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Stereoselective biodegradation of tricyclic terpanes in heavy oils from the Bolivar Coastal Fields, Venezuela
Organic Geochemistry 32 (2001) 181±191
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Stereoselective biodegradation of tricyclic terpanes in heavy oils from the Bolivar Coastal Fields, Venezuela M. Alberdi a,b, J.M. Moldowan a,*, K.E. Peters c, J.E. Dahl a a
Department of Geological and Environmental Sciences, Stanford University, Stanford, CA 94305-2115, USA b PDVSA-Intevep, Gerencia General, ExploracioÂn y ProduccioÂn, Caracas, Apdo. 76343, Venezuela c Mobil Technology Company, PO Box 650232, Dallas, TX 75265, USA Received 11 January 2000; accepted 30 August 2000 (returned to author for revision 10 April 2000)
Abstract Gas chromatography±mass spectrometry (GC±MS) and GC±MS±MS analyses of heavy oils from Bolivar Coastal Fields (Lagunillas Field) show a complete set of demethylated tricyclic terpanes. As is the case for the 25-norhopanes, the demethylated tricyclics are probably formed in reservoirs by microbially-mediated removal of the methyl group from the C-10 position, generating putative 17-nor-tricyclic terpanes. Diastereomeric pairs of tricyclic terpanes are resolved above C24 due to resolution of 22S and 22R epimers, but the elution order of the 22S and 22R epimers is unknown. Early-eluting diastereomers (EE) predominate over late-eluting diastereomers (LE) (C25±C29) in the heavily degraded oils, indicating a stereoselective preference for the LE stereoisomers during biodegradation. Conversely, the LE diastereomers predominate over the EE diastereomers in the 17-nor tricyclic series (C24±C28), indicating that tricyclic terpanes and 17-nor-tricyclic terpanes are directly linked as precursors and products, respectively. A good correlation exists between the destruction of steranes and the demethylation of hopanes and tricyclic terpanes. This suggests that terpane demethylation occurs during sterane destruction and hopane demethylation, although the rate is slower, indicating that tricyclic terpanes are more resistant to biodegradation. # 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction Tricyclic terpanes occur widely in petroleum and extracts of marine and lacustrine rocks, but those extended above C20 are typically absent in terrigenous oils and extracts dominated by higher-plant input. Extended tricyclic terpanes are associated with, and may originate from tasmanites, a possibly extinct planktonic algal group that is abundant in Permian tasmanites from Alaska and Tasmania (Simoneit et al., 1990). However, these associations do not prove an algal origin, because possible biosynthetic precursors in some bacteria have been identi®ed and suitable precursors have not been found in extant algae. Biochemical precursors such as hexaprenol are postulated to account for * Corresponding author. E-mail address: [email protected] (J.M. Moldowan).
the tricyclic terpanes up to C30 (Aquino Neto et al., 1982). Cyclization of higher polyprenols may account for the larger tricyclic terpanes, which have been reported up to C54 (De Grande et al., 1993). The structural similarity in the ABC ring system of the well-studied hopanes suggests they might be a useful model for the biodegradation of tricyclic terpanes. The microbially induced demethylation of extended hopanes to 25-norhopanes (Seifert & Moldowan, 1979; Volkman et al., 1983) occurs mainly during biodegradation of petroleum in reservoirs (Seifert and Moldowan, 1979; RullkoÈtter and Wendisch, 1982; Volkman et al., 1983; Cassani and Eglinton, 1986; Peters and Moldowan, 1991, 1993). Moldowan and McCarey (1995) have shown a quantitatively negative correlation between hopane and 25-norhopane abundance in core samples from a biodegraded oil ®eld which they interpreted as evidence for this process. This was con®rmed in several regional samplings by Peters et al. (1996), who showed a stereospeci®c
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M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191
attack on epimers related to conformational shape. It has also been suggested that in some cases demethylated hopanes occur as pre-existing biomarkers in source-rocks (Philp, 1983), and are subsequently concentrated in the associated crude by selective biodegradation of the more readily degradable hopanes (Blanc and Connan, 1992; Chosson et al., 1992). There appear to be two major biodegradation pathways. In the hopane demethylation pathway, 25-norhopanes begin to occur prior to destruction of the steranes. In the pathway where hopanes are destroyed without the formation of 25-norhopanes, steranes are destroyed ®rst (Moldowan et al., 1992). Although demethylated hopanes are widespread, demethylated tricyclic terpanes are rarely observed, with only a few occurrences reported from Venezuela (Cassani and Gallango, 1988) and West Africa (Blanc and Connan, 1992). To study the demethylation of tricyclic
terpanes in reservoirs, we analyzed ®fteen core extracts from a production well (20860 -27510 ) in the Lagunillas area, Bolivar Coastal Fields (Fig. 1). Understanding the biodegradation of tricyclic terpanes and hopanes and the relationship with their demethylated counterparts is important because: (a) demethylated hopanes are used as indicators of heavy biodegradation in the reservoir (Alexander et al., 1983; Volkman et al., 1983), (b) they are used as indicators of multiple phases of oil ®lling into reservoirs (Philp, 1983; Volkman et al., 1983; Talukdar et al., 1986), (c) tricyclic terpanes have been used to address thermal maturity in oils (Seifert and Moldowan, 1978; Ekweozor and Strausz, 1983; Cassani et al., 1987), and (d) both tricyclic terpanes and hopanes are widely used to indicate genetic characteristics of oils, even for samples aected by advanced biodegradation (Reed, 1977; Palacas et al., 1986).
