Structural investigation of nonpolar sulfur cross-linked macromolecules in petroleum

0016.7037/93/$6.00 t .oO

Geochmrca n Cosmmhmrcu ANa Vol. 57, pp. 3395-3419 Copyright C 1993 Pergamon Press Ltd.Printedin U.S.A.

Structural investigation

of nonpolar sulfur cross-linked

macromolecules

in petroleum

P. ADAM, ’ J. C. SCHMID, ’ B. MYCKE, ’ C. STRAZIELLE,’ J. CONNAN,~ A. Huc,~ A. RIVA,~ and P. ALBRECHT*” ‘Institut de Chimie, Universite Louis Pasteur, 1 rue Blaise Pascal, 67008 Strasbourg, France *Centre de Recherche sur les Macromolkules, Institut Charles Sadron, 6, rue Boussingault, 67000 Strasbourg, France 3Elf Aquitaine, 640 18 Pau, France 41nstitut Francais du PCtrole, l-4 avenue de Bois-Prkau, 92506 Rueil-Malmaison, France ‘AGIP Spa, 20097 San Donato Milanese-Milano, Italy (Received December 27, 1991; accepted in revisedform February 18, 1993)

Abstract-A novel hexane-soluble nonpolar macromolecular fraction (NPMF) has been found to occur in substantial amounts (up to 32%) in sulfur-rich crude oils and a rock extract. It is highly aliphatic and has a molecular weight culminating at several thousand mass units, as proven by spectroscopic and molecular weight studies. C-S bond hydrogenolysis of NPMF with Raney nickel as a catalyst yields high proportions of aliphatic hydrocarbons in which long linear, acyclic polyisoprenoid and carotenoid chains usually predominate (except in one case) over polycyclic structures, such as steroids and hopanoids. Hence, NPMF consists mainly of macromolecules composed of low molecular weight hydrocarbon subunits cross-linked with sulfide bridges. Use of deuterated Raney nickel indicated in one case (Rozel Point oil) that the long chains and some hopanoids are multiattached to the macromolecular network, whereas other structural subunits, such as steroids or gammacerane, are essentially monoattached. Detailed structural determinations of the hydrocarbon “building blocks” of NPMF give information on their origin and the mode of formation of these macromolecules in the subsurface. Indeed, most of the building blocks can be related to algal (e.g., long linear chains, steroids, p-carotene, and related carotenoids) or bacterial (e.g., acyclic and monocyclic carotenoids, long-chain acyclic isoprenoids) precursors which essentially exist in living organisms as monounsaturated or polyunsaturated species or are easily transformed into such species by diagenetic processes (e.g., steroids). It appears that these alkenes or polyenes become selectively trapped into a macromolecular network by reaction with inorganic sulfur species produced by bacteria in a kind of natural, low-temperature, vulcanization process. This process could start at early diagenesis already in the water column or eventually continue in the bottom sediment. Although its exact nature is yet unknown, it seems likely that the cross-linking reaction can be initiated by the cleavage of sulfur species in a radical type mechanism. The alkanes formed upon desulfurization of NPMF usually represent much higher amounts than the free alkanes of the samples and show a dramatically different composition. They may deliver very useful, complementary information in studies related to source and palaeoenvironment. However, by far the greater part of the organically bound sulfur of crude oils is usually present in high molecular weight fractions, in particular, in the resins and asphaltenes (IGNASIAKet al., 1977; SPEIGHT, 1984; TISSOT and WELTE, 1984; SCHMID, 1986; TRIFILIEFF, 1987; SINNINGHE DAMST~ et al., 1990), in which sulfur may act as a cross-linking agent. The structure and mode of formation of these high molecular weight species is still a matter of wide speculation. A better understanding of these questions is not only important for geochemical purposes but also for the problems they often pose in refining processes ( BODUSZYNSKI, 1987 ). Reported here are the results of investigations conducted in order to unravel the structure of a novel hexane-soluble non-polar macromolecular fraction (NPMF) commonly occurring in substantial amounts in sulfur-rich petroleums (SCHMID, 1986; SCHMID et al., 1987b). This material indeed provides a good, relatively simple model for understanding the origin and mode of formation of high molecular weight entities. Bulk measurements and chemical degradations show, in particular, that this fraction is mainly composed of low molecular weight hydrocarbon subunits cross-linked by sulfur in what appears to be the result of a natural vulcanization process.

INTRODUCTION DURING RECENT YEARS, increasing interest has been devoted to the geochemistry of organo-sulfur compounds. The latter are abundant in many crude oils and sediments from anoxic environments (in particular carbonates) where bacterial processes produce inorganic sulfur species (H$, polysulfides, elemental sulfur) susceptible to react with organic molecules from decaying organisms. A great number of structures of low molecular weight organo-sulfur components occurring in sediments and crude oils have been identified. In most cases, these structures have carbon skeletons based on nalkanes, long chain acyclic isoprenoid alkanes, polycyclic terpanes, steranes, and hopanes ( PAYZANT et al., 1983, 1986; VALISOLALAO et al., 1984; SCHMID, 1986; BRASSELL et al., 1986a; SCHMID et al., 1987a; SINNINGHE DAMST~ et al., 1987, 1989; SINNINGHE DAMST~ and DE LEEUW, 1990; and references therein). Their identification has brought a better understanding of their formation in the subsurface and, in some cases, a clearer view of the nature of the reacting species and of the mechanism of the reactions involved.

* Author to whom correspondence

should be addressed.

3395

P. Adam et al.

3396 DESCRIPTION

OF SAMPLES

The NPMF (Table 1) of the following six crude oils and one rock extract, all sulfur-rich, were studied.

1) Rozel Point seep oil is a surface sample from the Great Salt Lake area (Utah, USA; BORTZ, 1984) highly enriched in sulfur ( 15%S); the sample is a solid at room temperature. The source rock of this oil is thought to be a hypersaline deposit of Miocene age ( MEISSNERet al., 1984).

2) Maruejols crude oil (St. Jean de Maruejols 10 1, Al& basin,

3)

4)

5)

6)

7)

France; 6.5%S) is of Oligocene age and deposited in a carbonate evaporitic environment. Ponte Dirillo crude oil (Sicily, Italy; 6.6%S; 12.0” API; 3 100 m) is derived from a Late Rhaetien limestone reservoir, which is considered to be the source rock as well. Monterey 6-4 (Point Arena Harbor, California, USA) is an outcropping sandstone impregnated by asphalt which has undergone considerable biodegradation. Monterey 1509 is a crude oil of Miocene age from an offshore Santa Maria basin well (California, USA; 6.7%S; 7.5” API: 796.5-828.7 m). Monterey 1547 is another, slightly more mature crude oil of Miocene age from an offshore Santa Maria basin well (California, USA; 3.9%S; 20” API; 2302.7-2533.6 m). ORR ( 1986) provides a general description of Monterey oils from this basin. Sainte Cecile is a laminated anhydrite from an evaporitic basin of Oligocene age (Camargue, France). The sample is from the Sainte CCcile I well at 2027-2028 m depth; it contains 0.35% total organic carbon. EXPERIMENTAL

General Elemental analyses were carried out at the Service Central d’Analyse du CNRS (Vemaison. France). ‘H-NMR spectra were run in CDZC12 on a 400 MHz Bruker AM400 instrument. Gas chromatography(GC) was run on a Carlo Erba HRGC Fractovap 4160 equipped with an on-column injector and 25 m or 35 m X 0.3 mm i.d. SE54 glass capillary columns. Temperature program was 50-I 5O”C, lO”C/min; 150-300°C. 4”C/min: 20 min isothermal at 300°C. unless indicated in figure captions. Detector temperature was 3OO”C,with carrier gas HZ (0.7 kg/cm2). Gas chromatography-mass spectrometry (GC-MS) was run on the following instruments.

