 1988; DE LEEUW and SINNINGHEDAMST& 1990). The lower abundance of Crs homologues in the sterane distributions is consistent with our observations on recent
evaporite systems (BARBEet al., 1990) but is not exclusive of evaporite sourced crude oils (MOLDOWANet al., 1985; MELLOet al., 1988). The high proportion of 4cu-methylsteranes with respect to the steranes is a consequence of the diagenetic conversion of 4cY-methylsterols (DE LEEUW et al., 1983) and constitutes a recognized feature of marine evap oritic crude oils (PHILP and ZHAOAN, 1987; MELLO et al., 1988) microbial mats (BOONet al., 1983) and saline deposits (TEN HAVEN et al., 1985). Finally, Amposta oil has a high sulphur content (-5.8%; see Table 1) which is consistent with a marine evaporitic origin and the presence of a complex mixture of organic sul-
Table 2: Hopanes and steranes identified in the Amposta and Varadero oils. Numbers refer to peaks in the respective mass fragmentograms in Fig. 2. Peak
1 2 3 4 5 6 P 9 ;: l2 13 14 15 16 17 E E E
Assignment
Peak
C23 tricyclic terpane C24 tricyclic terpane C2s triqcfic terpanes C&stetqxyclic terpanes 18a(HM!2,29,30-trisnomeohopane 17a(H)_22,29,30&isnorhopane
17tKH)_22,29,30-trienorhopane 17a(H),218(H>30-norhopane 17B(H),21a(H)-30norhopane 17a(H1,21tNH)-hopane 2a-methyl-17a(H),21S(H)-hopane 173(HI,Pla(H)-hooane 17a(Hj,21tNH)_homohopane 22s 17a(H),21S(H)-homohopane 22R gammacerane (I) 17a(H),21B(H)-biahomohopane 22s 17a(H),2lp(H)-biehomohopane 22R 17a(H),21~(Hkrishomohopane 225 17~H),21~(Hkrishomohopane 22R 17a(H),21~(Hk.etrakiishomohopane 22s 17a(H),2lg(H>tetrakiahomohopane 22R 17a(H),21~(H>pantakiehomahomohopane 22s 17a(H),21~(H)_pantakiishomohopane 22R
9 10 ii 13
14 15
16 17 z 20
Assignment
13p(H),17a(H)_diacholeetane 20s 13B(H),17a(H)_diacholestane 20R Cs7 choleatane iaomer Css cholestane isomer C27 choieetane isomer c27 cholestane isomer 5a(H).14a(H).l7afHkholertane 205 5o(H1;14fNH);17e(Hkholeatane 20R + 2&e&l-138(H).17a(H!-diacholestana 20s 5a(H),i4ec~j,l;iWHkholeetane 20s 5cr(H),14a(H),l7o(H)-choleetane 20R 24-ethyl-13B(H),17a(H)_diachoIaatane 20R 4a-methyl-So(H), 14B(H),17B(Hkholrrtane 20R rla-methyl&(H), 14B(Hl,17B(Hkholertane 20s 24-methyl-5ofH),14a(H),17a(Hkholeatane 20s 24-methyl-5o(H),14B(H),17~(Hkholeatane 20R 24-methyl-5ar(H),14B(H),17~(Hkholeatane 20s 24-methyl-5a(H),14a(H),l7o(Hkholeatane 20R 24ethyl-5aIH),14a(H),l7a(Hkholaatane 205 4a,24-dimethyl-Sa(H),l4~H~,l7~Hkhole&.ane 20R 24-ethyl-5a(H1,14B(H),178(Hkholaatane 20R 24-ethyl-5a(H),14B(H),17B(H)-cholertane 20s 24-ethyl-5a(H),14afH),170o-eholeetana 20R 4~-methyl-24-ethyl-5a(H),l4afH),l7a~Hkholeatane 20s 4a-methyl-24-ethyl-5a(H),l4~~H),l7~Hkholeatana 20R 4a-methyl-24-ethyl-5a(H),l4~(H),l7~Hkholeatane 20s 4a-methyl-24-ethyl-5a(H),14a(H),l7a(Hkholastane 20R
Microbial degradation of crude oils phur species, namely, thiolanes, thianes, and benzo[blthiophenes. The composition of these compound classes will be described below. Controlled Microbial Degradation of Amposta Oil Diverse pure microbial cultures have been isolated from petroleum-rich environments where hydrocarbon degradation was observed. The strains selected for the experiments presented here have been used under different conditions, including various digestion times. As shown in Table 1, the sulphur content of the mixtures obtained after biodegradation with A104 and F21 strains (5.3-&O%) is approximately the same as that of the original Amposta oil (X8%), which corresponds to a low degree of microbial transformation (CONNAN, 1984). At the molecular level, the transformations observed in Amposta oil after laboratory bi~e~dation do not appear to be dependent on the bacterial strain used. Furthermore, no major differences were observed for transformation times longer than 15 days. We will focus our description of the results by referring to the compositional changes observed for one of these biodegradation experiments, namely that performed with strain A104 for 15 days. Aliphatic hydrocarbons
The GC traces corresponding to the column chromatography fractions of the original and biodegraded Amposta oil are compared in Fig. 1. The most significant differences are observed for the aliphatic hydrocarbon fraction (Fl). All the n-alkanes are removed by bi~e~dation. Pristane and phytane are preserved, but the lower molecular weight acyclic isoprenoid hydrocarbons, including norpristane, are depleted. Similar results have been obtained in the degradation of other oils, unrelated to an evaporitic origin, with the bacterial strains
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(i.e., Casablanca oil, Tarragona Basin, GRIMALT et al., un-
publ. results). In contrast, no ~gnificant changes in the composition of hopanes and steranes are observed (see Fig. 2). Therefore, at this level of biodegradation, the crude oil mixtures can still be easily recognized as having an evaporitic origin. The features mentioned above, such as a high proportion of CTs extended hopanes, high gammacerane content, depletion of CZS steranes, and abundance of 4a-methyl steranes, are preserved. These results parallel the changes in composition observed for the biodegradation of crude oils in reservoirs (CONNAN, 1984; MCKIRDY et al., 1983; VOLKMANet al., 1983). The n-alkane composition may be altered even by slight biodegradation whereas the acyclic isoprenoids are ~nemble to light-to-moderate degradation. According to the biodegradation scale used by SEIFERTet al. (1984), the mixtures obtained after these bi~e~a~tion experiments correspond to a level no. 4, indicating a moderate degree of transformation. Aryl hydr~arbons
The composition of the long-chain alkylbenzenes in Amposta oil has been described by ALBAIGESet al. (1986) and SINNINGHEDAMS& et al. (1988). These compounds are also represented in Fig. 3 using the following mass fragmentograms: m/z 91 + 92 for the alkylbenzenes, m/z 105 + 106 for the alkyltoluenes, m/z 119 + 120 for the alkyl C&enzenes, and m/z 133 + 134 for the alkyd C&enzenes. In all these types of ring-substituted hydrocarbons, series of CIXCZ, straight chain homologues predominate (ALBAIGESet al., 1986). In addition, aryl hy~oc~~ns with a phytanyl sidechain are present. They constitute a series ranging between CX and Czs where the CZ7 and CZs isomers predominate, namely 1-methyl-3-phytanytbnzene (II), 1,2-dimethyl-4ph~anyl~nzene (III) and 1,4~imethyl-2-ph~nyl~nzene
FIG. 3. Mass fragmentograms showing the distributions of alkyl benzenes (m/z 91 + 92), toluenes (m/z 105 + 106), xylenes (m/z 119 + 120), and trimethylbenzenes (m/z 133 + 134) in the original Amposta oil and in Amposta oil biodegraded in controlled experiments. II: I-methyl-3-phytanylbenzene; III: 1,2-dimethyl4phytanylbenzene; IV: 1,4dimethyl-2-ph~nyl~n~ne; V: 1,2,5,t~methyI4ph~nyl~n~ne; VI: ph~nyl~nzene.