Fig. 1. Geographic location of sidewall core and oil samples in Lagunillas oil ®eld, Venezuela (after Bockmeulan et al., 1983).
M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191
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Fig. 2. GC±MS±MS traces from analysis of the saturate fraction of the 25530 sidewall core sample showing (a) C26- C27- C28- and C29tricyclic terpanes with predominance of the early eluting (EE) peak in each doublet of 22R and 22S stereoisomers, and (b) C25- C26C27- and C28-demethylated tricyclic terpanes showing predominance of the late eluting (LE) peak in each doublet of 22R and 22S stereoisomers.
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2. Methodology Fifteen sidewall samples from a producing well in Lagunillas Field onshore (LS-5119 well, Fig. 1) were extracted with a mix of CH2Cl2:MeOH (80:20). The asphaltenes were precipitated with n-heptane and the maltene fraction was separated by HPLC. The n-alkanes and isoprenoids in the saturated fractions were analyzed using a HP-6890 gas chromatograph with a 12 m 25 mm 0.25 mm DB-1 column (J & W Scienti®c) with He carrier gas under the following conditions: initial temperature 70 C for 5 min, ramping 8 C/min, ®nal temperature 340 C for 15 min, detector temperature 360 C, injector temperature 300 C. Gas chromatography±mass spectrometry (GC±MS) of sidewall core extracts was completed using a VGTrio-quadrupole instrument. The GC was programmed as follows: 140 C for 5 min, 140±320 C for 2 C/min and isothermal at 320 C for 20 min, using hydrogen as carrier gas and a 60 m J&W DB-1 fused silica capillary column. Some analyses were repeated using the same conditions on a VG Micromass Autospec Q in SIM± GC±MS mode. The ratios of each tricyclic compound were measured from GC±MS chromatograms. Tricyclic terpanes show less co-elution problems than homohopanes. A correction
was made for co-elution of the C27-LE-demethylated tricyclic with the C27-EE-tricyclic. Some samples show low concentrations of unknown compounds that interfere slightly with the measurements of C28 and C29 tricyclic terpanes. They were not corrected because the coelution of overlapping peaks is estimated to account for less than 15% of the area. Response factors for each tricyclic homologue dier in GC±MS±MS analyses and the response factors decrease with increasing molecular weight, hence quanti®cation was performed using GC±MS data. GC±MS± MS data were applied to corroborate the presence of demethylated tricyclic terpanes in a qualitative to semiquantitative sense (e.g. Fig. 2). Transitions from m/z 346, 360, 374, 388, 402, 416 (parents) to m/z 191 and 177 (daughters) were used to monitor C25±C30 tricyclic terpanes and demethylated tricyclic terpanes, respectively. Nine samples were selected for analysis by MRM±GC±MS. Each sample was analyzed for tricyclic terpane and sterane (C26, C27, C28, C29 and C30) distributions. Numerous co-elutions create interference among the steranes in GC±MS, and low concentrations for C26 and C30 compounds restrict their measurement unless MRM±GC±MS is used. One oil sample from Lagunillas ®eld (oshore), Maracaibo Lake (well LS-2211), Eocene reservoir, 60000
Fig. 3. GC±MS traces of demethylated tricyclic terpanes in samples coming from the top and lower part of the oil column.
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depth), was analyzed by GC±MS for further comparison with the biodegraded extracts from the onshore well. All the n-alkanes are present and the biomarkers show no obvious biodegradation. Therefore, this oil was used as a non-biodegraded oil for comparison to the related biodegraded oils. 3. Results and discussion 3.1. Stereochemical control on tricyclic terpane demethylation The 15 samples analyzed in the Lagunillas onshore well from 20860 to 27510 lack n-alkanes and isoprenoids. Analyses by GC±MS mass chromatography indicate that biomarkers are partially altered (Fig. 3) and all of the extracts show a complete set of C-10 desmethyl hopanes and desmethyl tricyclic terpanes. Two stereoisomers are associated with the asymmetric carbon in the C-22 position of tricyclic terpanes (Fig. 4) and indeed two peaks can be recorded by GC±MS and GC±MS±MS (Figs. 2 and 3). For tricyclic terpanes, these presumed 22R and 22S diastereomers are resolved beginning with the C25 homologue, although generally the ®rst well-resolved isomers are C26 22S and 22R (Fig. 4a). The elution order of 22R or 22S in these doublets has not been established. A second asymmetric carbon
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at the C-27 position appears in the C30-tricyclic terpanes with additional 27S and 27R diastereomers expected for C30 and higher homologues, previously observed to be resolved only above C37 (Moldowan et al., 1983). Demethylation of tricyclic terpanes probably occurs by removal of a methyl group at C-10 (Fig. 4), like the analogous process in hopanes (RullkoÈtter and Wendisch, 1982; Trendel et al., 1990). If correct, the demethylated tricyclic terpanes are 17-nor-tricyclic terpanes according to the numbering system for these compounds (Chicarelli et al., 1988). GC±MS analysis of non-biodegraded oil (full suite of n-alkanes and isoprenoids present) from an Eocene reservoir in the ®eld showed no trace of the desmethyl tricyclic terpanes on the m/z 177 chromatogram. This observation supports that the mode of their formation is conversion from tricyclic terpanes, related to biodegradation of the oil, in agreement with additional evidence (below). Biodegraded Lagunillas extracts from side-wall cores (20860 -27510 ) show a lower abundance of the secondeluting stereoisomer for the C26-, C27-, C28-, and C29tricyclic terpanes (Figs. 2a and 5). For convenience we will designate the earlier-eluting stereoisomer as EE and the late-eluting stereoisomer as LE. The same extracts show more abundant LE stereoisomers for the C25-, C26-, C27-, and C28-desmethyl tricyclic terpanes (Figs. 4b and 5). C34 and C35 homohopanes show preferential biodegradation of the later-eluting 22R stereoisomer,
Fig. 4. Proposed microbial demethylation of tricyclic terpanes and fragments responsible for base peaks on mass spectra of tricyclic terpanes (m/z 191) and demethylated tricyclic terpanes (m/z 177).