1) KRATOS MS 80 coupled with a Carlo Erba HRGC Fractovap 4160 with on-column injector; data processing was done on a Data General NOVA 4X computer with DS55 software. Typical GC conditions were as follows: 80-3OO”C, 4”C/min; 20 min isothermal at 300°C; 25 m X 0.3 mm i.d. SE54 capillary columns. Typical MS conditions were as follows: electron energy 70 eV; source temperature 25O’C; scanning I s/decade. 2) Varian CH 7A MS coupled to a Carlo Erba HRGC Fractovap 4160 with on-column injector; data were also processed with a DS55 software. Gas chromatography and MS conditions were identical to ( I), except that a DB5 fused silica column was used. 3) Finnigan MAT Incas 50 quadrupole instrument coupled to a Varian 3400 GC with on-column injector; data processing was done on a Data General DGIO computer system with Incas 50 software. Typical GC conditions were as follows: 55-100°C. lO’C/ min; IOO-3OO”C,4”C/min; 20 min isothermal at 300°C; 30 m X 0.25 mm i.d.; 0.1 pm film thickness; DB5 fused silica column. Typical MS conditions were as follows: electron energy 70 eV, source temperature 150°C full scan mode 40-800 mass units, cycle time I I s

Molecular Weight Determinations Molecular weight measurements were made at the Centre de Recherche sur les MacromolCcules, Institut Charles Sadron, Strasbourg, using three independant methods. Vapor pressure osmometry was run on a Knauer tonometer; standardisation was achieved with polyoxyethylene of mass average number M, of 1000; the measurements were performed in toluene at a temperature of 50°C. Light scattering measurements were made in hexane on a photogoniodiffusometer FICA 42000 at 632 nm in vertically polarized light at ambient temperature. The refraction index was measured at 546 and 632 nm with a differential Brice Phoenix spectrometer. Size exclusion chromatography measurements were performed in tetrahydrofuran on two columns containing IO pm beads of polystyrenes (PS) of mixed porosity reticulated with divinylbenzene (Shodex type; 50-10,000 A). Calibration was realized with a series of linear PS with values of &fw(weight average molecular weight) ranging from 1500 to 1,300,OOO. The NPMFs of our samples were compared with a PS having a M,, value of 1500. Separation of Nonpolar Macromolecular Fractions The crude oils and rock extracts were chromatographed over silica gel (Merck, 40-63 pm) in two steps. In the first step, the crude oil or rock extract was dissolved in a minima1 volume of methylene chloride and loaded onto a silica gel column filled with hexane; elution was performed with hexane-ether (98:2) and more polar solvents. Typically, IO g of crude oil were dissolved in 50 mL of CH$& and deposited on top of a column containing 600 g of silica gel. Elution with 1500 mL of hexane-ether (98:2) afforded a non-polar to moderately polar fraction; the more polar fraction was eluted with chloroform-methanol-water (65:25:4). After evaporation of the solvent, the first fraction was redissolved in hexane and rechromatographed on another silica gel column (300 g). Extensive elution with hexane (4 L) afforded the saturated, unsaturated, and aromatic hydrocarbons, as well as the aliphatic sulfides (e.g., thiolanes). Further elution with about I L hexane-ether (98: 2) vielded a bulk NPMF. (7-32% of crude oil or rock extract) the elution of which could be followed by its slightly reddish color. The polarity range of this fraction is approximately between penta-aromatic hydrocarbons (e.g.. picene) and monocarboxylic acid methylesters. The more polar fraction was, as in the first step, eluted with chloroform-methanol-water (65:25:4). Synthesis of CA5Regular Nonaprenane, (2,6,10,14,18,22,26,30,34)-nonamethylhexatriacontane Solanesol (20 mg; Fluka) dissolved in 10 mL ethyl acetate was hydrogenated over Pd/C as a catalyst. The reaction product was purified by thin-layer chromatography over silicagel impregnated with 10% AgNOj, yielding the Cd5regular isoprenoid alkane as a mixture of diastereomers. MS was m/z (rel. int.) 630[M+-21 (
Nonpolar Table 1

macromolecular

fraction

Quantitative data on Non-Polar Macromolecular Fractions (NPMF) of sulfur rich crude oils and rock extract*. % NPMF in crude

% S in crude oil or

% Alkanes from RaNi

oil or rock extract

rock extract

degradation of NPMF

iozel Point

32.0

15.0

51.0

14.9

Mamejols

16.0

6.5

52.0

11.6

Ponte Dirillo

7.0

6.6

16.0

11.1

Monterey 6-4

7.3

Monterey 1509

8.2

Monterey1547

8.3

Sainte-CCcile*

20.7

Sample

NPMF: Non-Polar

Macromolecular

n.d.

with Deuterated

n.d.

6.7

25.0

n.d.

3.9

9.4

n.d.

26.0

n.d.

n.d.

Fraction

Raney Nickel

Deuteration of Raney nickel was achieved by a method slightly modified from that of DJERAssl and WILLIAMS ( I963), starting with Raney nickel alloy ( Merck-Schuchardt ). Five grams of alloy were digested with a diluted solution of NaOD (25 mL of a 16% NaOD solution in D,O) at 50°C in a beaker under continuous stirring; the beaker was cooled in an ice bath in order to keep temperature at 50°C. After the alloy had been added (x30 min), the mixture was allowed to stir for another 30 min. After cooling, the mixture was transferred into an Erlenmeyer flask, and the supernatant removed with a pipette. The Raney nickel was washed with DzO (x 150 mL) and finally with absolute EtOD. The washings were performed stepwise with small volumes of D20 and EtOD, followed by careful decantation of the solution. The final 20 mL of EtOD were retained; the mixture was stored under nitrogen in the freezer. RESULTS+

Separation and Bulk Characterization of a Novel Nonpolar Macromolecular Fraction Occurring in Crude Oils and Rock Extracts The crude oils, asphalt and rock extract, all sulfur-rich (see Table 1 ), were separated over silica gel, as described in the experimental section. After elution of the saturated and aromatic fractions with hexane, a non-polar fraction (which could usually be followed by its reddish color) was obtained by elution with a mixture of hexane:ether (98:2). This fraction, which represents the least polar part of the so-called resins, is hexane-soluble and represents a substantial proportion (7-32%) of the starting petroleum or rock extract. The first indication of the essentially macromolecular nature of this fraction came from its analyses by GC, which

’ See the Appendix for structures

cited within the text.

% S in NPMF

6.5

silica gel. The branched and cyclic alkanes were obtained from the total alkanes by adduction of the linear alkanes with 5 A molecular sieves (O’CONNOR et al., 1962). Desulfurization

3397

in S-rich petroleum

n.d. = not detenoinated

showed no discrete peaks up to 300°C. A complex unresolved mixture, presumably formed by the thermal breakdown of the fraction, started to appear at 260°C. Likewise, no significant molecular ion peaks were obtained by El, Cl, FL, FAB, or plasma desorption MS. Microanalysis of the NPMF of Rozel Point seep oil showed the following composition: 74.O%C, 10.3%H, and 14.9%S, from which it could be deduced that there are thirteen carbon atoms per sulfur atom and three degrees of unsaturation on average in the related hydrocarbon moiety. The non-polar molecular fraction of the more mature Maruejols crude oil had the following composition: 78.3%C, 10.3%H, and 11.6%S, from which it could be deduced that there are eighteen carbon atoms per sulfur atom and five degrees of unsaturation on average in the related hydrocarbon moiety. ‘H-NMR studies of Rozel Point and Maruejols NPMFs confirmed their highly aliphatic character and showed small proportions of aromatic protons of 2.5 and 4.0%, respectively. A broad signal centered around 2.7 ppm corresponded to protons on carbons vicinal to sulfur in aliphatic sulfides. A higher proportion of aromatic bound hydrogens ( 10%) was observed in the case of Ponte Dirillo crude oil. Molecular weight determinations were made on NPMFs of Rozel Point and Maruejols crude oils; they were based on three independant measurements by vapor pressure osmometry, light scattering, and size-exclusion chromatography. They confirmed the macromolecular nature of NPMFs. The number average molecular weight, M,, obtained by vapor pressure osmometry, is sensitive to the lighter components of the macromolecular fraction; whereas A4,, the weight average molecular weight obtained by light scattering, responds more to the higher molecular weight part of the fraction (Table 2). Size exclusion chromatography reflects the internal distribution of molecular weights within NPMFs (Fig. 1); in this case, it is not a quantitative measure of the molecular weight because it is both sensitive to molecular S:ze and shape and commonly

P. Adam et al.

3398 Table 2:

Determination of molecular weights of Non-Polar Macromolecular Fraction in the case of Rozel Point and Maruejols crude oils.

VP0

LS

Point oil, as corroborated sion index.

by the values of the polydisper-

Hydrogenolysis of Non-polar Macromolecular with Raney Nickel

SEC

Mll

Mw

Mp

Ma’

Mw*

I

815

3400

1300

795

1660

2.09

580

4100

1610

871

2210

2.54

VPO: Vapor Pressure Osmometry Light Scattering LS: SEC: Size Exclusion Chromatography Number average molecular weight M,: Weight average molecular weight M,: Mp: Peak in mass distribution from SEC M,‘: Number average molecular weight from SEC M,‘: Weight average molecular weight from SEC Polydispersion index calculated on halfpeak width I: in SEC chromatogram

Fractions

Hydrogenolysis of NPMFs was carried out with Raney nickel, which is known to cleave C-S bonds in aliphatic and aromatic sulfides ( PETTIT and VAN TAMELEN, 1962). This procedure led to varying amounts of saturated hydrocarbons. Except for two Monterey samples, the yields were high, culminating for Rozel Point and Maruejols oils at values above 50% (Table 1). If one makes the correction for the amount of sulfur in the original macromolecules, this means that nearly 60% of the carbon of the NPMFs of both oils were transformed into low molecular weight alkanes in this hydrogenolysis procedure. Rozel Point seep oil The distribution of the alkanes obtained by degradation of Rozel Point NPMF is shown in Fig. 2, along with the free alkanes of the oil (the latter represent 2% of the crude oil).

standardized by linear polymers (polystyrenes; see SPHGHT et al., 1985). Values of Mp and M, obtained by size exclusion chromatography suggested that the NPMF was probably composed of more globular shaped molecules. As can be seen in Table 2, a relatively good agreement was obtained for the two samples, indicating a molecular weight range varying approximately between M, and h4, values of 600 to 4000, respectively. The NPMF of Maruejols oil apparently contained more high molecular weight components and a wider range of molecular weights than Rozel

100

I-

1

5

log M

FIG. 1. Size exclusion chromatograms of NPMFs from Maruejols (A) and Rozel Point (B) crude oils. See the experimental section for conditions. Both fractions contain a small proportion of low molecular weight components.