J. 0. Grima dt et al.
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cially degraded Amposta oil only the isoprenoid alkylbenzenes are present. These changes are produced before any significant modification in the composition of the polycyclic aromatic hydro~ns, namely alkyl ~phth~en~ and phenanthrenes, can be observed by mass fragtnentography. LOW molecular weight organosulphur compounds (LMoscs)
FIG. 4. Mass fragmentograms illustrating the composition of thiolanes in Amposta oil. m/z 101: 2-n-alkyl-S-methyl; m/z 115: 2-nalkyl-4-ethyl; and m/z 87: mid-chain 2,5-di-n-alkyl-thiolanes. c = cis and I = trans isomers.
(IV) (Fig. 3). The mixture also contains 1,2,5-trimethyl-4phytanylbenzene (Czs; V) and minor amounts of phytanylbenzene (C&; VI). The structural identification of these isoprenoid hydrocarbons is given in SINNINGHE DAMSTBet al. (1988). These dist~butions strongfy contrast with the composition of the microbially degraded Amposta oil, where the straightchain components have been removed paralleling the selective elimination of n-alkanes (see Fig. 3). This transformation is in agreement with previously reported experiments on the fungal degradation of synthetic linear long-chain alkylbenzenes (FEDORAKand WESTLAKE, 1986). Thus, in the artiti-
The LMOSCs present in Amposta oil have been described in SINNING~E DAMSTBet al. (1989). This crude oil is characterized by the high abundance of alkyl~nzo[b]thiophenes, which constitute the major species together with alkylthiolanes and alkylthianes. The 2,5-di-n-alkylthiolanes encompass various isomeric series of C&C35 homologues maximizing at CM, that include 2-n-alkyl-S-methyl~io~nes (m/z fOl), cis and tram 2+alkyl5-ethyl-thiolanes (m/z 1t 5), and a complex mixture of cis and tram mid-chain 2,5-di-n-alkylthiolanes (m/z 87) (Fig. 4). 2,6-Di-n-alkylthianes are present in minor proportions. These compounds have been identified according to the mass spectral and retention time data reported by SCHMIDet al. (1987) and SINNINGHEDAMSTBet al. f 1989). All these compounds are removed in the microbial de~a~tion experiments of Amposta oil, which parallel the processes of elimination of other straight-chain compounds described above. This transformation at low biodegradation stages, before any significant alteration of the polycyclic aromatic hydrocarbons, is in agreement with the laboratory experiments reported by FEDORAKet al. ( 1988). Details of the dist~butions of the alkyl~nzo[b]thiophenes in Amposta oil are given in Fig. 5. These compounds constitute a complex mixture composed of several series of &CX homologues. One group of series encompasses 2-n-alkylbenzo[b]~iophenes without C-4 substitution (m/z 147) or with methyl (m/z 16 1I, ethyl (m/z 175), propyl (m/z 189),
FIG. 5. Mass fragmentograms ihustrating the composition of unsubstituted (m/z 147) and methyl (m/z 16l), ethyl (m/z 175), Propyl (m/z 189), and butyl (m/z 203) substituted n-alkylhenzo[b]thiophenes in the original Amposta oil and in Amposta oil biodegraded in controlled experiments. 0 indicates C-2 Me, Et, Pr, or BU substituted 4-n-alkyl isomersand l correspondsto unsubstituted or C-4 Me, Et, FY,or Bu substituted 2-n-alkydisomers. A in&ate iwp=noid ~nzo[b]thiopbenes
(see Fig. g).
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Microbial degradation of crude oils 7
428
FIG. 6. Mass fragmentograms showing the composition ofthe isoprenoid alkylbenzo[b]thiophenes in Amposta oil. For each group of homologues both the molecular weight and the base ion have been selected: CZ7(m/z 400 and 175); Cl8 (m/z 414 and 189); and CZ9(m/ z 428 and 203). Further info~ation on the structures of these isomers is given in Fig. 8.
or butyl (m/z 203) groups at the C-4 position. Another group of series includes 4-n-alkylbenzo[b]thiophenes with C-2 methyl (m/z 16if, ethyl (m/z 175), propyl (m/z 189), or butyl (m/z 203) substituents. These compounds have been identified by comparison with the mass spectral and retention time data reported for Rozel Point oil (SINNINGHEDAMSTB et al., 1987). In addition to these ~-~kyl~nzo[b]thiophen~, the profiles displayed in Fig. 5, namely the m/z 175, 189, and 203 mass fragmentograms, show the presence of branched chain isomers. Both mass spectral and retention time data are consistent with isoprenoid substituted benzo[b]thiophene molecules. The occurrence of this group of compounds in Amposta oil and other oils has been previously indicated (SINNINGHE DAMST~~et al., 1989). Their isomeric composition is now shown in detail by their diagnostic ions (Fig. 6). CZ7and CZ8constitute the isomers present in higher concentration (see mass spectra in Fig. 7) and CZy homologues are also found. In fact, an interesting feature of their distribution is that it parallels that of the isoprenoid aryl hydro-
carbons described above, which suggests a close genetic relationship between the two types of compounds. The correspondence between isoprenoid aryl and ~nzo[b]thiophene compounds is illustrated in Fig. 8. iMethyl-3-phytanylbenzene (II) is genetically related to two benzo[b]thiophene isomers: 7-methyl-2-( 1,5.9,I3-tetramethyltetradecyl)-benzo[b]thiophene (VII), and S-methyl-2-( 1, 5,9,13-tet~methyltet~de~yl)~nzo~b]thiophene (VIII). 1,2Dimethyl-4-ph~anylbenzene (III) is also related to two isomers, with methyl substitutions at positions 6,7 (IX) or 56 (X). However, 1,4-dimethyl-2-phytanylbenzene (IV) and 1,2,5-trimethyl-l-phytanyl-benzene (V) are related to a single isomer because of hindering by their ortho methyl substituents. Two, three, and one major doublets are effectively observed in the m/z 400, 414, and 428 mass fragments. respectively (see Fig. 6). In the case of m/z 414 (CZs isomers) one ofthese doublets is present in a minor proportion, which is consistent with the lower concentmtion of the possible aryl precursor ( 1,4-dimethyl-2-ph~nyl~nzene (IV)). The occurrence of these doublets may correspond to the partial resolution of the S and R isomers of the tertiary carbon of the isoprenoid chain linked to the thiophene ring (Fig. 8). The parallelism in the isomeric com~sition of the isoprenoid aryl and benzo[bJthiophene compounds indicates that they derive from common precursors or that the aryl hydrocarbons are formed by desulphurization of the benzo(b]thiophenes. The benzo[b]thiophene distributions are also modified when subjected to microbial de~adation. Two main changes are observed (Fig. 5). First, there is a general depletion of all the compounds containing linear carbon-chain substituents, which results in a higher proportion of the isoprenoid benzo[b]thiophenes. This parallels the changes reported above for the ahphatic and aryl hydr~rbons, although in this case a substantial proportion of ~-alkyl~nzo[b]thiophenes is preserved. Second, there is a selective elimination of specific nalkyl isomers. As can be observed in the m/z 147 and m/z 161 profiles of Fig. 5, the C-4 unsubstituted n-alkyl~nzo[bJthiophenes and the 2-~-alkyl-4-methyl~nzo[b]thiophenes are not present in the biodegraded oil. However, the remaining C-4 substituted 2+alkylbenzo[b]thiophenes and the 4-n-alkylbenzo[b]thiophenes are preserved. In principle, the resistence of these compounds to microbial degradation suggests that they can be used together with the isoprenoid isomers for oil-oil correlation of biodegraded crude oils. la
