Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol32.Issue1.2001:

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: moldowan@pangea.stanford.edu (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 McCa€rey (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 a€ected 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

183

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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M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

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 di€er
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 (o€shore), 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.

M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

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

185

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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M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

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.

M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

187

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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M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

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 e€ects 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.

M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

189

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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M. Alberdi et al. / Organic Geochemistry 32 (2001) 181±191

results suggest that scales of biodegradation are not
universal because the relative rates of biodegradation of
di€erent 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
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