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 aDepartment of Geological and Environmental Sciences, Stanford University, Stanford, CA 94305-2115, USAbPDVSA-Intevep, Gerencia General, ExploracioÂn y ProduccioÂn, Caracas, Apdo. 76343, Venezuela cMobil 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 C24due 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 dia-stereomers predominate over the EE diadia-stereomers in the 17-nor tricyclic series (C24±C28), indicating that tricyclic
ter-panes and 17-nor-tricyclic terter-panes 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, indi-cating 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 fromtasmanites, 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 ori-gin, because possible biosynthetic precursors in some bacteria have been identi®ed and suitable precursors have not been found in extant algae. Biochemical pre-cursors such as hexaprenol are postulated to account for
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 repor-ted 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 biode-graded oil ®eld which they interpreted as evidence for this process. This was con®rmed in several regional sam-plings by Peters et al. (1996), who showed a stereospeci®c
0146-6380/01/$ - see front matter#2001 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 3 0 - 3
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* Corresponding author.
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 bio-degradation pathways. In the hopane demethylation pathway, 25-norhopanes begin to occur prior to destruc-tion of the steranes. In the pathway where hopanes are destroyed without the formation of 25-norhopanes, ster-anes 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 (Cas-sani 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 ter-panes and hoter-panes 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 char-acteristics of oils, even for samples aected by advanced biodegradation (Reed, 1977; Palacas et al., 1986).
Fig. 2. GC±MS±MS traces from analysis of the saturate fraction of the 25530sidewall core sample showing (a) C26- C27- C28- and
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.
Then-alkanes and isoprenoids in the saturated frac-tions were analyzed using a HP-6890 gas chromato-graph with a 12 m25mm0.25mm DB-1 column (J & W Scienti®c) with He carrier gas under the following conditions: initial temperature 70C for 5 min, ramping
8C/min, ®nal temperature 340C for 15 min, detector
temperature 360C, injector temperature 300C.
Gas chromatography±mass spectrometry (GC±MS) of sidewall core extracts was completed using a VG-Trio-quadrupole instrument. The GC was programmed as follows: 140C for 5 min, 140±320C for 2C/min and
isothermal at 320C for 20 min, using hydrogen as
car-rier 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 inter-fere slightly with the measurements of C28and C29
tri-cyclic terpanes. They were not corrected because the co-elution 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 quan-ti®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 semi-quantitative sense (e.g. Fig. 2).
Transitions from m/z 346, 360, 374, 388, 402, 416 (parents) to m/z191 and 177 (daughters) were used to monitor C25±C30 tricyclic terpanes and demethylated
tricyclic terpanes, respectively. Nine samples were selec-ted for analysis by MRM±GC±MS. Each sample was analyzed for tricyclic terpane and sterane (C26, C27, C28,
C29and C30) distributions. Numerous co-elutions create
interference among the steranes in GC±MS, and low concentrations for C26and C30compounds restrict their
measurement unless MRM±GC±MS is used.
One oil sample from Lagunillas ®eld (oshore), Mar-acaibo Lake (well LS-2211), Eocene reservoir, 60000
depth), was analyzed by GC±MS for further compar-ison with the biodegraded extracts from the onshore well. All then-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 20860to 27510 lackn-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 C25homologue, although generally
the ®rst well-resolved isomers are C2622S and 22R (Fig.
4a). The elution order of 22R or 22S in these doublets has not been established. A second asymmetric carbon
at the C-27 position appears in the C30-tricyclic terpanes
with additional 27S and 27R diastereomers expected for C30and 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 Wen-disch, 1982; Trendel et al., 1990). If correct, the deme-thylated tricyclic terpanes are 17-nor-tricyclic terpanes according to the numbering system for these com-pounds (Chicarelli et al., 1988). GC±MS analysis of non-biodegraded oil (full suite of n-alkanes and iso-prenoids present) from an Eocene reservoir in the ®eld showed no trace of the desmethyl tricyclic terpanes on them/z177 chromatogram. This observation supports that the mode of their formation is conversion from tri-cyclic 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
second-eluting stereoisomer for the C26-, C27-, C28-, and C29
-tricyclic 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). C34and C35homohopanes show preferential
biodegradation of the later-eluting 22R stereoisomer,
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 25530sidewall core sample (dots) and the non-biodegraded oil (squares) in Lagunillas oil ®eld. Note
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 ter-panes, and a similar molecular mechanics treatment of these compounds has been carried out (Peters, 2000).
The reconstructed relative concentration of the tri-cyclic terpanes (normalized peaks at m/z177+m/z191 for each isomer) shows a similar pattern to the normal-ized concentrations of tricyclic terpanes (m/z191 peaks present, m/z 177 peaks absent) in the related non-biodegraded oil (Fig. 6). This can be used in the same sense as that of Peters et al. (1996) suggesting a precursor-to-product relationship between hopanes and 25-norho-panes in examples from various basins. Thus, the pattern of the summed parent and demethylated tricyclic ter-panes 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 C28and C29tricyclics.
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 deme-thylated hopanes decrease relative to their unaltered
counterparts (Fig. 7). It is also seen that C21+C22
pregnanes/C27steranes 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/tri-cyclic 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 biode-gradation, 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. C 22-3D/C23-3) are plotted against demethylated hopane
ver-sus hopane parent (i.e. 25,dinorhopane verver-sus 30-norhopane). 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 ter-panes appear highly resistant to biodegradation, surviv-ing even when hopanes are removed. Interestsurviv-ingly, 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. 9. Relationship between alteration of tricyclic terpanes (demethylated/parent tricyclic terpane ratio) and alteration of hopanes (demethylated /parent hopane ratio) in biodegraded Lagunillas reservoirs.
results suggest that scales of biodegradation are not universal because the relative rates of biodegradation of dierent compound classes depend upon speci®c envir-onmental 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 early-eluting (EE) stereoisomers at C-22 of the C26-, C27-, C28
-and C29- tricyclic terpanes. Conversely, the EE
de-methylated tricyclic terpanes (C25, C26, C27and C28) are
formed preferentially compared to LE during biode-gradation. 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 com-pared to the creation of 25-norhopanes from hopanes and the destruction of steranes. C21and C22pregnanes/
C27+C28bbsteranes has been found to correlate with
established biodegradation parameters suggesting the pregnanes could be relatively biodegradation resistant.
Acknowledgements
Laboratory assistance at PDVSA-Intevep was pro-vided by C. Rodriguez, O. Rada and A. Gonzales and at Stanford by F. Fago and P. Lipton. A. Iraldi provided the sidewall core samples. EP and PDVSA-Intevep are thanked for ®nancial support and permis-sion to publish. Helpful comments in reviews by F. R. Aquino Neto, S. George and an unidenti®ed reviewer are acknowledged.
Associate EditorÐS. George
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