150

200

250

300

‘C

FIG. 2. Gas chromatograms of total alkanes of Rozel Point crude oil (a) and of total alkanes obtained by Raney nickel desulfurization of the NPMF of Rozel Point crude oil (b). Conditions: SE54, 30 m X 0.3 mm X 0.25 firn; 40-IOO”C, IO”C/min: IOO-3OO”C, 4’C/ min; 20 min isothermal at 300°C. Czz, C26, etc.: n-alkanes. Ph: phytane. Car: fi-carotane.

Nonpolar

macromolecular

Both fractions bear many similarities which probably suggest an origin from the same biological precursors. The main features are a predominance of steranes and phytane and an even predominance of n-alkanes. However, a few substantial differences could be observed. In the alkanes formed upon hydrogenolysis of Rozel Point NPMF, there is a higher proportion of n-alkanes. Their high even carbon number predominance is often typical of evaporitic carbonate environments ( WELTE and WAPLES, 1973; ALBAIG& and TORRADAS, 1974; DEMBICKI et al., 1976; CONNAN et al., 1986) and confirms the immature character of Rozel Point seep oil, which is further reflected in the distribution of the steranes and of the hopanes, as will be discussed below. Another difference appears at higher carbon numbers where the alkanes released by hydrogenolysis are enriched in /3-carotane, as well as in a series of linear alkanes maximizing at C3,. The latter are probably derived from long-chain unsaturated aliphatic components (in particular, ketones) which appear frequently in substantial amounts in certain planktonic Prymnesiophyceue algae (DE LEEUW et al., 1980; VOLKMAN et al., 1980; BRASSELLet al., 1986b). Furthermore, the highly branched CZO isoprenoid alkane (compound X; see YON et al., 1982), which is one of the major components

fraction

3399

in S-rich petroleum

//(a’

100

150

200

250

300

“C

1(b)

Hopanes

,230 .

a)

I

.

,

RI/Z.191

a!3 Hopanes

0 Gammacerane 100

150

200

250

300

“C

FIG. 4. Gas chromatograms of total alkanes (a) and branched and cyclic alkanes (b) of Maruejols crude oil. Conditions: DB5, 30 m X 0.25 mm X 0.1 pm; 40-LOO”C, lO”C/min; IOO-3OO”C,4”C/ min; 20 min isothermal at 300°C. C,,, C20, etc.: n-alkanes. Ph: phytane.

250

260

270

290°C

280

1

(W flvz=191

C35

h&v 250

260

270

280

of the free alkanes, could barely be detected in the S-bound alkanes. On the other hand, squalane is significant in the latter but not in the free alkanes. The distributions of steranes are very similar to each other in both alkane fractions. The strong predominance of 20R ~cIH, 14aH, 17cuH-steranes (C27-C29) confirms the unusual immaturity of Rozel Point seep oil. Their study is reported in detail by ADAM et al. ( 1992). The triterpanes of the free alkanes are dominated by the C3,, and Cx5 17aH,2lflH-hopanes and by gammacerane, a compound which frequently occurs in hypersaline deposits ( MOLDOWAN et al., 1985)) as can be noticed in the m/z = 19 1 fragmentogram (Fig. 3). The triterpanes newly formed upon hydrogenolysis show a different distribution in which gammacerane is the dominant peak, the C3,, hopane being substantially smaller. The C3s 17aH,2 l@H-hopanes in the hydrogenolysis products are dominated by the 22R isomer, contrary to the free alkanes, a point which will be discussed in the text to follow.

290°C

m/z = 19 1 showing the distribution of pentacyclic triterpanes in Rozel Point crude oil (a) and in Raney nickel degradation products of the NPMF of Rozel Point crude oil

FIG. 3. Mass fragmentograms

(b). Conditions: Finnigan MAT INCOS 50; EI, 70eV, CC-MS. See experimental section for detailed conditions.

Maruejols crude oil The distribution of the free alkanes and those formed upon hydrogenolysis of Maruejols crude oil NPMFs are shown in Figs. 4 and 5. In this case, the two fractions display great

P. Adam et al.

3400 (a) 1

ii

150

ZOO

250

300

“C

LYC

( (b)

I

150

too

250

3bo

“C

FIG. 5. Gas chromatograms of total alkanes (a) and branched and cyclic alkanes (b) obtained by Raney nickel desulfurization of the NPMF from Maruejols crude oil. Conditions are identical to those listed in Fig. 2. CZZ,Cj4, etc.: n-alkanes. iC,: regular isoprenoid. Pr: pristane. Ph: phytane. Sq: squalane. Lye: lycopane. Hop: hopane. Car: B-carotane.

differences, contrary to Rozel Point crude oil. Although both fractions are strongly dominated by normal alkanes, the distribution maximizes in the lower carbon number range (around C,5) in the case of the free alkanes, whereas it is highly enriched in high molecular weight n-alkanes in the hydrogenolysis products (maximum around &). Predominance of even carbon numbered n-alkanes is more pronounced in the free alkanes ( C2&34) than in those obtained by hydrogenolysis. The branched and cyclic alkanes, obtained by selective inclusion of the linear components into 5A molecular sieves, are totally different in the free and in the S-bound alkanes. In the free alkanes, the distribution is frequently encountered in oils from source rocks deposited in evaporitic environments: acyclic isoprenoids as well as series ofbranched alkanes dominate at lower carbon numbers; steranes ( C27-C29) and hopanes ( C27-C35, with C35 predominant among the extended hopanes) in the higher molecular weight range. Equilibrium is apparently reached at C-20 in steranes and C-22 in hopanes. In the S-bound branched and cyclic alkanes, steranes can barely be detected, whereas hopanes appear in small amounts and are dominated by the Cj5 17aH,2 1@H homologs (22 R diastereoisomer predominant as in Rozel Point oil). With the exception of these hopanes, of /3-carotane and of Czj

isualkane (a compound frequently encountered in evaporitic series; see CONNAN et al., 1986; HUSSLER, 1985 ), the distribution of the branched and cyclic alkanes formed upon hydrogenolysis is almost exclusively composed of long-chain acyclic isoprenoid hydrocarbons ranging from Cl8 to Cd5. The occurrence of the regular CZs isoprenoid alkane (typical of evaporitic series; e.g., WAPLES et al., 1974) and of squalane ( RISATTI et al., 1984) could be interpreted in terms of their origin from archaebacteria (probably methanogenic bacteria, although a contribution from halophiles cannot be excluded; see DE ROSA et al., 1982). The major peak of the chromatogram has been identified as lycopane. the acyclic C, carotane, which has only rarely been found as a free alkane in geological samples (e.g., Messel oil shale, KIMBLE et al., 1974; Cariaco Trench recent sediments, ALBRECHT et al., 1983; and surface sediments off Peru, MCCAFFREY et al., 1991; but never in a crude oil). The source of this compound is not yet clear. Recently, METZGER and CASADEVALL ( 1987) showed the existence of a lycopadiene in a Botryococcus braunii race. In our case, it is more probable that lycopane derives from acyclic carotenoids typical of photosynthetic prokaryots, such as purple sulfur or non-sulfur bacteria (LIAAEN-JENSEN, 1976, 1979). The regular Ch5 acyclic isoprenoid alkane ( nonaprenane), which also occurs in substantial amount in the branched and cyclic S-bound alkanes, has been identified by comparison with a standard (mixture of diastereoisomers) obtained by hydrogenation of solanesol, a common higher plant isoprenoid alcohol ( JSARRER et al., 1976; see the experimental section here). Fig. 6 shows the mass spectra of both the geological and the reference compound, which are almost identical. Identification was confirmed by coelution of both compounds in high resolution GC (DB5, 30 m X 0.25 mm X 0.1 hm). The major peak eluting after the Cd5 nonaprenane was attributed tentatively to a regular Cd7 isoprenoid alkane. These compounds probably derive from regular Cbj, C5,,, and Css polyprenols which have been reported from various sources (higher plants, microorganisms: see discussion to follow). Ponte Dirillo crude oil The Ponte Dirillo free alkanes (Fig. 7) show some analogies with those of Rozel Point seep oil. They are indeed relatively poor in n-alkanes and dominated by phytane. However, the high molecular weight range is dominated by hopanes instead of steranes. Contrary to Rozel Point oil, the n-alkanes display a slight odd carbon number predominance eventually reflecting a higher plant influence. The distributions of hopanes ( C27-C35 ) and steranes ( C27-C29 ) correspond to a rather mature situation. Both classes of compounds were indeed at equilibrium at C-22 and C-20, respectively. The distribution of hopanes culminating at Cj5 and the predominance of phytane were again typical of organic matter from an evaporitic environment. Also characteristic is the occurrence of a small amount of @-carotane. A completely different picture was obtained in the alkanes formed upon hydrogenolysis ( 16% of NPMF; Fig. 8a). The n-alkanes show a pronounced odd carbon number predominance at C1,, Czs, and C3, ; their overall importance in the saturated hydrocarbon fraction is relatively low. Major com-