n5
I00
200 FIG.
100
400
lea
2Bc3
300
7. Representative mass spectra of two isoprenoid benzo[b]thiophene isomers.
w0
J. 0. Grim& et al.
1910
e \ 0
7-
II
and sterane distributions of these oils are remarkably similar to those of Amposta oil. As shown in Fig. 2, the m/z 191 profiles are characterized by high relative amounts of the C35 extended 17oI(H),Z1@(H)hopanes and ~rnrn~mne (I). The steranes are also depleted in C& homologues and contain significant amounts of 4cY-methylsteranes. The high sulphur content of these oils (4.4-5.6%, Table 1) is also consistent with an evaporitic origin. The CZ profiles of the aliphatic hyphen fractions (see Fig. 9) of Varadero oils show that they are depleted in nalkanes and acyclic isoprenoid hydrocarbons, including pristane and phytane. This is indicative of microbial degradation and represents a feature different from Amposta oil. Other differences include the lack of o~nosulphur species and longchain aryl hydrocarbons. The absence of LMOSCs is particularly noteworthy due to the high sulphur content of these crude oils.
P-Y 0
S
’ *
&
I
s’ + FY & x
I
I
0 0”” IV
--
I
h 0
s’ *
XI
xjr?----&
maturity and biodegradation constrains~orLMOSC occurrence The disparity in LMOSC compositions of Varadero and Amposta oils can be attributed to source rock differences, bi~~tion, or matumtion. Maturity can be assessed from several molecular marker and aromatic hydrocarbon ratios (Table 3). Some hopane (17a(H),2la(H)- vs. 17j3(H),21a(H)hopanes: CW(UB/(LU/~ + Ba), 225 vs. 22R 17cu(H),21/3(H)bi~omoho~ne~ C3,22S/(S+R)) and aromatic (methyldi~nzothiophene, DBT) ratios are not very informative because they have already reached the equilibrium values (SEIFERT and MOLDOWAN, 1981; MACKENZIEet al., 1981). In the case of the 18ar(H)- and 17ti(H)- CZ7 hopane ratio (Ts/ (Ts + Tm)), similar values corresponding to an immature stage are found for all the oils. Since this ratio is both source and maturity controlled (SEIEERTand MOLDOWAN, 1978) these values give further support to arguments for a similar
R:
Fro. 8. Structural relationships between the phytanyl benzene and benzo[b]thiophene compounds in Amposta oil. Compound names are given in Fig. 3 and in the text. *denotes a chiral carbon giving rise to S and R isomers apparently resolved as doublets in Fig. 6.
Compared Biomarker Composition of Amposta and Varadero Oils The Varadero crude oils correspond to the Varadero-Cardenas region (North Cuba). They are presumably sourced by a Cretaceous evaporitic-carbonate facies located in relatively shallow (- 1500 m) reservoir rocks (Neogene). The hopane
Table 3: Maturity parametera of Amp&a Compounds*
~tzZ
and Varadero oils
Amposta
Varadero-Y
Varadero-S
0.60 1.10
0.61 0.80
0.62 0.75
1 Hopane and sterane numbers refer to assignments
in Table 2 and peaks in Fig. 2. 2 2-~/l-~, calculated from peak areas in m/z 192 mass chromatograms. 3 2- + 3-DBT/2 x I-DBT, calculated from peak areas in m/z 198 mass chromatograms.
1Yll
Microbial degradation of crude oils a
--Y
VARADERO-S
FIG. 9. Gas chromatograms of the aliphatic hydrocarbon fraction (Fl) of Varadero oils. Numbered peaks refer to the hopanes listed in Table 2.
depositional setting for these oil source rocks, an interpretation consistent with the affinities in their sterane and hopane compositions. Some sterane ratios, namely 20s vs. 20R 24-ethyl%(H), 14c~(H),17c~(H)-cholestanes (CZ9+~20S/(S+R)) and Scu(H),14@(H),178(H)- vs. 5a((H),14a(H), 17cy(H)-24-ethylcholestanes 20R (C*~20~~/(~~ + SKY)), as well as the methyl phen~threne ratio (MPR) (CLAYTONand KING, 1987), indicate that the Varadero oils are less mature than Amposta oil (SEIFERT and MOLDOWAN, 1981; LEYTHAEUSERet al., 1988). In fact, Amposta oil represents one of the most mature oils in which LMOSC compounds have been identified (SINNINGHE DAMST~ et al., 1989). Therefore, the lack of di-nalkylthiolanes and di-n-alkylthianes and the low abundance of alkylbenzo[b]thiophenes in Varadero oils cannot be attributed to maturity effects. This assessment seems to be contradicted by the values of the i 313(H),17~(H)~iacholes~ne 20R vs. S&(H), lrtcu(H), 17Lu(H)-cholestane 2OR ratio (C2,20Rrr/(rr + sd)), given that diasterane enrichment is maturity dependent (SEIFERTand MOLDOWAN,1978). However, this ratio may also be controlled by source or reflect biodegradation. Source rock differences can be excluded in the case of Varadero oils but their diasterane ratios show major discrepancies, 0.13 and 0.34 for Varadero-Y and Varadero-S, respectively. The aliphatic fraction of Varadero-S contains almost no n-alkanes (see Fig. 9) and therefore corresponds to a higher degree of biodegradation than Varadero-Y. Similarly, the differences in the C2,-20Rrr/(rr + sd) ratio between Amposta and Varadero oils are consistent with the differences in their compositions of n-alkanes and aliphatic isoprenoids, suggesting a correspondence with biodegradation. In fact, these crude oils can be rated according to the biodegradation scale used by SEIFERTet al. ( 1984), giving values of 0,6, and 8 for the Amposta, Varadero-Y, and Varadero-S oils, respectively. CONCLUSIONS Low levels of microbial degradation of crude oil such as those involving only n-alkane removal are also sufficient for the removal of straight-chain aryl hydrocarbons and LMOSCs
such as 2,5~i~kylthiolanes
and 2,6-di~~l~ianes.
Isopren-
oid aryl hydrocarbons and both n-alkyl and isoprenoid benzo[b]thiophenes are more resistant, although biodegradation may lead to the selective removal of some isomers such as the 2-n-alkyl and the 2-n-alkyl-4-methylbenzo[blthiophenes. However, both aryl isoprenoids and alkyl benzo[b]thiophenes are absent from marine evaporitic crude oils in which microbial degradation has reached the level at which slight m~ification of the steroid com~sition has occurred. Hence, long-chain alkyl benzenes and LMOSCs are chemical species liable to microbial attack. They may be completely removed at a stage where microbial activity has not achieved any significant modification of steranes, hopanes, or aromatic hydrocarbons. These observations lead us to question whether the limit for the reiatively restricted occurrence of LMOSCs in ancient en~ronmen~ is dependent on conditions of preservation rather than on conditions of formation. In this respect, the results presented here show that the removal of these compounds does not involve a significant modification of the bulk sulphur content in the biodegraded crude oils. In addition to maturity, microbial decomposition processes must be considered when studying sulphur-rich crude oils in which LMOSCs are absent. Acknowied~enr~-V~dero crude oil samples were kindly provided by Dr. P. Campus (Institute de Investigaciones Quimicas, La Havana, Cuba). We thank Prof. J. W. de L.eeuwand Dr. J. S. Sinninghe Dam& (Delfl University of Technology) and Prof. Pierre Albrecht (University of Strasboug) for helpful discussions. Technical assistance from R. Alonso @C-MS analyses) and P. Domenech (elemental analyses) is gratefully acknowledged. Editorial h~ndi~~g:S. C. Brassell
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Geochimrca d Cosmochimrca Ada Vol. 55, pp. 1903-1913 Copyright 0 1991 Pergamon Press pk. Printed in U.S.A.