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Fig. 5. Amounts normalized to 100% of each parent and demethylated tricyclic terpane analyzed by SIM±GC±MS for representative sidewall core sample from 2086, 2553 and 2751 foot depths.
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Fig. 6. Reconstructed tricyclic terpane distributions from the sum of the parent (C# shown) and the demethylated (C# 1 less than shown) tricyclic terpane for the 25530 sidewall core sample (dots) and the non-biodegraded oil (squares) in Lagunillas oil ®eld. Note that demethylated tricyclic terpanes are absent (0) in the non-biodegraded oil.
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which has been attributed to the C-10 position being sterically protected by the n-alkyl chain in the 22S extended hopanes (scorpion conformation, Peters et al., 1996). Similar eects could be involved for tricyclic terpanes, and a similar molecular mechanics treatment of these compounds has been carried out (Peters, 2000). The reconstructed relative concentration of the tricyclic terpanes (normalized peaks at m/z 177+m/z 191 for each isomer) shows a similar pattern to the normalized concentrations of tricyclic terpanes (m/z 191 peaks present, m/z 177 peaks absent) in the related nonbiodegraded oil (Fig. 6). This can be used in the same sense as that of Peters et al. (1996) suggesting a precursorto-product relationship between hopanes and 25-norhopanes in examples from various basins. Thus, the pattern of the summed parent and demethylated tricyclic terpanes in the biodegraded oil matches that of the tricyclic terpanes in related nonbiodegraded oil. This suggests a microbially-mediated demethylation of tricyclic terpanes under reservoir conditions without signi®cant generation of other products. Slight variations in the match are attributed to instrumental errors and co-elution of minor components with the C28 and C29 tricyclics. 3.2. Comparison of biodegradation of tricyclic terpanes, hopanes and steranes For the core extracts in Lagunillas onshore wells, steranes increase relative to diasteranes with increasing depth, while demethylated tricyclic terpanes and demethylated hopanes decrease relative to their unaltered
counterparts (Fig. 7). It is also seen that C21+C22 pregnanes/C27 steranes correlate with this trend (Fig. 7) and the strength of this correlation is seen to be very strong when the ratio of a demethylated tricyclic/tricyclic parent (i.e. C22-3D/C23-3) is plotted against C21+ C22 pregnanes/C27 steranes (Fig. 8). This correlation suggests that pregnanes are highly resistant to biodegradation, although to our knowledge this observation has not been reported. This result also suggests that demethylation of tricyclics occurs concurrently with sterane destruction. Similarly, a relationship is found when ratios of a demethylated tricyclic versus tricyclic parent (i.e. C223D/C23-3) are plotted against demethylated hopane versus hopane parent (i.e. 25,30-dinorhopane versus 30norhopane). In this case, the relationship suggests that demethylation of tricyclics occurs simultaneously with that of hopanes, although more slowly (Fig. 9). There is a deviation from a straight line suggesting some other process has also occurred. It is possible that tricyclic terpanes in some samples are altered by biodegradation but without quantitative demethylation. Several scales have been proposed to assess the extent of biodegradation in oils, almost all using alkanes (Volkman et al., 1983; Connan, 1984; Moldowan et al., 1992; Peters and Moldowan, 1993). The tricyclic terpanes appear highly resistant to biodegradation, surviving even when hopanes are removed. Interestingly, the tricyclic terpanes in our Venezuelan oils appear to be altered simultaneously with hopanes and steranes, although the rate of tricyclic alteration is slower. Our
Fig. 7. General trends of tricyclic terpane demethylation, sterane destruction and hopane alteration with depth in biodegraded core extracts from Lagunillas reservoirs.
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Fig. 8. Relationship between alteration of tricyclic terpanes (demethylated/parent tricyclic terpane ratio) and biodegradation of steranes (C21+C22 pregnanes/C27+C28 bb steranes) in biodegraded Lagunillas reservoirs.
Fig. 9. Relationship between alteration of tricyclic terpanes (demethylated/parent tricyclic terpane ratio) and alteration of hopanes (demethylated /parent hopane ratio) in biodegraded Lagunillas reservoirs.