3401

Nonpolar macromolecular fraction in S-rich petroleum

6

462 476

500

600

(b) Synthetic 2,6,10,t4,18,22,26,30,34-nonamethylhexatriacontane

462 4,76

FIG. 6. Mass spectrum (EI, 70 eV) of regular C,, isoprenoid (i&) obtained by Raney nickel desulfurization of the NPMF from Maruejols crude oil (a) and of the synthesized product (b). pounds are clearly the long-chain isoprenoid hydrocarbons, in particular, at Cd0 and above. A more detailed study of the branched and cyclic alkane fraction (Fig. 8b) again revealed a complete series of regular isoprenoid alkanes ranging from

Cl8 to the. regular Ca5 nonaprenane already described in the case of Maruejols crude oil. Lycopane. p-carotane, and squalane are again significant peaks. The major peak, however, from its mass spectrum appears to be a CeOmonocyclic

P. Adam et al.

3402

Ph

Hopanes -c Steranes ) cao HOP

____. 100

200

300

~-. “C

A

FIG. 7. Gas chromatogram of total alkanes of Ponte Dirillo crude oil. Conditions are identical to those listed in Fig. 2. For abbreviations, see Fig. 5.

isoprenoid hydrocarbon (M+ = 560) related to the carotcnoids; further studies are required for its full identification. It is interesting to note that monocyclic carotenoids are abundant in photosynthetic bacteria (e.g., Chlorofexus) and yeasts ( BROCKand MADIGAN, 1988 ).

The three crude oil samples from Monterey which were analyzed for their NPMFs were different in their behaviour towards Raney nickel hydrogenolysis. Althou~ the amounts of NPMF were comparable, the yields of alkanes obtained by desuIfu~zation showed great differences. As the Monterey source rocks have been deposited under varying conditions ( ORR, 1986; ISAAC& 1983 ), these differences may be due to changes in palaeoenvironmental conditions, in maturity, or in secondary alteration (e.g., biodegradation). Some of these differences are reflected in the amount and composition of the free alkanes of the three samples (DE LEMOSSCOFIELD, 1990). Indeed, the presumably most mature sample, 1547, contains, as expected, the highest percentage of alkanes ( 15.7%) as compared to sample 1.509(5.7%) and biodegraded sample 6-4 (5.1%). A comparison of their respective distributions (Fig. 9) shows that n-alkanes are relatively weak in the less mature sample, 1509, and display a rather narrow range with a maximum around Ci4. In sample 1547, they cover the complete carbon number range (CIz-C38) with a maximum around Cl6 and a slight even carbon number predominance between Cl6 and C24, whereas they are absent due to selective biodegradation in sample 6-4. Phytane appears as the major peak of the alkanes in sample 1509; it is

also the largest isoprenoid alkane in sample I547 (but less

predominant over pristane) and absent in asphalt 6-4 due to biodegradation. Biphytane, which is probably derived from polar lipidic membrane constituents occurring in methanogenie archaebacteria ( CHAPPEet al., 1982)) appears as a major peak in both samples 1509 and 1547 but not in asphalt 6-4, perhaps as a result of the bi~e~adation process undergone by this sample. The three samples display very similar m/z = 191 fragmentograms dominated by a series of 17cuH,21/3H-hopanes ranging from CZ7to CX5(equilib~um is reached at C-22 in the higher homologs). All samples contain a series of shortchain (CZ,-CZ,) and long-chain steranes (basically C27-C29). A detailed analysis of the m/z = 2 17 fragmentograms confirmed that oil sample 1509 was less mature than sample 1547 (20R diastereoisomers more predominant; 5olH, 14PH, 17PH-steranes weakerascompared to their 5aH, 14aH, 17nH counterparts in sample 1509). In asphalt 6-4, the sterane pattern has been altered drastically due to the biodegradation process; short-chain steranes appear unchanged, whereas in the long-chain constituents, only some diasteranes have survived the process, which is in agreement with the most generally accepted biodegradation scheme of this class of compounds (DE LEMOSSCOFIELD, 1990; SEIFERT and MOLDOWAN, 1979). The distributions of the alkanes obtained upon Raney nickel hydrogenolysis of NPMF of the three samples were again totally different from those of the free alkanes (Fig. IO). The S-bound alkanes of the three samples showed great similarities, except for the n-alkanes, which were much weaker in biodegraded asphalt 6-4, implying that the bio-

3403

Nonpolar macromolecular fraction in S-rich petroleum

‘b)

Ii0

200

zio

300

*c

FIG. 8. Gas chromatograms of total alkanes (a) and of branched and cyclic alkanes (b) obtained by Raney nickel desulfurization of the NPMF from Ponte Dirillo crude oil, Conditions: SES4, 30 m X 0.3 mm X 0.25 pm: 50-i WC, ~~*C/min; f50-3#*C, 4”Cimin: 20 min isothermal at 3W.K’. n: n-alkane. 0: Ca monocylic car&me. For abbreviations, see Fig. 5. de~adatjau processmust have affected the macrorn~iecu~~r

fraction as well, a hypothesis which we are now checking. The branched and cyclic S-bound alkanes were quite similar for the three samples and, furthermore, showed many analogies with those from Ma~ejols and Ponte Dirillo crude oils. Acyclic isoprenoid hydrocarbons were again the major constituents. Phytane, squalane, lycopane, and especially the regular Cqgisoprenoid component were clearly the dominant

peaks. @-carotane also occurred as a s~gnj~cant compound. Hopanes and steranes could not he detected in significant amounts. Sir&e CXi(f ~~~y~ri~~ The NPMF of the Sake Gkile anhydrite sample (20.7% of total rock extract) delivered a fairly good yield of saturated

P. Adam et al.

3404

Ph

I

II

I

loo

I

l

200

300

- Biphytane

“C

FIG. 9. Gas chromatograms of total alkanes of three Monterey crude oils. Conditions: DBS, 30 X 0.25 pm; 40-1OO”C, lO’C/min; lOO-315”C,4”C/min; 25 min isothermal at 315°C.

hydrocarbons upon Raney nickel desulfurization ( 26.0% ) . Distribution of the free alkanes (6.5% of total extract) confirmed the immatu~ty of the organic matter (Fig. 11). The n-alkanes indeed showed a strong even carbon number preference ( C16-C32) frequently observed in evaporitic deposits. Further chamcte~stics of such deposits appeared in the strong predominance of phytane, in the presence of a series of I7cuH,2 1PH-hopanes dominated by the C35homologs (equilibrium reached at C-22), as well as in the occurrence of gammacerane. The CZs acyclic isoprenoid alkane was again the regular isomer (as shown by its mass spectrum) and was present in significant amount, suggesting a probable contribution from methanogenic or halophilic archaebacteria. The m/z = 2 17 fragmentogram showed that steranes (essentially C&&) were dominated by the CZ9 homologs; clear predominance of the 20R diastereoisomers confirmed the low maturity of the organic matter of this rock. The distribution of the alkanes obtained by Raney nickel hydrogenolysis of the NPMF of this sample (Fig. 12 ) was

m X 0.35 mm

clearly different from that of the free aikanes. Indeed, it was dominated by a series of n-alkanes ranging from Cl1 to over CW and displaying an even carbon number predominance between Ct6 and C,, , as well as between C,, and C32(it was, however, generally less pronounced than in the free alkanes). Phytane, steranes, and the CS5 hopanes were again present as major components; but other products occurred. in addition, as significant constituents. This was, in particular, the case of the CZ3isoalkane which had been observed previously as a major constituent of evaporitic series from Guatemala (CONNAN et al., 1986; HUSSLER, 1985). A yet unidentified Cz4acyclic component eluted slightly after the CZ3isoalkane. Besides squalane, major peaks appeared in the high molecular weight range: P-carotane, accompanied by a yet unidentified monounsaturated counterpart, as well as the regular acyclic Cd5nonaprenane previously observed in all the other analyzed samples, except Rozel Point seep oil. The steranes were also dominated by the CZs homologs: they showed a higher predominance of the 5aH, 14~uH,17aH

3405

Nonpolar macromolecular fraction in S-rich petroleum

Ph

ic38

e3

ic35

iCz8

iC25

I

I I 1

Ph

150

200

250

300

“C

FIG. 10.