+ MI
Microbial degradation of marine evaporitic crude oils J. 0. GRIMALT,' M. GRIFOLL,'A. M. SOLANAS,’and J. ALBAIG~S’ ‘Department of Environmental Chemistry (CID-CSIC), Jordi Girona, 18,08034-Barcelona, Spain *Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain (Received February 1, 1990; accepted in revised form April 9, 199 I)
Abstract-The composition of alkylbenzenes and organosulphur compounds in crude oils is strongly modified by biodegradation, even at low levels of transformation where the composition of steranes, hopanes, alkylnaphthalenes, and alkylphenanthrenes has not been altered. These conclusions emerge from the comparison of preserved and biodegraded crude oils of marine evaporitic origin (Amposta and Varadero oils) and controlled experiments of microbial transformation of oils containing high amounts of aryl hydrocarbons and organic sulphur species. The compositions of straight chain compounds, such as n-alkanes, n-alkylbenzenes, 2,5-di-n-alkylthiolanes, 2,6,-di-n-alkylthianes and 2,4dialkylbenzo[blthiophenes are more easily modified than those of compounds with isoprenoid side-chains. The experiments also show that benzo[b]thiophenes are markedly more resistent to microbial attack than thiolanes and thianes. INTRODUCTION
ecules and the stability of the organosulphur compounds upon
AN UNDERSTANDINGOF THE COMPOSITIONAL changes produced during the microbial degradation of crude oils is important to petroleum geochemistry. This topic has been addressed in terms of biomarkers (CONNAN, 1984; SEIFERTand MOLDOWAN, 1979; MCKIRDY et al., 1983; RUBINSTEINet al., 1977; SEIFERTet al., 1984; SOFERet al., 1986; VOLKMAN et al., 1983, 1984; WEHNER et al., 1986; WILLIAMSet al., 1986), aflording valuable information on the transformations of acyclic isoprenoids, hopanes and steranes, used for oil-oil and oil-source rock correlations (SEIFERTand MOLDOWAN, 1979; MCKIRDY et al., 1983; VOLKMAN~~al., 1983; SEIFERT et al., 1984). Stable isotope ratios (SOFER et al., 1986; WEHNER et al., 1986) and other techniques such as 13CCPJMAS nuclear magnetic resonance (LANDAISet al., 1988), fluorescence spectroscopy (DUMKE and TESCHNER,1988), and hydrous pyrolysis (JONES et al., 1988) have also been used in the study of biodegraded oils. Nevertheless, some questions remain to be addressed. One of them concerns marine evaporitic crude oils, which have received limited attention in biodegradation studies. This gap is important in terms of molecular marker geochemistry because most of the oils reported to contain significant amounts of organosulphur compounds were generated from marine evaporitic source rocks (SINNINGHEDAMSTB et al., 1989). Marine evaporitic environments (also quoted as hypersaline environments) are anoxic with the amount of hydrogen sulphide far exceeding the amounts of available iron and other heavy metals; they therefore provide good conditions for the formation of organosulphur molecules (DE LEEUW and SINNINGHEDAMST& 1990). Accordingly, studies on sedimentary sequences including marine evaporite deposits have shown an enrichment of organosulphur compounds in this type of facies (SINNINGHEDAMSTBet al., 1986; TEN HAVEN et al., 1987). However, many aspects related to the occurrence of these compounds still need to be clarified (e.g., the geochemical pathways of sulphur incorporation into many organic mol1903
diagenesis and catagenesis). In this respect, we have found several crude oils of marine evaporitic origin with high sulphur content (4-6%) in which no low molecular weight organic sulphur compounds (LMOSC), those amenable to gas chromatographic analysis, can be observed. Biodegradation was suspected to account for the absence of these molecular species. Another common feature of many marine evaporitic crude oils is their high content of long-chain alkylbenzenes (ALBAIGBSet al., 1986), especially those with an isoprenoid side chain (SUMMONSand POWELL, 1987; SINNINGHEDAMST~ et al., 1988). However, we have observed that these hydrocarbons are absent (or present at only trace levels) in partially biodegraded evaporitic crude oils. To date, no data are available to relate the lack of these compounds to microbial decomposition processes. This paper concerns the comparison of crude oils of marine evaporitic origin from Amposta (Tarragona Basin, Catalonia, NE Spain) and Varadero (Varadero-Cardenas region, N Cuba). The major differences in their composition lie in the strong depletion of n-alkanes, aryl hydrocarbons, and LMOSC in Varadero oils. In principle, these differences were attributed to microbial degradation, a hypothesis tested by means of degradation experiments of Amposta oil using microbial strains isolated from freshwater and marine petroleum-rich environments. Hence, the major compositional features of Varadero oils and the original and artificially biodegraded Amposta oil are investigated, and their differences are discussed in terms of possible biodegradation processes. EXPERIMENTAL Organisms The isolation of strain F2 1 is described in SOLANASet al. ( 1984). In short, ballast water from an oil tanker was used as inoculum of sterilized artificial seawater (100 mL) supplemented with KN03 (0.0 1 M) and Na2HFQ (0.00035 M). Arabian light crude oil residues were used as the only source of carbon and energy. The incubation was
1904
J. 0. Grim& et al.
performed at 30°C with continuous stirring (120rpm) for five days. The resulting mixture was transferred to another flask containing the same fresh medium and the operation was repeated. The samples were plated in the same medium with agar. After several successive incubations the isolated cultures were identified as Pseudomonasspp. This was based on morphological characterization and on a series of biochemical tests that used the culture media described by AUSTIN etal. ( 1977). The bacterial strain A 104 was isolated from a waste water treatment system of a petroleum refinery in Tarragona (Catalonia, NE Spain). Batch enrichment was carried out by inoculating sludge samples on the minimal salts medium described below supplemented with Arabian crude oil fO.l%, w/v). The cultures were incubated at 22°C with shaking. After several transfers different bacterial strains were isolated by making serial dilutions and plating on Tripticase Soy Agar (ADSA, Spain). Strain A 104 was selected for the quick growth in mineral medium containing Arabian light crude oil, and for the early production of crude oil emulsions. This isolate is a non-motile, gram negative coccoid rod, catalase positive, cytochrome oxidase negative and uses glucose only in aerobic conditions. Biodegradation Assays of the Crude Oil The biodegradation assays were perhormed with two different media depending on the bacterial strain. In tbe experiences with Al04 strain a minimal salts medium (a solution of Na2S04 ( 1 g), MgSO, *7H20 (0.2 g), K2 HPQ - 3HzO (0.65 g), NH&l (1 g), KNOr (2 g), and FeSO, (traces) in 1 liter of distilled water) was used. The assays with F21 were performed in artificial seawater (ADSA, Spain) supplemented with KNOs (0.01 M) and Na*HPO., (0.~35 M). Crude oil was added to both media aseptically to 0.1% of final concentration. The starter cultures were obtained by growing the strains in 250 mL Erlenmeyer flasks containing 50 mL of the respective mineral media and hexadecane (O.l%, w/v) as source of carbon. AAer 72 hours of incubation the cultures were centrifuged, washed twice, and resuspended in the same volume of mineral media. 5 mL of these suspensions were used to inoculate 250 mL Erlenmeyer flasks contaming 50 mL of the same medium and 0.1% of Amposta oil. All flasks were incubated at 22°C on an orbital shaker (150 mm) durine 15 or 40 days. Duplicate cultures sterilized before the addition of the crude oil and incubated under the same conditions were used as blanks. Crude Oil Analyses The flask contents from each incubation were blended and the homogenate extracted with n-hexane (3 X 30 mL). The extracts were vacuum evaporated to about 0.5 mL. The crude oils were also extracted with n-hexane using a mechanical stirrer; the n-hexane soluble fraction was decanted and vacuum evaporated to 0.5 mL. All concentrated extracts were fractionated follo~ng previously established methods (ACEVES et al., 1988). About 0.5 g were introduced into a column filled with 8 g each of 5% water-deactivated alumina (70230 mesh, Merck) (top) and silica (70-230 mesh, Merck) (bottom). The following fractions were collected: (Fl), 20 mL of n-hexane (aliphatic hydrocarbons); (FZ), 20 mL of 10% dichloromethane in nhexane (monocyclic aromatic hydrocarbons); and (F3), 40 mL of 20% dichloromethane in n-hexane (polycyclic aromatic hydrocarbons). With this method the organosulphur compounds are separated into thiophenes (Fl), ~n~~ophen~ (F2) and thiolanes and thianes (F3). These fractions were concentrated to small volumes by vacuum rotary evaporation and submitted to instrumental analysis.