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results suggest that scales of biodegradation are not universal because the relative rates of biodegradation of dierent compound classes depend upon speci®c environmental conditions. The demethylation of tricyclic terpanes during biodegradation is an ongoing process in the Lagunillas oil ®eld, a process that has not been documented previously. 4. Conclusions Lagunillas oils from Bolivar Coastal Fields show a complete series of demethylated tricyclic terpanes resulting from heavy biodegradation that occurred in the reservoir. There is a preference for demethylation of the late-eluting compound (LE) compared to the earlyeluting (EE) stereoisomers at C-22 of the C26-, C27-, C28and C29- tricyclic terpanes. Conversely, the EE demethylated tricyclic terpanes (C25, C26, C27 and C28) are formed preferentially compared to LE during biodegradation. Our data represent a snapshot of the ongoing microbially-mediated demethylation of tricyclic terpanes in the reservoir. Demethylation of tricyclic terpanes in Lagunillas Field occurs concurrently, though at a slower rate compared to the creation of 25-norhopanes from hopanes and the destruction of steranes. C21 and C22 pregnanes/ C27+C28 bb steranes has been found to correlate with established biodegradation parameters suggesting the pregnanes could be relatively biodegradation resistant. Acknowledgements Laboratory assistance at PDVSA-Intevep was provided by C. Rodriguez, O. Rada and A. Gonzales and at Stanford by F. Fago and P. Lipton. A. Iraldi provided the sidewall core samples. PDVSA-EP and PDVSAIntevep are thanked for ®nancial support and permission to publish. Helpful comments in reviews by F. R. Aquino Neto, S. George and an unidenti®ed reviewer are acknowledged. Associate EditorÐS. George References Alexander, R., Kagi, R.I., Woodhouse, G.W., Volkman, J.K., 1983. The geochemistry of some biodegraded Australian oils. Australian Petroleum Exploration Association Journal 23, 53±63. Aquino Neto, F.R., Restle, A., Connan, J., Albrecht, P., Ourisson, G., 1982. Novel tricyclic terpanes (C19, C20) in sediments and petroleums. Tetrahedron Letters 23, 2027±2030. Blanc, P., Connan, J., 1992. Origin and occurrence of 25-norhopanes: a statistical study. Organic Geochemistry 18 (6), 813±828.
Bockmeulen, H., Barker, C., Dickey, P.A., 1983. Geology and geochemistry of crude oils, Bolivar Coastal Fields, Venezuela. American Association of Petroleum Geologists Bulletin 67, 242±270. Cassani, F., Eglinton, G., 1986. Organic geochemistry of Venezuelan extra-heavy oils-1 (Molecular assessment of biodegradation.). Chemical Geology 91, 315±333. Cassani, F., Gallango, O., 1988. Organic geochemistry of Venezuelan heavy and extra heavy crude oils. Fourth UNITAR/UNDP International Conference on Heavy Crude and Tar sands. Paper. 165, pp. 543±553. Cassani, F., Gallango, O., Talukdar, S., Vallejos, C., Ehrmann, U., 1987. Methylphenanthrene maturity index of marine source rock extracts and crude oils from the Maracaibo Basin. Organic Geochemistry 13, 73±80. Chicarelli, M.I., Aquino Neto, F.R., Albrecht, P., 1988. Occurence of four stereoisomeric tricyclic terpane series in immature Brazilian shales. Geochimica et Cosmochimica Acta 52, 1955±1959. Chosson, P., Connan, J., Dessort, D., Lanau, C., 1992. In vitro biodegradation of steranes and terpanes: a clue to understanding geological situations. In: Moldowan, J.M., Albrecht, P., Philp, R.P (Eds.), Biological Markers in Sediments and Petroleum. Prentice Hall, Englewood Clis, NJ, pp. 320±349. Connan, J., 1984. Biodegradation of crude oils in reservoirs. In: Brooks, J., Welte, D.H. (Eds.), Advances in Petroleum Geochemistry, 1. Academic Press, London, pp. 299±335. De Grande, S.M.B., Aquino Neto, F.R., Mello, M.R., 1993. Extended tricyclic terpanes in sediments and petroleum. Organic Geochemistry 20, 1039±1047. Ekweozor, C.M., Strausz, O.P., 1981. Tricyclic terpanes in the Athabasca oil sands: their geochemistry. In: Bjorùy, M. et al. (Eds.), Advances in Organic Geochemistry 1983. Wiley, Chichester, pp. 746±766. Moldowan, J.M., Lee, C.Y., Sundararaman, P., Salvatori, R., Alajbeg, A., Gjukic, B., Demaison, G.J., Slougui, N.E., Watt, D.S., 1992. Source correlation and maturity assessment of select oils and rocks from the Central Adriatic basin (Italy and Yugoslavia). In: Moldowan, J.M., Albrecht, P., Philp, R.P. (Eds.), Biological Markers in Sediments and Petroleum. Prentice Hall, Englewood Clis, NJ, pp. 370±401. Moldowan, J.M., McCarey, M.A., 1995. A novel microbial hydrocarbon degradation pathway revealed by hopane demethylation in a petroleum reservoir. Geochimica et Cosmochimica Acta 59, 1891±1894. Moldowan, J.M., Seifert, W.K., Gallegos, E.G., 1983. Identi®cation of an extended series of tricyclic terpanes in petroleum. Geochimica et Cosmochimica Acta 47, 1531±1534. Palacas, J.G., Monopolis, D., Nicolaou, C.A., Anders, D.E., 1986. Geochemical correlation of surface and subsurface oils, western Greece. Organic Geochemistry 10, 417±423. Peters, K.E., 2000. Petroleum tricyclic terpanes predicted physicochemical behavior from molecular mechanics calculations. Organic Geochemistry 31, 497±507. Peters, K.E., Moldowan, J.M., 1991. Eects of source, thermal maturity, and biodegradation on the distribution and isomerization of homohopanes in petroleum. Organic Geochemistry 17, 47±61. Peters, K.E., Moldowan, J.M., 1993. The Biomarker Guide. Interpreting Molecular Fossils in Petroleum and Ancient Sediments. Prentice Hall, Englewood Clis (363 pages).