Gas chromatograms of total alkanes from Raney nickel desulfurization of NPMF from three Monterey crude oils. Conditions arc identical to those listed in Fig. 2. For abbreviations, see Fig. 5.

series (a~) over their a@@equivalents as compared to the steranes of the free alkanes; the 20R N(YLY steranes were Iikewise the major diastereoisomers. The m/z = 191 fragmentogram showed the occurrence of a series of 17otH,2 1/3Hhopanes largely dominated by the C35 homoiogs which were not at equilibrium at C-22. Gammacerane further appeared as a major component; it was only observed previously as a hydrogenolysis product from the NPMF of Rozel Point seep oil. Desulfurization of the Nonpolar Macromolecular Fraction of Rozel Point Seep 03 with Deuterated Raney Nickel Deuterated Raney nickel (see experimental section) was utilized for the reductive cleavage of C-S bonds in NPMF of Rozel Point seep oil in order to obtain information on the number of C-S linkages by which the hydrocarbon subunits

were attached to the macromolecular network. In general, the use of the deuterated catalyst led to a smaller recovery (about 10% lower) of low molecular weight alkanes as compared to the non-deutcrated reagent. A comparison of the distributions of the alkanes obtained upon degradation of the NPMF of Rozel Point seep oil with RaNi (H) and RaNi (D) is shown in Fig. 13. It cleady indicates a selectivity in the deuteration reaction since steranes and n-alkanes appear more predominant in the alkanes obtained with RaNi (D) as compared to long-chain isoprenoid alkanes (phytane, squalane) or p-carotane. This could be due to the fact that the activity of RaNi (D) is lower than that of RaNi (H) with the consequence that probably only less tightly bound or less hindered molecules are liberated from the macromolecules with the deuterated catalyst. The results would in this case imply that the long-chain isoprenoids and &carotane are bound more tightly to the macromolecular

P. Adam et al.

3406 Ph

100

100

200

200

300

300

‘C

*C

FIG. I 1. Gas chromatograms of total alkanes (a) and branched and cyclic alkanes (b) of Sainte C&cilerock extract. Conditions are identical to those listed in Fig. 4.

network than the n-alkanes and especially the steranes (for the latter, this is obvious from the results of deuterium incorporation described in the text to follow). It is, however, also possible that polydeuteration of the isoprenoid alkanes has widened their peaks to such an extent that they appear smaller in the gas chromatogram. A similar broadening effect of polydeuterated hydrocarbons in gas chromatographic analyses has been described previously ( HOERING, 1984). In this respect, a careful comparison of both chromatograms clearly shows a widening of the n-alkane peaks in the deuterated fraction as compared to the nondeuterated one. The distribution of steranes was very similar in both cases, as shown by GC-MS studies. The latter further indicated that the steranes had essentially incorporated one deuterium atom on ring A or B. A more complete study comprising other selective degradations (LiAlHJ and synthesis confirmed this conclusion and further demonstrated that the steroid skeletons were attached to the macromolecules at position 2cr or 3p (ADAM et al., 1991, 1992). It should be noticed that deuteration was found not to be completely effective. A careful examination ofthe mass spectra of the steranes, which were essentially monodeuterated, indicated a l/ 1 mixture of deuterated and nondeuterated

compounds (the percentage of deuteration is even lower in the case of gammacerane; see the following text). The results could be explained by an incomplete deuteration of the catalyst, along with the higher reactivity of hydrogen vs. deuterium. In any case, it will complicate the interpretation of polydeuterated species. The m/z = 191 fragmentogram confirmed the presence of small amounts of hopanes ( Cl5 homologs in particular f, and of gammacerane. A detailed study of the mass spectra (in particular of ions M ‘, M +- 15, and 19 1) in the deuterated and non-deuterated alkane fraction showed that gammacerane had incorporated only one deuterium, implying one point of attachment to the macromolecular network (Fig. 14). By analogy with the steroids (ADAM et al., 1991, 1992), the gammacerane skeleton may also be attached Eo the macromolecular matrix at position 2 or 3, by reaction between its A* counterpart and active sulfur species (Fig. 15a; see discussion to follow). The situation was less clear in the case of the Cs5 hopanes where several deute~um atoms appeared to be incorporated exclusively into the side chain, as can be seen from the fragment ion at W/Z = 369 (Fig. 16). This probably implies several points of attachment ofthe side chain to the macromolecular network via C-S bonds (Fig. 15b). The n-alkanes and p-carotane were found to be polydeuterated (incorporation of up to SD for n-Clo), reflecting several points of attachment to the macromolecular network. A clear inte~retation of polydeute~~ion (>2D) is, however, difficult because our approach cannot distinguish between cleavage of intramolecular and intermolecular C-S bonds. It would, for example, in the case of 3D incorporation, not be possible to distinguish between a mixture of cyclic sulfides (thiolanes or thianes) attached to the macromolecular network via one C-S linkage and a mixture of linear sulfides attached via three C-S bonds. This situation may be complicated even further in the case of alkylthiophenes since Raney nickel reduction of a thiophene will by itself introduce 6D, this class of compounds is, however, not present in great amounts in Rozel Point NPMF as can be judged from the NMR data. A more detailed interpretation of polydeuteration would necessitate a more refined approach implying various selective chemical transformations. DISCUSSION General Structural Aspects of Non-polar Macromolecular Fractions

The results of the bulk characterization and of Raney Nickel hydrogenolysis of NPMFs show that this fraction is essentially macromolecular and composed mainly of low molecular weight hydrocarbon entities cross-linked with sulfur in the form of monosulfide or polysulfide bridges. Hydrocarbon subunits are mostly aliphatic, especially in the case of Rozel Point and Maruejols crude oils. A detailed study of the mode of attachment of the sterane skeletons to the macromolecular matrix showed that a small proportion of these structures was attached at one single position (2n or 3@)via disulfide or polysulfide bridges but that the main part was probably bound by monosulfide linkages (ADAM et al., 199 1,

3407

Nonpolar macromolecular fraction in S-rich petroleum

n (4

(b)

Ph C24 alkane steranes

Car I

iC45

I_ loo

L 200

300

"C

FIG. 12. Gas chromatograms of total alkanes (a) and branched and cyclic alkanes (b) obtained by Raney nickel desulfurization of the NPMF of Sainte Ctcile rock extract. Conditions for (a) are identical to those listed in Fig. 4; for (b), they are identical to those listed in Fig. 9. For abbreviations, see Fig. 5.

1992). Long-chain linear or isoprenoid and some polycyclic subunits may be attached to the matrix at several positions. Non-polar macromolecular fractions are complex mixtures of macromolecules varying in molecular weights from about 600 to over 4000 mass units. Incorporation of sulfur is likely to occur by reaction between inorganic sulfur species formed by microbial activity and functionalized molecules, such as alkenes or polyenes, a process which might be complicated further by the superposing of hydrogen transfer reactions (see the following text). The variety of hydrocarbon subunits and of the positions of the functionalities (e.g., double bonds) probably gives rise to such a great number of cross-linking possibilities that almost all the molecules in NPMFs are likely to be different from each other. This fact would explain the lack of any significant peaks in MS nor at high-temperature in GC. The proportion of NPMFs is likely to decrease in crude oils with increasing maturity, as suggested by the results of laboratory thermal treatment. The amount of the NPMF of West Rozel, a crude oil related to Rozel Point seep oil, indeed diminished by half after two weeks at 300°C ( SCHMID, 1986).