Gas chromatography (GC) was performed with a Cario Erba FTV 4 160 GC instmment equipped with a flame ionization detector and a splitless injector. A column of 25 m X 0.25 mm id. coated with CP-Sil CB (Chrompack) was used (film thickness 0.13 pm). Hydrogen was the carrier gas (50 cm-s-‘). The oven temperature was programmed from 60 to 300°C at 6°C * min-‘. Injector and detector temperatures were 300 and 330-C, respectively. The injection was in the splitlessmode (solvent &octane, hot needle technique) keeping the split valve closed for 35 s. GC coupled to mass spectrometry (GC-MS) was performed with a Hewie~-icky 5995 inst~ment equipped with an HP 300 data
system. A column of 2.5 m X 0.2 mm i.d. coated with HP-S (Hewlett Packard) was used (film thickness 0.11 pm). Helium was used as carrier gas. The oven temperature was programmed from 60 to 3OO’C at 4°C - min-‘. The injection conditions were the same as described above, MS temperatures were transfer line 3OO”C,ion source 2OO”C, and analyzer 230°C. Data were acquired in the electron impact mode (70 eV), scanning range m/z 50-650 at 1 s per decade. Carbon and sulphur determinations were performed with a Carlo Erba elemental analyzer Model 1500. RESULTS AND DISCUSSION
The Biomarker Composition of Amposta Oit Amposta oil occurs in Miocene reservoirs of the Tarragona Basin. Most of the crudes of this basin originate from the Casablanca Fo~ation (Miocene carbonate; DEMAISONand BOURGEOIS, 1985). However, Amposta is the most representative example of a group of sulphur-rich oils from the basin (Table 1) which does not originate from these deposits (ALBAIGES et al., 1986). The source rock of this group of oiis remains to be identified, but their molecular markers are characteristic of oils generated from marine evaporitic sediments. Diverse authors have contributed to the study of the composition of Amposta oil (ALBAIGBSand TORRADAS,1974, 1977; ALBAIG& et al., 1986; SEIFERTet al., 1983; SINNINGHE DAMSTE:et al., 1989), and several new molecular markers have been described as a consequence of these studies; namely, long chain acyclic isoprenoid hydrocarbons (ALBAIG~ZS, 1980; ALBAIGB et al., 1985), isoprenoid cyclohexanes (BARBE et al., 1988), and isoprenoid alkyl~nzenes (SINNINGHEDAMSTBet al., 1988). The gas chromatograms corresponding to the aliphatic and aromatic fractions of Amposta oil are presented in Fig. 1. The tr-alkanes range between Cr.&& showing the characteristic C22-C32 even-to-odd carbon number predominance (ALBAIG&and TORRADAS,1974). This feature, together with the pr~ominan~ of phytane over pristane, is indicative of marine evaporitic deposits (CONNAN et al., 1986; Fu JIAMO et al., 1986; MELLO et al., 1988; TEN HAVEN et al., 1985, 1987, 1988). The distributions of hopanes and steranes are shown in Fig. 2. Peak identifications are given in Table 2. These compounds were identified by comparison of the m/z 19 1,205, 2 17, and 23 1 mass ~~mento~arns with previously reported mass fragmentographic data (SEIFERT and MOUXIWAN, 1979) and with those of crude oils of known sterane and hopane composition (ALBAIGI?Set al., 1986). The position of the A-ring methyl substituent in the hopane compound no. 11 was assigned according to the mass spectral and retention time data reported by SUMMONSand JAHNKE(1990). The position and stereochemistry of the A-ring methyi substituents in the sterane compounds nos. 12, 13, 19, 23, 24, 25, and 26 were determined by comparison of selected mass fmgmentograms(m/z 217,231,372,286,400, and414)with those reported by SUMMONSet al. (1987,1988) and SUMMONS and CAPON ( 1988). Absence of 24-n-propylcholestanes was deduced by mass spectral examination of the m/z 414 molecular weight steranes and from the lack of a response in the m/z 304 mass fragmentographic trace (MOLDOWANet al., 1990). Examination ofthe m/z 358 mass fmgmentogram showed that only two Ga steranes are present. They represent two minor ~rn~un~, one coeluting with the C2, sterane
1905
Microbial degradation of crude oils
WPOSTA
5KxEGRADu) AIWOSTA
F2
F3
F3
I
FIG. 1. Gas chromatographic profiles of the aliphatic (FL) and aromatic (F2 and F3) hydrocarbons fractions of the origina Amposta oil and Amposta oil biodegraded in controlled experiments. Numbers refer to chain length of nafkanes: a, nor&Sane; b, pristane; c, phytane. M and F3 are mainly composed of afkyfbenzenes and poiycyclic aromatic hydrocarbons, respectively.
IARADERO-S
8 lx1
FIG. 2. Hopane (m/z 19 1)and sterane (m/z 217) distributions of the original Amposta oit, Amposta oil biodegaded in co~troi~~ experiments, Varadero-Y, and Varadero-S oiis. Peak identiftcations are given in Tabie 2.
J. 0. Grimalt et al.
1906
Tablel: SuIphur content in Amposta and Varadero oils, and in Amposta oil transformed by A104 and F21 strains. The weight losses observed tier laboratory biodegradation are also given.
Amposta Varadero Varadero Amposta* Amposta* Amposta*
Weight losses (%) after biodegradation F2 F3 Total Fl
%S
Sample
Y S A104 (15d) A104 (40d) F21(15d)
n.a. n.a. n.a. 18 al 19
5.8 4.4 5.6 6.0 5.3 5.6
n.a. n.8. n.a. 78 80 78
n.a. n.a. n.a. : 6
n.a. n.a. n.a. 1 1 1
n.a. not applicable. * Mixtures obtained after degradation of Amposta oil with F21 (Pseudomona spp.) and A104 strains.