M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191 Peters, K.E., Moldowan, J.M., McCarey, M.A., Fago, F.J., 1996. Selective biodegradation of extended hopanes to 25norhopanes in petroleum reservoirs (Insights from molecular mechanics ). Organic Geochemistry 24, 765±783. Philp, R.P., 1983. Correlation of crude oils from the San Jorges Basin, Argentina. Geochimica et Cosmochimica Acta 47, 267±275. Reed, W.E., 1977. Molecular compositions of weathered petroleum and comparison with its possible source. Geochimica et Cosmochimica Acta 41, 237±247. RullkoÈtter, J., Wendisch, D., 1982. Microbial alteration of 17a(H)-hopane in Madagascar asphalts: removal of C-10 methyl group and ring opening. Geochimica et Cosmochimica Acta 46, 1543±1553. Seifert, W.K., Moldowan, J.M., 1978. Applications of steranes, terpanes and monoaromatics to the maturation migration and source of crude oils. Geochimica et Cosmochimica Acta 42, 77±95. Seifert, W.K., Moldowan, J.M., 1979. The eect of biodegradation
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on steranes and terpanes in crude oils. Geochimica et Cosmochimica Acta 43, 11±126. Simoneit, B.R.T., Leif, R.N., Aquino Neto, F.R., Azevedo, D.A., Pinto, A.C., Albrecht, P., 1990. On the presence of tricyclic terpane hydrocarbons in permian tasmanite algae. Naturwissenschaften 77, 80±383. Talukdar, S., Gallango, O., Chin-A-Lien, M., 1986. Generation and migration of hydrocarbons in the Maracaibo Basin, Venezuela: an integrated basin study. Organic Geochemistry 10, 261±279. Trendel, J.-M., Guilhem, J., Crisp, P., Repeta, D., Connan, J., Albrecht, P., 1990. Identi®cation of two C-10 demethylated C28 hopanes in biodegraded petroleum. Journal of the Chemical Society, Chemical Communications 5, 424± 425. Volkman, J.K., Alexander, R., Kagi, R.I., Woodhouse, G.W., 1983. Demethylated hopanes in crude oils and their applications in petroleum geochemistry. Geochimica et Cosmochimica Acta 47, 785±794.
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Stereoselective biodegradation of tricyclic terpanes in heavy oils from the Bolivar Coastal Fields, Venezuela M. Alberdi a,b, J.M. Moldowan a,*, K.E. Peters c, J.E. Dahl a a
Department of Geological and Environmental Sciences, Stanford University, Stanford, CA 94305-2115, USA b PDVSA-Intevep, Gerencia General, ExploracioÂn y ProduccioÂn, Caracas, Apdo. 76343, Venezuela c Mobil Technology Company, PO Box 650232, Dallas, TX 75265, USA Received 11 January 2000; accepted 30 August 2000 (returned to author for revision 10 April 2000)
Abstract Gas chromatography±mass spectrometry (GC±MS) and GC±MS±MS analyses of heavy oils from Bolivar Coastal Fields (Lagunillas Field) show a complete set of demethylated tricyclic terpanes. As is the case for the 25-norhopanes, the demethylated tricyclics are probably formed in reservoirs by microbially-mediated removal of the methyl group from the C-10 position, generating putative 17-nor-tricyclic terpanes. Diastereomeric pairs of tricyclic terpanes are resolved above C24 due to resolution of 22S and 22R epimers, but the elution order of the 22S and 22R epimers is unknown. Early-eluting diastereomers (EE) predominate over late-eluting diastereomers (LE) (C25±C29) in the heavily degraded oils, indicating a stereoselective preference for the LE stereoisomers during biodegradation. Conversely, the LE diastereomers predominate over the EE diastereomers in the 17-nor tricyclic series (C24±C28), indicating that tricyclic terpanes and 17-nor-tricyclic terpanes are directly linked as precursors and products, respectively. A good correlation exists between the destruction of steranes and the demethylation of hopanes and tricyclic terpanes. This suggests that terpane demethylation occurs during sterane destruction and hopane demethylation, although the rate is slower, indicating that tricyclic terpanes are more resistant to biodegradation. # 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction Tricyclic terpanes occur widely in petroleum and extracts of marine and lacustrine rocks, but those extended above C20 are typically absent in terrigenous oils and extracts dominated by higher-plant input. Extended tricyclic terpanes are associated with, and may originate from tasmanites, a possibly extinct planktonic algal group that is abundant in Permian tasmanites from Alaska and Tasmania (Simoneit et al., 1990). However, these associations do not prove an algal origin, because possible biosynthetic precursors in some bacteria have been identi®ed and suitable precursors have not been found in extant algae. Biochemical precursors such as hexaprenol are postulated to account for * Corresponding author. E-mail address: [email protected] (J.M. Moldowan).