The nature of the aliphatic hydrocarbon subunits of NPMFs varies depending on the crude oil. Except for Rozel Point oil, the alkanes formed by hydrogenolysis with Raney nickel display a very different distribution as compared to the free alkanes of the crude oil or of the rock extract, which implies that their functionalized precursors must have been selectively trapped into the sulfur cross-linked macromolecules at some stage during sedimentation. This is especially true for the branched and cyclic alkanes, which are dominated mostly by long-chain isoprenoid hydrocarbons (e.g., phytane, regular CZ5 isoprenoid alkane, squalane, C45 regular isoprenoid alkane) and carotenoid-type C4a hydrocarbons (e.g., lycopane, /3-carotane, monocyclic carotane). The branched and cyclic free alkanes, on the other hand, display a classical distribution which is typical of evaporitic or other anoxic deposits: dominance of phytane, non-rearranged steranes, and hopanes. Except for Rozel Point crude oil and Sainte CCcile rock extract, steranes, and hopanes are usually small in the alkanes obtained by Raney nickel desulfurization. In Rozel Point oil, however, the steranes are the major components of the free and of the S-bound alkanes, whereas the hopanes

P. Adam et al.

3408

h

150

200

250

300

"C

FIG. 13. Gas chromatograms of total alkanes obtained by Raney nickel desulfurization (a) and by deuterated Raney Nickel desulfurization (b) of the NPMF from Rozel Point crude oil. Conditions are identical to those listed in Fig. 8.

are subordinate. In the Sainte Cecile rock extract, hopanes and steranes occur in about equal proportions. Gammacerane is also a significant component of the NPMF alkanes of both samples. The n-alkane chains are important subunits of several NPMFs, in particular of Maruejols crude oil and of Sainte CCcile rock extract. In the other samples, they are usually less important in comparison with the branched and cyclic alkanes. Even carbon number predominance appears in the NPMF n-alkanes of Rozel Point, Maruejols, and Sainte Cecile samples; whereas Ponte Dirillo and the Monterey crude oils rather show an odd carbon number predominance. Although in varying proportions, the same features appear in the free alkanes of these samples. Based on the considerations mentioned in the preceding text, Figure 17 shows the structural variety of molecules typically occurring in NPMFs. These molecules eventually appear to be composed of long-chain linear and isoprenoid (mostly regular polyprenane or carotenoid-type) alkane sub-

units cross-linked by monosulfide bridges and a rather small percentage of disulfide or polysulfide bonds (e.g., about 2 to 3% for Rozel Point crude oil; ADAM et al., 1992). The linear and isoprenoid chains may vary considerably in relative proportions. Steroid and terpane skeletons may be attached to the macromolecular network in variable but usually rather small proportions, except for Rozel Point oil, where steroids represent about one-third of the hydrocarbon framework of NPMF macromolecules. The long straight and isoprenoid chains are likely to be attached to the network at several positions. In the Rozel Point NPMF, steroids are attached at one single position on ring A; in Sainte Cecile, however, further points of attachment could exist on the side-chain as inferred from the occurrence of unusual structural isomers in the hydrogenolysis products. Gammacerane is monoattached in the Rozel Point oil NPMF, whereas the CXshopanes appear to be multiattached via the side-chain. Aromatic structures may occur in NPMFs, either as discrete subunits or as intramolecular moieties (e.g., thiophenes) of long chain

3409

Nonpolar macromolecular fraction in S-rich petroleum .M

l-

‘“T

(a)

91

I

412 398 I,$ I” I” “I”‘/-“’

(b)

1

91 I

FIG. 14. Mass spectrum of gammacerane obtained by deuterated Raney nickel desulfurization (a) and by Raney nickel desulfurization (b) of the NPMF from Rozel Point crude oil. Conditions: KRATOS MS80, EI, 70eV, GC-MS.

or cyclic skeletons. From the NMR data, they appear to be rather small in the NPMFs of Rozel Point and Maruejols oils, but significantly higher in that of Ponte Dirillo oil. They have not been represented on the schematic drawing since their structures and mode of linkage to the macromolecular matrix are not yet elucidated. Preliminary results from selective chemical degradation indicate, however, the occur-

rence of small amounts of thiophenes Point NPMF (ADAM, 199 1) . Differences Alkanes

in the case of the Rozel

in Maturity Parameters of Free and S-bound

Differences appear in free and S-bound alkane fractions in various maturity parameters. Indeed, in Maruejols crude

P. Adam et al.

3410

(b)

FIG. 15. Possible modes of attachment of gammacerane skeleton (a) and of Cu hopane skeleton (b) to maeromoiecular matrix of NPMF (C-S linkages attached to positions Czz, C29-C35in the case of hopane).

oil, for example, the even carbon number predominance is higher (C22-C34) in free n-alkanes than in the NPMF n-alkanes. In Ponte Dirillo, on the other hand, odd carbon number preference appears more strongly in the NPMF alkanes. In the Monterey samples, strong odd carbon number n-alkane predominance appears in the NPMF alkanes; whereas in Sainte CCcile NPMF alkanes, it is the other way around. In summary, aithough the n-alkanes obtained from NPMF are usually different in distribution from the free n-alkanes, no clear trend can be observed in the variation of carbon number preference. In Rozel Point oil, the steranes from the NPMF alkanes display approximately the same immature distribution as in

the free alkanes. In Sainte Cecile, the only other sample containing significant amounts of steranes in NPMF alkanes, however, the dis~~bution in the latter appeared slightly more immature. Hopanes generally appeared more immature in NPMF alkanes than in free alkanes (Rozel Point crude oil, Maruejols, Sainte kile). Two main explanations could account for these differences in isomer ratios between biomarkers occurring in the free alkanes and those obtained from hydrogenolysis of macromolecular species, a feature which has been observed previously (SEIFERT, 1978; RUB-

INSTEINet al.. 1979; TRIFILIEFF et al., 1992). First, it is possible that there is a protecting efYectdue to steric hindrance in macromolecular

species,

thus preventing,

for example,

Nonpolar macromolecular fraction in S-rich petroleum

a)

191

I

,9t

I

80.

60.

j

40.

II 207

261

0

FIG. 16. Mass spectrum of C,, hopane obtained by deuterated Rauey nickel desulfurisation (a) and by Raney nickel desu~fu~~t~ou f b) of the NPMF from Rozet Point crude oil. Conditions: KRATOS MS80, EI, 7OeV, GC-MS.

3411

P. Adam et al.

3412

-

n-alkyl chains

or polyisoprenoid

FIG. 17. Schematic representation of macromolecules occurring in NPMFs as deduced from Raney nickel and lithium aluminium hydride hydrogenolysis products (at least 2% of disulfide or polysulfide bridges in the case of the Rozel Point seep oil).

catalysis to be operative at certain sites. The second, more probable explanation lies in variations in the relative stabilities of the stereoisomers at C-20 in steranes or C-22 in hopanes. This would be particularly true when these centers are involved in a sulfur-containing cycle or are located near a C-S linkage, which is generally the case of hopanoids, and could hold, in particular, for steranes of the Sainte-Cecile NPMF which seem to contain sulfur in the side chain (ADAM, 1991). mineral

Origin of the Hydrocarbon Subunits of Nonpolar Macromolecular Fractions and Geochemical Implications

A striking feature in the composition of the alkanes formed upon hydrogenolysis with Raney nickel of most NPMFs is the high proportion of long-chain regular isoprenoid hydrocarbons (Cl8 to above &), of squalane, and of carotanes (lycopane, @carotane, monocyclic carotane). The biological precursors of these compounds are polyenes (squalene, lycopene, and related acyclic carotenes; P-carotene; monocyclic carotenes) or polyprenols (e.g., C&& ). The polyunsaturated structures of these compounds, as well as of some of their diagenetic degradation products, could explain, to a large extent, their selective trapping by active sulfur species into the macromolecular network of the NPMF (see the text to follow). Several other types of structural subunits of the NPMF could equally derive from monounsaturated or polyunsaturated precursors occurring as such in living organisms or formed by defunctionalization (e.g., dehydration) of biological molecules during early diagenesis. This would, in particular, be the case of some long-chain linear monoalkenes or polyalkenes, of sterenes and of hopenes. Long-chain regular isoprenoid hydrocarbons are dominant components of the branched and cyclic alkanes from reductive cleavage of NPMFs of most studied samples. They culminate around Cq5, which appears as a major component in several samples. Their most likely precursors are long-chain regular polyprenols which are widespread lipidic constituents

of many living organisms (e.g., higher plants, microorganisms) where they commonly appear with carbon numbers up to Cloo (RIP et al., 1985). Bacteria are often enriched in C55 undecaprenols which play a key role in the transport of carbohydrate units to the cell wall of these organisms (HEMMING, 1973). Rather than deriving from higher plants, the contribution of which seems rather scarce in our sampling, it is more likely that the long-chain regular isoprenoids are the products of diagenetic transformations of higher polyprenols of bacterial origin, formed prior to their incorporation in macromolecular entities. Lower carbon number isoprenoid subunits of NPMFs could have a more specific source. Indeed, predominance of phytane and of the Cz5 regular isoprenoid may be related to methanogenic or halophilic archaebacteria ( LANGWORTHY et al., 1982; DE ROSA et al., 1982). Squalane may likewise be related to these types of organisms ( LANGWORTHYet al., 1982; KATE& 1978), although the widespread occurrence of squalene in living organisms makes precise source assignments rather delicate. In the Monterey samples, which come from sediments deposited under upwelling conditions, but also in the Sainte CCcile evaporitic anhydrite, a more precise relationship with a methanogenic source can be established since biphytane derived from typical methanogenic lipidic membrane constituents is a significant component ofthe free alkanes. Its near absence in the NPMF alkanes is consistent with its origin from fully saturated dibiphytanyltetraethers ( LANGWORTHYet al., 1982) which do not seem to be available for interaction with sulfur species and appear mostly in the more polar fractions of crude oils or rock extracts, as well as incorporated into kerogens ( CHAPPEet al., 1980, 1982). Carotenoid-type hydrocarbon subunits of NPMF (lycopane, /3-carotane, monocyclic carotane) may also give useful information concerning their source. Indeed, p-carotene and related compounds, the likely precursors of @-carotane in NPMFs, are widespread constituents of photosynthetic eukaryotes (e.g., algae) and also occur in substantial amounts in quite a few photosynthetic prokaryotes (e.g., green non-