compound no. 3 and the other corresponding to the unlabelled m/z 2 17 peak that elutes before this compound. The high abundance of gammacerane (I) and Cj4 and Cjs 17a(H),2 1/3(H) extended hopanes (Cx., > Cs3 and C35> &) represents a distinct feature of Amposta oil (ALBAIGBSet al., 1986) which is commonly observed in many evaporitic crude oils (CONNAN et al., 1986; Fu JIAMOet al., 1986; TEN HAVEN et al., 1985, 1987), although it may also be encountered in oils sourced from normal marine salinity environments (MELLO et al., 1988; DE LEEUW and SINNINGHEDAMST& 1990). The lower abundance of Crs homologues in the sterane distributions is consistent with our observations on recent
evaporite systems (BARBEet al., 1990) but is not exclusive of evaporite sourced crude oils (MOLDOWANet al., 1985; MELLOet al., 1988). The high proportion of 4cu-methylsteranes with respect to the steranes is a consequence of the diagenetic conversion of 4cY-methylsterols (DE LEEUW et al., 1983) and constitutes a recognized feature of marine evap oritic crude oils (PHILP and ZHAOAN, 1987; MELLO et al., 1988) microbial mats (BOONet al., 1983) and saline deposits (TEN HAVEN et al., 1985). Finally, Amposta oil has a high sulphur content (-5.8%; see Table 1) which is consistent with a marine evaporitic origin and the presence of a complex mixture of organic sul-
Table 2: Hopanes and steranes identified in the Amposta and Varadero oils. Numbers refer to peaks in the respective mass fragmentograms in Fig. 2. Peak
1 2 3 4 5 6 P 9 ;: l2 13 14 15 16 17 E E E
Assignment
Peak
C23 tricyclic terpane C24 tricyclic terpane C2s triqcfic terpanes C&stetqxyclic terpanes 18a(HM!2,29,30-trisnomeohopane 17a(H)_22,29,30&isnorhopane
17tKH)_22,29,30-trienorhopane 17a(H),218(H>30-norhopane 17B(H),21a(H)-30norhopane 17a(H1,21tNH)-hopane 2a-methyl-17a(H),21S(H)-hopane 173(HI,Pla(H)-hooane 17a(Hj,21tNH)_homohopane 22s 17a(H),21S(H)-homohopane 22R gammacerane (I) 17a(H),21B(H)-biahomohopane 22s 17a(H),2lp(H)-biehomohopane 22R 17a(H),21~(Hkrishomohopane 225 17~H),21~(Hkrishomohopane 22R 17a(H),21~(Hk.etrakiishomohopane 22s 17a(H),2lg(H>tetrakiahomohopane 22R 17a(H),21~(H>pantakiehomahomohopane 22s 17a(H),21~(H)_pantakiishomohopane 22R
9 10 ii 13
14 15
16 17 z 20
Assignment
13p(H),17a(H)_diacholeetane 20s 13B(H),17a(H)_diacholestane 20R Cs7 choleatane iaomer Css cholestane isomer C27 choieetane isomer c27 cholestane isomer 5a(H).14a(H).l7afHkholertane 205 5o(H1;14fNH);17e(Hkholeatane 20R + 2&e&l-138(H).17a(H!-diacholestana 20s 5a(H),i4ec~j,l;iWHkholeetane 20s 5cr(H),14a(H),l7o(H)-choleetane 20R 24-ethyl-13B(H),17a(H)_diachoIaatane 20R 4a-methyl-So(H), 14B(H),17B(Hkholrrtane 20R rla-methyl&(H), 14B(Hl,17B(Hkholertane 20s 24-methyl-5ofH),14a(H),17a(Hkholeatane 20s 24-methyl-5o(H),14B(H),17~(Hkholeatane 20R 24-methyl-5ar(H),14B(H),17~(Hkholeatane 20s 24-methyl-5a(H),14a(H),l7o(Hkholeatane 20R 24ethyl-5aIH),14a(H),l7a(Hkholaatane 205 4a,24-dimethyl-Sa(H),l4~H~,l7~Hkhole&.ane 20R 24-ethyl-5a(H1,14B(H),178(Hkholaatane 20R 24-ethyl-5a(H),14B(H),17B(H)-cholertane 20s 24-ethyl-5a(H),14afH),170o-eholeetana 20R 4~-methyl-24-ethyl-5a(H),l4afH),l7a~Hkholeatane 20s 4a-methyl-24-ethyl-5a(H),l4~~H),l7~Hkholeatana 20R 4a-methyl-24-ethyl-5a(H),l4~(H),l7~Hkholeatane 20s 4a-methyl-24-ethyl-5a(H),14a(H),l7a(Hkholastane 20R
Microbial degradation of crude oils phur species, namely, thiolanes, thianes, and benzo[blthiophenes. The composition of these compound classes will be described below. Controlled Microbial Degradation of Amposta Oil Diverse pure microbial cultures have been isolated from petroleum-rich environments where hydrocarbon degradation was observed. The strains selected for the experiments presented here have been used under different conditions, including various digestion times. As shown in Table 1, the sulphur content of the mixtures obtained after biodegradation with A104 and F21 strains (5.3-&O%) is approximately the same as that of the original Amposta oil (X8%), which corresponds to a low degree of microbial transformation (CONNAN, 1984). At the molecular level, the transformations observed in Amposta oil after laboratory bi~e~dation do not appear to be dependent on the bacterial strain used. Furthermore, no major differences were observed for transformation times longer than 15 days. We will focus our description of the results by referring to the compositional changes observed for one of these biodegradation experiments, namely that performed with strain A104 for 15 days. Aliphatic hydrocarbons
The GC traces corresponding to the column chromatography fractions of the original and biodegraded Amposta oil are compared in Fig. 1. The most significant differences are observed for the aliphatic hydrocarbon fraction (Fl). All the n-alkanes are removed by bi~e~dation. Pristane and phytane are preserved, but the lower molecular weight acyclic isoprenoid hydrocarbons, including norpristane, are depleted. Similar results have been obtained in the degradation of other oils, unrelated to an evaporitic origin, with the bacterial strains
1907
(i.e., Casablanca oil, Tarragona Basin, GRIMALT et al., un-
publ. results). In contrast, no ~gnificant changes in the composition of hopanes and steranes are observed (see Fig. 2). Therefore, at this level of biodegradation, the crude oil mixtures can still be easily recognized as having an evaporitic origin. The features mentioned above, such as a high proportion of CTs extended hopanes, high gammacerane content, depletion of CZS steranes, and abundance of 4a-methyl steranes, are preserved. These results parallel the changes in composition observed for the biodegradation of crude oils in reservoirs (CONNAN, 1984; MCKIRDY et al., 1983; VOLKMANet al., 1983). The n-alkane composition may be altered even by slight biodegradation whereas the acyclic isoprenoids are ~nemble to light-to-moderate degradation. According to the biodegradation scale used by SEIFERTet al. (1984), the mixtures obtained after these bi~e~a~tion experiments correspond to a level no. 4, indicating a moderate degree of transformation. Aryl hydr~arbons
The composition of the long-chain alkylbenzenes in Amposta oil has been described by ALBAIGESet al. (1986) and SINNINGHEDAMS& et al. (1988). These compounds are also represented in Fig. 3 using the following mass fragmentograms: m/z 91 + 92 for the alkylbenzenes, m/z 105 + 106 for the alkyltoluenes, m/z 119 + 120 for the alkyl C&enzenes, and m/z 133 + 134 for the alkyd C&enzenes. In all these types of ring-substituted hydrocarbons, series of CIXCZ, straight chain homologues predominate (ALBAIGESet al., 1986). In addition, aryl hy~oc~~ns with a phytanyl sidechain are present. They constitute a series ranging between CX and Czs where the CZ7 and CZs isomers predominate, namely 1-methyl-3-phytanytbnzene (II), 1,2-dimethyl-4ph~anyl~nzene (III) and 1,4~imethyl-2-ph~nyl~nzene
FIG. 3. Mass fragmentograms showing the distributions of alkyl benzenes (m/z 91 + 92), toluenes (m/z 105 + 106), xylenes (m/z 119 + 120), and trimethylbenzenes (m/z 133 + 134) in the original Amposta oil and in Amposta oil biodegraded in controlled experiments. II: I-methyl-3-phytanylbenzene; III: 1,2-dimethyl4phytanylbenzene; IV: 1,4dimethyl-2-ph~nyl~n~ne; V: 1,2,5,t~methyI4ph~nyl~n~ne; VI: ph~nyl~nzene.