the tricyclic terpanes up to C30 (Aquino Neto et al., 1982). Cyclization of higher polyprenols may account for the larger tricyclic terpanes, which have been reported up to C54 (De Grande et al., 1993). The structural similarity in the ABC ring system of the well-studied hopanes suggests they might be a useful model for the biodegradation of tricyclic terpanes. The microbially induced demethylation of extended hopanes to 25-norhopanes (Seifert & Moldowan, 1979; Volkman et al., 1983) occurs mainly during biodegradation of petroleum in reservoirs (Seifert and Moldowan, 1979; RullkoÈtter and Wendisch, 1982; Volkman et al., 1983; Cassani and Eglinton, 1986; Peters and Moldowan, 1991, 1993). Moldowan and McCarey (1995) have shown a quantitatively negative correlation between hopane and 25-norhopane abundance in core samples from a biodegraded oil ®eld which they interpreted as evidence for this process. This was con®rmed in several regional samplings by Peters et al. (1996), who showed a stereospeci®c
0146-6380/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00130-3
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attack on epimers related to conformational shape. It has also been suggested that in some cases demethylated hopanes occur as pre-existing biomarkers in source-rocks (Philp, 1983), and are subsequently concentrated in the associated crude by selective biodegradation of the more readily degradable hopanes (Blanc and Connan, 1992; Chosson et al., 1992). There appear to be two major biodegradation pathways. In the hopane demethylation pathway, 25-norhopanes begin to occur prior to destruction of the steranes. In the pathway where hopanes are destroyed without the formation of 25-norhopanes, steranes are destroyed ®rst (Moldowan et al., 1992). Although demethylated hopanes are widespread, demethylated tricyclic terpanes are rarely observed, with only a few occurrences reported from Venezuela (Cassani and Gallango, 1988) and West Africa (Blanc and Connan, 1992). To study the demethylation of tricyclic
terpanes in reservoirs, we analyzed ®fteen core extracts from a production well (20860 -27510 ) in the Lagunillas area, Bolivar Coastal Fields (Fig. 1). Understanding the biodegradation of tricyclic terpanes and hopanes and the relationship with their demethylated counterparts is important because: (a) demethylated hopanes are used as indicators of heavy biodegradation in the reservoir (Alexander et al., 1983; Volkman et al., 1983), (b) they are used as indicators of multiple phases of oil ®lling into reservoirs (Philp, 1983; Volkman et al., 1983; Talukdar et al., 1986), (c) tricyclic terpanes have been used to address thermal maturity in oils (Seifert and Moldowan, 1978; Ekweozor and Strausz, 1983; Cassani et al., 1987), and (d) both tricyclic terpanes and hopanes are widely used to indicate genetic characteristics of oils, even for samples aected by advanced biodegradation (Reed, 1977; Palacas et al., 1986).
Fig. 1. Geographic location of sidewall core and oil samples in Lagunillas oil ®eld, Venezuela (after Bockmeulan et al., 1983).
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Fig. 2. GC±MS±MS traces from analysis of the saturate fraction of the 25530 sidewall core sample showing (a) C26- C27- C28- and C29tricyclic terpanes with predominance of the early eluting (EE) peak in each doublet of 22R and 22S stereoisomers, and (b) C25- C26C27- and C28-demethylated tricyclic terpanes showing predominance of the late eluting (LE) peak in each doublet of 22R and 22S stereoisomers.
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2. Methodology Fifteen sidewall samples from a producing well in Lagunillas Field onshore (LS-5119 well, Fig. 1) were extracted with a mix of CH2Cl2:MeOH (80:20). The asphaltenes were precipitated with n-heptane and the maltene fraction was separated by HPLC. The n-alkanes and isoprenoids in the saturated fractions were analyzed using a HP-6890 gas chromatograph with a 12 m 25 mm 0.25 mm DB-1 column (J & W Scienti®c) with He carrier gas under the following conditions: initial temperature 70 C for 5 min, ramping 8 C/min, ®nal temperature 340 C for 15 min, detector temperature 360 C, injector temperature 300 C. Gas chromatography±mass spectrometry (GC±MS) of sidewall core extracts was completed using a VGTrio-quadrupole instrument. The GC was programmed as follows: 140 C for 5 min, 140±320 C for 2 C/min and isothermal at 320 C for 20 min, using hydrogen as carrier gas and a 60 m J&W DB-1 fused silica capillary column. Some analyses were repeated using the same conditions on a VG Micromass Autospec Q in SIM± GC±MS mode. The ratios of each tricyclic compound were measured from GC±MS chromatograms. Tricyclic terpanes show less co-elution problems than homohopanes. A correction
was made for co-elution of the C27-LE-demethylated tricyclic with the C27-EE-tricyclic. Some samples show low concentrations of unknown compounds that interfere slightly with the measurements of C28 and C29 tricyclic terpanes. They were not corrected because the coelution of overlapping peaks is estimated to account for less than 15% of the area. Response factors for each tricyclic homologue dier in GC±MS±MS analyses and the response factors decrease with increasing molecular weight, hence quanti®cation was performed using GC±MS data. GC±MS± MS data were applied to corroborate the presence of demethylated tricyclic terpanes in a qualitative to semiquantitative sense (e.g. Fig. 2). Transitions from m/z 346, 360, 374, 388, 402, 416 (parents) to m/z 191 and 177 (daughters) were used to monitor C25±C30 tricyclic terpanes and demethylated tricyclic terpanes, respectively. Nine samples were selected for analysis by MRM±GC±MS. Each sample was analyzed for tricyclic terpane and sterane (C26, C27, C28, C29 and C30) distributions. Numerous co-elutions create interference among the steranes in GC±MS, and low concentrations for C26 and C30 compounds restrict their measurement unless MRM±GC±MS is used. One oil sample from Lagunillas ®eld (oshore), Maracaibo Lake (well LS-2211), Eocene reservoir, 60000
Fig. 3. GC±MS traces of demethylated tricyclic terpanes in samples coming from the top and lower part of the oil column.