Nonpolar macromolecular fraction in S-rich petroleum sulfur bacteria, cyanobacteria) . Lycopane, an important constituent of most alkane fractions from NPMFs, probably finds its origin in lycopene and related acyclic carotenoids which occur mainly in photosynthetic purple sulfur and nonsulfur bacteria ( BROCK and MADIGAN, 1988). An alternative source of this compound could be lycopadiene, which occurs as an important constituent of some algae ( METZGER and CASADEVALL, 1987 ). Monocyclic carotenoids occurring mainly in green non-sulfur bacteria (e.g., Chloroflexus; see LIAAEN-JENSEN,1976, 1979) could give rise to monocyclic carotane, which is the major single component from the Ponte Dirillo NPMF. Furthermore, it may be interesting to note that a Cd0 monoaromatic carotenoid has been tentatively identified (M+ = 554; m/z = 133; see the Appendix) in a fraction slightly more polar than the alkanes obtained by hydrogenolysis of the Rozel Point NPMF. Such a compound could originate from aromatic carotenoids typical of green sulfur bacteria ( BROCKand MADIGAN, 1988; SUMMONSand POWELL, 1987). The carotenoid content is quite rich in geochemical information concerning the source of organic matter of NPMFs. Some of these carotenoids (e.g., /?-carotane) could come from algae or cyanobacteria living in the photic oxic zone of the water column. However, the major part of the carotenoids would probably originate from anaerobic photosynthetic prokaryots, in particular, from sulfur bacteria (e.g., Chlorobacteriaceae, Chromatiaceae) which may be an important source of primary productivity in anoxic basins, as shown by REPETA et al. ( 1989). These bacteria play an active role in the sulfur cycle and may produce sulfur species reactive in cross-linking of hydrocarbon units (see the text to follow). n-alkane subunits appear in varying proportions of the NPMFs of the studied samples. They are major components in the alkanes from the NPMF of Maruejols crude oil and Sainte Cecile anhydrite, where they extend from around CIZ to over Cd0and display some even carbon number predominance. In Maruejols crude oil, they show a maximum around C&, but they show a bimodal distribution with maxima at Ct6 and Czs in Sainte Cecile anhydrite. They are less important in the other samples as compared to the branched and cyclic alkanes. In Rozel Point oil, they show a bimodal distribution with maxima at Cl8 and at C3,, as well as a high even carbon number predominance between Cl6 and Cz6. In the nonbiodegraded Monterey samples, the n-alkanes of the NPMF show a pronounced odd carbon number predominance with C2,, Czs, Cj, , and CXsbeing particularly dominant. In Ponte Dirillo crude oil, this is also the case with a predominance of the C2,, CZs, and Cj, n-alkanes. The source of most of these linear alkane subunits is not clearly understood. They might originate in part from longchain polyenes occurring in microscopic algae ( GELPI et al., 1968, 1970; YOUNGBLOODet al., 1971; MOLDOWANet al., 1985 ) ; compounds with shortened chains could come from degradation intermediates formed during early diagenetic processes. The odd carbon number predominance displayed by some of the samples could be due to some specific algal contribution or to a higher plant input, since long-chain alkenes (or precursor alcohols) occur widely in these living organisms (EGLINTON and HAMILTON, 1963; SORM et al., 1964). The maximum shown at CX7in the alkanes from the

3413

NPMF of Rozel Point could be consistent with an origin from certain Prymnesiophyceae algae which contain substantial amounts of long-chain polyunsaturated components (e.g., unsaturated ketones) in that carbon range (DE LEEUW et al., 1980; VOLKMAN et al., 1980), an observation also made by ROLLKOTTERand MICHAELIS( 1990) in the case of a Monterey oil. Except for the Sainte CCcile anhydrite, hopanes generally appear only in small amounts in the NPMF alkanes, whereas they are usually major components in the free alkanes of our samples. This could mean that hopanoids derive mostly from cyanobacteria or aerobic bacteria active in the reworking of organic matter in the oxic zone of the water column (OURISSONet al., 1979 ). Diagenetic processes would only partially transform the hopanoids into monounsaturated or polyunsaturated counterparts susceptible to react with inorganic sulfur species in the underlying anoxic zone. In the latter, indeed, many polyunsaturated components are selectively preserved from oxidation and may therefore be trapped by active sulfur species. Gammacerane is frequently found in the free alkanes of evaporitic samples ( MOLDOWANet al., 1985) where it is most likely derived from tetrahymanol (TEN HAVEN et al., 1989, VENKATESAN,1989) which finds its source in various microorganisms (protozoae and bacteria; see KLEEMANNet al., 1990; HARVEYand MCMANUS, 199 1). This compound has only been detected in significant amounts in Rozel Point oil and Sainte Cecile anhydrite, which shows that, like the hopanoids, tetrahymanol is probably only partially transformed into unsaturated counterparts able to interact with sulfur species in the anoxic zone. Steranes appear in fairly high amounts in Sainte CCcile and especially in the Rozel Point NPMF alkanes but are almost absent in the other samples. The precursor sterols are widespread constituents of planktonic algae which are one of the main sources of organic matter in our samples (DICKSONet al., 1979). Their high degree of preservation, in particular in Rozel Point, could be explained by the low degree of bacterial reworking undergone by massive inputs of algal material which would be rapidly transferred into the anoxic zone due to a shallow oxic/anoxic interface. This explanation is in agreement with the relatively low amounts of hopanes in Rozel Point oil, in comparison to the steranes. The sterols and their early diagenetic transformation products (e.g., sterenes; see DASTILLUNGand ALBRECHT, 1977; MACKENZIE et al., 1982) would then undergo further alteration comprising, in particular, incorporation into macromolecular entities due to cross-linking with sulfur species (see the following text )

Mode of Formation of Nonpolar Macromolecular

Fractions

The structures of the obvious biological precursors of many of the alkyl subunits occurring in the NPMFs give a clue to some aspects of its mode of formation. Indeed, most of these precursors appear as monounsaturated or polyunsaturated species either inherited as such from living organisms (e.g., carotenoids, long-chain polyprenols, long-chain linear polyenes) or formed by geochemical transformations of biological

3414

P. Adam et al.

molecules during early diagenetic processes (e.g., sterenes, hopenes, long-chain alkenes, de~adation products of the above). Many of these monoun~tumted and polyunsaturated species may be provided by rapidly sedimented algal or bacterial material coming from the photic zone. Most of our samples, except for those of Monterey formation, are from carbonate evaporitic sequences which are usually deposited under relatively shallow waters which are often anoxic except for the very upper layers. The Monterey samples have been deposited under upwelling conditions, probably under a much greater depth of water ( CWRIALEand ODERMATT, 1989, and references therein); however, here again, we have to deal with a stratified water column due to the existence of an oxygen-depleted zone. Whatever the case, rapid sedimentation of the organic matter coming from the surface below the oxic/anoxic boundary will preserve many unsaturated molecules from oxidative degradation, the degree of preservation becoming higher as the interface becomes more shaflow. This probation should even be more efficient for polyunsaturated molecules produced in the anoxic (photic) zone, in particular, by anaerobic photosynthetic bacteria. In the anoxic zone of marine environments, there is usually a high activity of sulfate-reducing bacteria producing hydrogen sulfide, I&S (POSTGATE? 1979), which is itself used in other bacterial processes, in particular, by photosynthetic sulfur bacteria which live in the photic zone under the oxic/ anoxic interface when this boundary is relatively shallow ( BROCKand MADICAN, 1988 ). The latter produce elemental sulfur or related catenated sulfur species; polysulfides may be formed by oxidation of H2S at the oxic f anoxic interface or by reaction between HIS and elemental sulfur ( FRANCOIS, 1987). In any case, some of the sulfur species may interact with reactive unsaturated or polyunsaturated molecules present in decaying organic matter, either already in the water column or later in the bottom sediments. The process would especially occur to a large extent in carbonate-rich environments which are usually depleted in reactive iron species; in other environments, such as the Monterey upwelling, the competition between pyrite formation and incorporation of sulfur species into organic matter will be an important factor. The end result will depend largely upon the relative amounts of iron and sulfur present ( BERNER, 1984, 1985 ). As mentioned above, it is likely that most reactive organic species are monounsaturated or polyunsaturated molecules. This conclusion can be deduced from the structures of the hydrocarbon subunits of NPMFs, as well as from the experiment with deuterated Raney nickel. It is clearly illustrated in the case of the steroids from Rozel Point seep oil NPMF, in which incorporation of deuterium and analysis of the thiols formed upon selective cleavage of disulfide or pofysulfide bonds have shown that A*-sterenes could be the reactive intermediates ( AUAM et al., 1991, 1992). The nature of the reactive sulfur species is still a matter of speculation. It has been shown previously in the case of thienylhopanes occurring in immature Cretaceous black shales that these compounds could derive from bacteriohopanetetrol or related molecules (e.g., 1,4-ketoaldehyde) by an acid-catalyzed reaction with H2S (VALISOLALAOet al., 1984). It is, however, not very likely that ionic addition of HzS to alkenes is operative in