J. 0. Grima dt et al.
1908
cially degraded Amposta oil only the isoprenoid alkylbenzenes are present. These changes are produced before any significant modification in the composition of the polycyclic aromatic hydro~ns, namely alkyl ~phth~en~ and phenanthrenes, can be observed by mass fragtnentography. LOW molecular weight organosulphur compounds (LMoscs)
FIG. 4. Mass fragmentograms illustrating the composition of thiolanes in Amposta oil. m/z 101: 2-n-alkyl-S-methyl; m/z 115: 2-nalkyl-4-ethyl; and m/z 87: mid-chain 2,5-di-n-alkyl-thiolanes. c = cis and I = trans isomers.
(IV) (Fig. 3). The mixture also contains 1,2,5-trimethyl-4phytanylbenzene (Czs; V) and minor amounts of phytanylbenzene (C&; VI). The structural identification of these isoprenoid hydrocarbons is given in SINNINGHE DAMSTBet al. (1988). These dist~butions strongfy contrast with the composition of the microbially degraded Amposta oil, where the straightchain components have been removed paralleling the selective elimination of n-alkanes (see Fig. 3). This transformation is in agreement with previously reported experiments on the fungal degradation of synthetic linear long-chain alkylbenzenes (FEDORAKand WESTLAKE, 1986). Thus, in the artiti-
The LMOSCs present in Amposta oil have been described in SINNING~E DAMSTBet al. (1989). This crude oil is characterized by the high abundance of alkyl~nzo[b]thiophenes, which constitute the major species together with alkylthiolanes and alkylthianes. The 2,5-di-n-alkylthiolanes encompass various isomeric series of C&C35 homologues maximizing at CM, that include 2-n-alkyl-S-methyl~io~nes (m/z fOl), cis and tram 2+alkyl5-ethyl-thiolanes (m/z 1t 5), and a complex mixture of cis and tram mid-chain 2,5-di-n-alkylthiolanes (m/z 87) (Fig. 4). 2,6-Di-n-alkylthianes are present in minor proportions. These compounds have been identified according to the mass spectral and retention time data reported by SCHMIDet al. (1987) and SINNINGHEDAMSTBet al. f 1989). All these compounds are removed in the microbial de~a~tion experiments of Amposta oil, which parallel the processes of elimination of other straight-chain compounds described above. This transformation at low biodegradation stages, before any significant alteration of the polycyclic aromatic hydrocarbons, is in agreement with the laboratory experiments reported by FEDORAKet al. ( 1988). Details of the dist~butions of the alkyl~nzo[b]thiophenes in Amposta oil are given in Fig. 5. These compounds constitute a complex mixture composed of several series of &CX homologues. One group of series encompasses 2-n-alkylbenzo[b]~iophenes without C-4 substitution (m/z 147) or with methyl (m/z 16 1I, ethyl (m/z 175), propyl (m/z 189),
FIG. 5. Mass fragmentograms ihustrating the composition of unsubstituted (m/z 147) and methyl (m/z 16l), ethyl (m/z 175), Propyl (m/z 189), and butyl (m/z 203) substituted n-alkylhenzo[b]thiophenes in the original Amposta oil and in Amposta oil biodegraded in controlled experiments. 0 indicates C-2 Me, Et, Pr, or BU substituted 4-n-alkyl isomersand l correspondsto unsubstituted or C-4 Me, Et, FY,or Bu substituted 2-n-alkydisomers. A in&ate iwp=noid ~nzo[b]thiopbenes
(see Fig. g).
1909
Microbial degradation of crude oils 7
428
FIG. 6. Mass fragmentograms showing the composition ofthe isoprenoid alkylbenzo[b]thiophenes in Amposta oil. For each group of homologues both the molecular weight and the base ion have been selected: CZ7(m/z 400 and 175); Cl8 (m/z 414 and 189); and CZ9(m/ z 428 and 203). Further info~ation on the structures of these isomers is given in Fig. 8.
or butyl (m/z 203) groups at the C-4 position. Another group of series includes 4-n-alkylbenzo[b]thiophenes with C-2 methyl (m/z 16if, ethyl (m/z 175), propyl (m/z 189), or butyl (m/z 203) substituents. These compounds have been identified by comparison with the mass spectral and retention time data reported for Rozel Point oil (SINNINGHEDAMSTB et al., 1987). In addition to these ~-~kyl~nzo[b]thiophen~, the profiles displayed in Fig. 5, namely the m/z 175, 189, and 203 mass fragmentograms, show the presence of branched chain isomers. Both mass spectral and retention time data are consistent with isoprenoid substituted benzo[b]thiophene molecules. The occurrence of this group of compounds in Amposta oil and other oils has been previously indicated (SINNINGHE DAMST~~et al., 1989). Their isomeric composition is now shown in detail by their diagnostic ions (Fig. 6). CZ7and CZ8constitute the isomers present in higher concentration (see mass spectra in Fig. 7) and CZy homologues are also found. In fact, an interesting feature of their distribution is that it parallels that of the isoprenoid aryl hydro-
carbons described above, which suggests a close genetic relationship between the two types of compounds. The correspondence between isoprenoid aryl and ~nzo[b]thiophene compounds is illustrated in Fig. 8. iMethyl-3-phytanylbenzene (II) is genetically related to two benzo[b]thiophene isomers: 7-methyl-2-( 1,5.9,I3-tetramethyltetradecyl)-benzo[b]thiophene (VII), and S-methyl-2-( 1, 5,9,13-tet~methyltet~de~yl)~nzo~b]thiophene (VIII). 1,2Dimethyl-4-ph~anylbenzene (III) is also related to two isomers, with methyl substitutions at positions 6,7 (IX) or 56 (X). However, 1,4-dimethyl-2-phytanylbenzene (IV) and 1,2,5-trimethyl-l-phytanyl-benzene (V) are related to a single isomer because of hindering by their ortho methyl substituents. Two, three, and one major doublets are effectively observed in the m/z 400, 414, and 428 mass fragments. respectively (see Fig. 6). In the case of m/z 414 (CZs isomers) one ofthese doublets is present in a minor proportion, which is consistent with the lower concentmtion of the possible aryl precursor ( 1,4-dimethyl-2-ph~nyl~nzene (IV)). The occurrence of these doublets may correspond to the partial resolution of the S and R isomers of the tertiary carbon of the isoprenoid chain linked to the thiophene ring (Fig. 8). The parallelism in the isomeric com~sition of the isoprenoid aryl and benzo[bJthiophene compounds indicates that they derive from common precursors or that the aryl hydrocarbons are formed by desulphurization of the benzo(b]thiophenes. The benzo[b]thiophene distributions are also modified when subjected to microbial de~adation. Two main changes are observed (Fig. 5). First, there is a general depletion of all the compounds containing linear carbon-chain substituents, which results in a higher proportion of the isoprenoid benzo[b]thiophenes. This parallels the changes reported above for the ahphatic and aryl hydr~rbons, although in this case a substantial proportion of ~-alkyl~nzo[b]thiophenes is preserved. Second, there is a selective elimination of specific nalkyl isomers. As can be observed in the m/z 147 and m/z 161 profiles of Fig. 5, the C-4 unsubstituted n-alkyl~nzo[bJthiophenes and the 2-~-alkyl-4-methyl~nzo[b]thiophenes are not present in the biodegraded oil. However, the remaining C-4 substituted 2+alkylbenzo[b]thiophenes and the 4-n-alkylbenzo[b]thiophenes are preserved. In principle, the resistence of these compounds to microbial degradation suggests that they can be used together with the isoprenoid isomers for oil-oil correlation of biodegraded crude oils. la