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depth), was analyzed by GC±MS for further comparison with the biodegraded extracts from the onshore well. All the n-alkanes are present and the biomarkers show no obvious biodegradation. Therefore, this oil was used as a non-biodegraded oil for comparison to the related biodegraded oils. 3. Results and discussion 3.1. Stereochemical control on tricyclic terpane demethylation The 15 samples analyzed in the Lagunillas onshore well from 20860 to 27510 lack n-alkanes and isoprenoids. Analyses by GC±MS mass chromatography indicate that biomarkers are partially altered (Fig. 3) and all of the extracts show a complete set of C-10 desmethyl hopanes and desmethyl tricyclic terpanes. Two stereoisomers are associated with the asymmetric carbon in the C-22 position of tricyclic terpanes (Fig. 4) and indeed two peaks can be recorded by GC±MS and GC±MS±MS (Figs. 2 and 3). For tricyclic terpanes, these presumed 22R and 22S diastereomers are resolved beginning with the C25 homologue, although generally the ®rst well-resolved isomers are C26 22S and 22R (Fig. 4a). The elution order of 22R or 22S in these doublets has not been established. A second asymmetric carbon
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at the C-27 position appears in the C30-tricyclic terpanes with additional 27S and 27R diastereomers expected for C30 and higher homologues, previously observed to be resolved only above C37 (Moldowan et al., 1983). Demethylation of tricyclic terpanes probably occurs by removal of a methyl group at C-10 (Fig. 4), like the analogous process in hopanes (RullkoÈtter and Wendisch, 1982; Trendel et al., 1990). If correct, the demethylated tricyclic terpanes are 17-nor-tricyclic terpanes according to the numbering system for these compounds (Chicarelli et al., 1988). GC±MS analysis of non-biodegraded oil (full suite of n-alkanes and isoprenoids present) from an Eocene reservoir in the ®eld showed no trace of the desmethyl tricyclic terpanes on the m/z 177 chromatogram. This observation supports that the mode of their formation is conversion from tricyclic terpanes, related to biodegradation of the oil, in agreement with additional evidence (below). Biodegraded Lagunillas extracts from side-wall cores (20860 -27510 ) show a lower abundance of the secondeluting stereoisomer for the C26-, C27-, C28-, and C29tricyclic terpanes (Figs. 2a and 5). For convenience we will designate the earlier-eluting stereoisomer as EE and the late-eluting stereoisomer as LE. The same extracts show more abundant LE stereoisomers for the C25-, C26-, C27-, and C28-desmethyl tricyclic terpanes (Figs. 4b and 5). C34 and C35 homohopanes show preferential biodegradation of the later-eluting 22R stereoisomer,
Fig. 4. Proposed microbial demethylation of tricyclic terpanes and fragments responsible for base peaks on mass spectra of tricyclic terpanes (m/z 191) and demethylated tricyclic terpanes (m/z 177).
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Fig. 5. Amounts normalized to 100% of each parent and demethylated tricyclic terpane analyzed by SIM±GC±MS for representative sidewall core sample from 2086, 2553 and 2751 foot depths.
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Fig. 6. Reconstructed tricyclic terpane distributions from the sum of the parent (C# shown) and the demethylated (C# 1 less than shown) tricyclic terpane for the 25530 sidewall core sample (dots) and the non-biodegraded oil (squares) in Lagunillas oil ®eld. Note that demethylated tricyclic terpanes are absent (0) in the non-biodegraded oil.