the cross-linking leading to NPMFs, as previously proposed ( KOHNEN et al., 199 1). Indeed, ionic addition of HzS to alkenes requires strong acid catalysis (PRILEZHAEVA and SHOSTAKOVSIUI, 1963) which seems a priori unlikely to occur in natural environments. The same would hold for polysul-

fides which are equally inactive as anionic species towards double bonds, unless the latter are highly activated. Sulfides (HS) or polysulfides (HS;) may indeed react with activated double bonds (e.g., unsaturated ketones, aldehydes or acids) in a Michael-type addition, which might explain incorporation of sulfur and subsequent cross-linking reactions in some cases ( LALONDEet al., 1987, VAIRAVAMURTHY and MOPPER, 1987). However, such a reaction would certainly not be operative in the more general case of isolated or conjugated, non-activated double bonds. It appears more likely therefore that we have to deal with reactions involving sulfur species which would react by a radical-type mechanism ( FRANCOIS, 1987; AIZENSHTATet al., 1983). Radicals could be formed by homolytic cleavage of H2S or S-S bonds in polysulfides or elemental sulfur taking place in the reorganization of sulfur species produced by microbial processes or be induced by radical promoters existing in natural environments. Such reactions would yieId a network (Fig. 18) in which polysulfide bridges might predominate at first, as confirmed by KOHNEN et al. ( 1991) in an immature shale. The latter would be gradually eliminated due to reorganization in favor of monosulfide linkages during increasing maturation. In Rozel Point oil, the NPMF monosulfide bridges are indeed largely predominant, accompanied by only a small propo~ion ofdisulfide or pofysulfide linkages (ADAM et al., 1992). Hydrogen transfer would take place, and there would be a constant competition between intramolecular and intermolecular incorporation of sulfur. Intramolecular incorporation of sulfur woutd lead to low molecular weight organosulfur com~nents. It is probable that both inte~olecular and intramolecular incorporation has taken place in NPMFs. Depending on the ease of hydrogen transfer possibilities within the mixture, cross-linking will take place to varying extents (C-C bond formation by reaction between a carbon radical and an alkene may also occur). Polyenes will have increased chances of becoming attached by multiple C-S linkages (e.g., carotenes, long-chain isoprenoids, or linear polyenes). However, even starting from a monoene, multiple incorporation of sulfur species could be envisaged (Fig. 18 ). Various laboratory experiments aiming at a better elucidation of some of these reactions will be reported elsewhere. Whatever the mode of formation of NPMF, it clearly appears from these results that a great part of the organic matter of sediments and crude oils has become selectively trapped into non-polar macromolecular species due to cross-linking with sulfur. Its geochemically contained information (source, palaeoenvironment) is rich and very complemental to that of the more classically studied free alkanes. CONCLUSIONS Structural investi~tion of a novel hexane soluble NPMF from six crude oils and one rock extract, all sulfur-rich, have been carried out by using bulk measurements, as well as se-

3415

Nonpolar macromolecular fraction in S-rich petroleum Low molecular components (e.g. thiolanes,

weight

thiophenes) H transfer

I

cleavage

H transfer H-S or S-S cleavage

Formation reaction

of macromolecules by further with alkenes or sulfur chains

FIG. 18. Possible mode of formation of sulfur cross-linked macromolecules and of low molecular weight organosulfur compounds by a radical type mechanism initiated by monosulfide or polysultide radicals (. S, . could be another reacting species). I: polyene, mainly linear or polyisoprenoid. lective C-S bond cleavage. Nuclear magnetic resonance studies and molecular weight determinations have shown that the NPMF is highly aliphatic and, most importantly, that it is essentially macromolecular, its molecular weight ranging from about 600 to over 4000 mass units. Carbon-sulfur bond hydrogenolysis of NPMFs with Raney nickel (H or D) yields substantial amounts of low molecular weight alkanes, showing that the macromolecules of NPMFs are composed mostly of aliphatic subunits reticulated by sulfide and disulfide or polysulfide bridges. The aliphatic moieties comprise mainly linear and polyisoprenoid (acyclic isoprenoid- or carotenoid-type) alkanes. Polycyclic skeletons (steroids, triterpanes) usually occur in rather low proportions, except for the Rozel Point seep oil NPMF, where steroids are major components. Hydrogenolysis of NPMFs with deuterated Raney nickel has shown, in the case of Rozel Point,

that the linear and polyisoprenoid chains, as well as some hopanes, are multiattached to the macromolecular network. Steroids, however, are attached at a single position on ring A, which is probably also the case for gammacerane. Structural determination of the hydrocarbon “building blocks” of NPMFs gives clues to their origin and to the mode of formation of NPMF macromolecules. Most of the subunits originate in algal and bacterial organic matter. The algal contribution would mostly be provided by long linear chains, as well as by steroids and P-carotene-related carotenoids. Bacterial input seems reflected more specifically in carotenoids from anaerobic photosynthetic bacteria, in long polyisoprenoid chains, as well as in some shorter chain isoprenoids typical of archaebacteria (e.g., methanogens). Compared to the free alkanes, the alkanes formed by desulfurization of NPMFs usually represent much larger amounts and show, in most

P. Adam et al.

3416

cases, a quite different composition. They may therefore be complementary as geochemical indicators and of great use for studies of source and palaeoenvironment. A common feature of most building blocks is that they are derived from monounsaturated or polyunsaturated biological precursors. The latter become selectively trapped (and therefore preserved) by reaction with inorganic sulfur species produced by bacterial processes taking place in the anoxic zone, in what appears to be a naturally occurring vulcanization process. A cross-linking takes place which produces macromolecules composed of hydrocarbon building blocks reticulated by monosulfide, disulfide, or polysulfide bridges. This reticulation would take place mainly at early diagenesis in the water column or in the bottom sediment. The exact mechanism of this low-temperature vulcanization process is yet unknown. It seems likely, however, that there is a radical mechanism initiated by the cleavage of H-S or S-S bonds and accompanied by hydrogen transfer reactions. These results show that some sulfur-rich crude oils may essentially be composed of macromolecular material (over 80% in the case of Rozel Point oil). Although their sulfur content is similar to that of NPMFs (TRIFILIEFF, 1987), in the more polar fractions (resins, C7 asphaltenes), the relative amounts of low molecular weight material hydrocarbons liberated upon Raney nickel hydrogenolysis are reduced drastically. This decrease could be due, of course, to higher steric hindrance; it is more probable that in these more polar fractions, additional reticulation may have been inherited from the biological precursors, for example, as esters or ethers, or may be due to the occurrence of other cross-linking reactions (e.g., C-C bond formation, estetification ). AcknoM,Iedg~nents-We thank the SocittC Nationale Elf Aquitaine (Production) and the CNRS for a doctoral fellowship (P. Adam) and financial support: the European Economic Community for financial support (Contract No. ST2-0225 ); G. Ryback, Shell Research Limited, Sittingbourne, UK, for a sample of Rozel Point seep oil; W. Michaelis, Universitlt Hamburg, Germany, for mass spectral analysis; H. Kjosen, University of Trondheim, Norway, for helpful information on carotenoids; E. Krempp. Universite Louis Pasteur, Strasbourg, for NMR analvsis: E. Galleaos. Chevron Research Comoanv. Richmond, CA, USA, for FI ma; spectral analysis; and M.C. Schweigert, G. Teller. and P. Wehrung, Universite Louis Pasteur, Strasbourg, for mass spectral measurements. Edimrrul handling: S. A. Macko

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Appendix Main

structures

cited

in text

R

R = H. CH,. C,HS

R

R= H, CH3, C2H5 qH-(CH,),-CH3 CHJ

Hopanes (c27-%)

Gammacerane

Carotane

“= o-5

Nonpolar macromolecular fraction in S-rich petroleum

Monoaromatic carotenoid (tentative)

Phytane

Pristane

Squalane

Regular nonaprenane, Cd5

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