n5
I00
200 FIG.
100
400
lea
2Bc3
300
7. Representative mass spectra of two isoprenoid benzo[b]thiophene isomers.
w0
J. 0. Grim& et al.
1910
e \ 0
7-
II
and sterane distributions of these oils are remarkably similar to those of Amposta oil. As shown in Fig. 2, the m/z 191 profiles are characterized by high relative amounts of the C35 extended 17oI(H),Z1@(H)hopanes and ~rnrn~mne (I). The steranes are also depleted in C& homologues and contain significant amounts of 4cY-methylsteranes. The high sulphur content of these oils (4.4-5.6%, Table 1) is also consistent with an evaporitic origin. The CZ profiles of the aliphatic hyphen fractions (see Fig. 9) of Varadero oils show that they are depleted in nalkanes and acyclic isoprenoid hydrocarbons, including pristane and phytane. This is indicative of microbial degradation and represents a feature different from Amposta oil. Other differences include the lack of o~nosulphur species and longchain aryl hydrocarbons. The absence of LMOSCs is particularly noteworthy due to the high sulphur content of these crude oils.
P-Y 0
S
’ *
&
I
s’ + FY & x
I
I
0 0”” IV
--
I
h 0
s’ *
XI
xjr?----&
maturity and biodegradation constrains~orLMOSC occurrence The disparity in LMOSC compositions of Varadero and Amposta oils can be attributed to source rock differences, bi~~tion, or matumtion. Maturity can be assessed from several molecular marker and aromatic hydrocarbon ratios (Table 3). Some hopane (17a(H),2la(H)- vs. 17j3(H),21a(H)hopanes: CW(UB/(LU/~ + Ba), 225 vs. 22R 17cu(H),21/3(H)bi~omoho~ne~ C3,22S/(S+R)) and aromatic (methyldi~nzothiophene, DBT) ratios are not very informative because they have already reached the equilibrium values (SEIFERT and MOLDOWAN, 1981; MACKENZIEet al., 1981). In the case of the 18ar(H)- and 17ti(H)- CZ7 hopane ratio (Ts/ (Ts + Tm)), similar values corresponding to an immature stage are found for all the oils. Since this ratio is both source and maturity controlled (SEIEERTand MOLDOWAN, 1978) these values give further support to arguments for a similar
R:
Fro. 8. Structural relationships between the phytanyl benzene and benzo[b]thiophene compounds in Amposta oil. Compound names are given in Fig. 3 and in the text. *denotes a chiral carbon giving rise to S and R isomers apparently resolved as doublets in Fig. 6.
Compared Biomarker Composition of Amposta and Varadero Oils The Varadero crude oils correspond to the Varadero-Cardenas region (North Cuba). They are presumably sourced by a Cretaceous evaporitic-carbonate facies located in relatively shallow (- 1500 m) reservoir rocks (Neogene). The hopane
Table 3: Maturity parametera of Amp&a Compounds*
~tzZ
and Varadero oils
Amposta
Varadero-Y
Varadero-S
0.60 1.10
0.61 0.80
0.62 0.75
1 Hopane and sterane numbers refer to assignments
in Table 2 and peaks in Fig. 2. 2 2-~/l-~, calculated from peak areas in m/z 192 mass chromatograms. 3 2- + 3-DBT/2 x I-DBT, calculated from peak areas in m/z 198 mass chromatograms.
1Yll
Microbial degradation of crude oils a
--Y
VARADERO-S
FIG. 9. Gas chromatograms of the aliphatic hydrocarbon fraction (Fl) of Varadero oils. Numbered peaks refer to the hopanes listed in Table 2.
depositional setting for these oil source rocks, an interpretation consistent with the affinities in their sterane and hopane compositions. Some sterane ratios, namely 20s vs. 20R 24-ethyl%(H), 14c~(H),17c~(H)-cholestanes (CZ9+~20S/(S+R)) and Scu(H),14@(H),178(H)- vs. 5a((H),14a(H), 17cy(H)-24-ethylcholestanes 20R (C*~20~~/(~~ + SKY)), as well as the methyl phen~threne ratio (MPR) (CLAYTONand KING, 1987), indicate that the Varadero oils are less mature than Amposta oil (SEIFERT and MOLDOWAN, 1981; LEYTHAEUSERet al., 1988). In fact, Amposta oil represents one of the most mature oils in which LMOSC compounds have been identified (SINNINGHE DAMST~ et al., 1989). Therefore, the lack of di-nalkylthiolanes and di-n-alkylthianes and the low abundance of alkylbenzo[b]thiophenes in Varadero oils cannot be attributed to maturity effects. This assessment seems to be contradicted by the values of the i 313(H),17~(H)~iacholes~ne 20R vs. S&(H), lrtcu(H), 17Lu(H)-cholestane 2OR ratio (C2,20Rrr/(rr + sd)), given that diasterane enrichment is maturity dependent (SEIFERTand MOLDOWAN,1978). However, this ratio may also be controlled by source or reflect biodegradation. Source rock differences can be excluded in the case of Varadero oils but their diasterane ratios show major discrepancies, 0.13 and 0.34 for Varadero-Y and Varadero-S, respectively. The aliphatic fraction of Varadero-S contains almost no n-alkanes (see Fig. 9) and therefore corresponds to a higher degree of biodegradation than Varadero-Y. Similarly, the differences in the C2,-20Rrr/(rr + sd) ratio between Amposta and Varadero oils are consistent with the differences in their compositions of n-alkanes and aliphatic isoprenoids, suggesting a correspondence with biodegradation. In fact, these crude oils can be rated according to the biodegradation scale used by SEIFERTet al. ( 1984), giving values of 0,6, and 8 for the Amposta, Varadero-Y, and Varadero-S oils, respectively. CONCLUSIONS Low levels of microbial degradation of crude oil such as those involving only n-alkane removal are also sufficient for the removal of straight-chain aryl hydrocarbons and LMOSCs
such as 2,5~i~kylthiolanes
and 2,6-di~~l~ianes.
Isopren-
oid aryl hydrocarbons and both n-alkyl and isoprenoid benzo[b]thiophenes are more resistant, although biodegradation may lead to the selective removal of some isomers such as the 2-n-alkyl and the 2-n-alkyl-4-methylbenzo[blthiophenes. However, both aryl isoprenoids and alkyl benzo[b]thiophenes are absent from marine evaporitic crude oils in which microbial degradation has reached the level at which slight m~ification of the steroid com~sition has occurred. Hence, long-chain alkyl benzenes and LMOSCs are chemical species liable to microbial attack. They may be completely removed at a stage where microbial activity has not achieved any significant modification of steranes, hopanes, or aromatic hydrocarbons. These observations lead us to question whether the limit for the reiatively restricted occurrence of LMOSCs in ancient en~ronmen~ is dependent on conditions of preservation rather than on conditions of formation. In this respect, the results presented here show that the removal of these compounds does not involve a significant modification of the bulk sulphur content in the biodegraded crude oils. In addition to maturity, microbial decomposition processes must be considered when studying sulphur-rich crude oils in which LMOSCs are absent. Acknowied~enr~-V~dero crude oil samples were kindly provided by Dr. P. Campus (Institute de Investigaciones Quimicas, La Havana, Cuba). We thank Prof. J. W. de L.eeuwand Dr. J. S. Sinninghe Dam& (Delfl University of Technology) and Prof. Pierre Albrecht (University of Strasboug) for helpful discussions. Technical assistance from R. Alonso @C-MS analyses) and P. Domenech (elemental analyses) is gratefully acknowledged. Editorial h~ndi~~g:S. C. Brassell
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