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which has been attributed to the C-10 position being sterically protected by the n-alkyl chain in the 22S extended hopanes (scorpion conformation, Peters et al., 1996). Similar eects could be involved for tricyclic terpanes, and a similar molecular mechanics treatment of these compounds has been carried out (Peters, 2000). The reconstructed relative concentration of the tricyclic terpanes (normalized peaks at m/z 177+m/z 191 for each isomer) shows a similar pattern to the normalized concentrations of tricyclic terpanes (m/z 191 peaks present, m/z 177 peaks absent) in the related nonbiodegraded oil (Fig. 6). This can be used in the same sense as that of Peters et al. (1996) suggesting a precursorto-product relationship between hopanes and 25-norhopanes in examples from various basins. Thus, the pattern of the summed parent and demethylated tricyclic terpanes in the biodegraded oil matches that of the tricyclic terpanes in related nonbiodegraded oil. This suggests a microbially-mediated demethylation of tricyclic terpanes under reservoir conditions without signi®cant generation of other products. Slight variations in the match are attributed to instrumental errors and co-elution of minor components with the C28 and C29 tricyclics. 3.2. Comparison of biodegradation of tricyclic terpanes, hopanes and steranes For the core extracts in Lagunillas onshore wells, steranes increase relative to diasteranes with increasing depth, while demethylated tricyclic terpanes and demethylated hopanes decrease relative to their unaltered
counterparts (Fig. 7). It is also seen that C21+C22 pregnanes/C27 steranes correlate with this trend (Fig. 7) and the strength of this correlation is seen to be very strong when the ratio of a demethylated tricyclic/tricyclic parent (i.e. C22-3D/C23-3) is plotted against C21+ C22 pregnanes/C27 steranes (Fig. 8). This correlation suggests that pregnanes are highly resistant to biodegradation, although to our knowledge this observation has not been reported. This result also suggests that demethylation of tricyclics occurs concurrently with sterane destruction. Similarly, a relationship is found when ratios of a demethylated tricyclic versus tricyclic parent (i.e. C223D/C23-3) are plotted against demethylated hopane versus hopane parent (i.e. 25,30-dinorhopane versus 30norhopane). In this case, the relationship suggests that demethylation of tricyclics occurs simultaneously with that of hopanes, although more slowly (Fig. 9). There is a deviation from a straight line suggesting some other process has also occurred. It is possible that tricyclic terpanes in some samples are altered by biodegradation but without quantitative demethylation. Several scales have been proposed to assess the extent of biodegradation in oils, almost all using alkanes (Volkman et al., 1983; Connan, 1984; Moldowan et al., 1992; Peters and Moldowan, 1993). The tricyclic terpanes appear highly resistant to biodegradation, surviving even when hopanes are removed. Interestingly, the tricyclic terpanes in our Venezuelan oils appear to be altered simultaneously with hopanes and steranes, although the rate of tricyclic alteration is slower. Our
Fig. 7. General trends of tricyclic terpane demethylation, sterane destruction and hopane alteration with depth in biodegraded core extracts from Lagunillas reservoirs.
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Fig. 8. Relationship between alteration of tricyclic terpanes (demethylated/parent tricyclic terpane ratio) and biodegradation of steranes (C21+C22 pregnanes/C27+C28 bb steranes) in biodegraded Lagunillas reservoirs.
Fig. 9. Relationship between alteration of tricyclic terpanes (demethylated/parent tricyclic terpane ratio) and alteration of hopanes (demethylated /parent hopane ratio) in biodegraded Lagunillas reservoirs.
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results suggest that scales of biodegradation are not universal because the relative rates of biodegradation of dierent compound classes depend upon speci®c environmental conditions. The demethylation of tricyclic terpanes during biodegradation is an ongoing process in the Lagunillas oil ®eld, a process that has not been documented previously. 4. Conclusions Lagunillas oils from Bolivar Coastal Fields show a complete series of demethylated tricyclic terpanes resulting from heavy biodegradation that occurred in the reservoir. There is a preference for demethylation of the late-eluting compound (LE) compared to the earlyeluting (EE) stereoisomers at C-22 of the C26-, C27-, C28and C29- tricyclic terpanes. Conversely, the EE demethylated tricyclic terpanes (C25, C26, C27 and C28) are formed preferentially compared to LE during biodegradation. Our data represent a snapshot of the ongoing microbially-mediated demethylation of tricyclic terpanes in the reservoir. Demethylation of tricyclic terpanes in Lagunillas Field occurs concurrently, though at a slower rate compared to the creation of 25-norhopanes from hopanes and the destruction of steranes. C21 and C22 pregnanes/ C27+C28 bb steranes has been found to correlate with established biodegradation parameters suggesting the pregnanes could be relatively biodegradation resistant. Acknowledgements Laboratory assistance at PDVSA-Intevep was provided by C. Rodriguez, O. Rada and A. Gonzales and at Stanford by F. Fago and P. Lipton. A. Iraldi provided the sidewall core samples. PDVSA-EP and PDVSAIntevep are thanked for ®nancial support and permission to publish. Helpful comments in reviews by F. R. Aquino Neto, S. George and an unidenti®ed reviewer are acknowledged. Associate EditorÐS. George References Alexander, R., Kagi, R.I., Woodhouse, G.W., Volkman, J.K., 1983. The geochemistry of some biodegraded Australian oils. Australian Petroleum Exploration Association Journal 23, 53±63. Aquino Neto, F.R., Restle, A., Connan, J., Albrecht, P., Ourisson, G., 1982. Novel tricyclic terpanes (C19, C20) in sediments and petroleums. Tetrahedron Letters 23, 2027±2030. Blanc, P., Connan, J., 1992. Origin and occurrence of 25-norhopanes: a statistical study. Organic Geochemistry 18 (6), 813±828.
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