Aerobic biodegradation of hopanes and norhopanes
in Venezuelan crude oils
F.D. Bost
a, R. Frontera-Suau
a,1, T.J. McDonald
b,
K.E. Peters
c,2, P.J. Morris
a,d,*
aDepartment of Microbiology and Immunology, Medical University of South Carolina, 221 Fort Johnson Rd.,
Charleston, SC 29412, USA
bB & B Laboratories, 1902 Pinon Dr., College Station, TX 77845, USA cMobil Technology Company, PO Box 650232, Dallas, TX 75265, USA
dMarine Biomedicine and Environmental Sciences, Medical University of South Carolina, 221 Fort Johnson Rd.,
Charleston, SC 29412, USA
Received 12 January 2000; accepted 25 September 2000 (returned to author for revision 25 March 2000)
Abstract
The microbial degradation of two Venezuelan crude oils enriched in 25-norhopanes was examined after a 5-week aerobic incubation using a microbial enrichment culture. Analysis of the oils using gas chromatography±mass spec-trometry revealed degradation of the C28 tricyclic terpane, the C29±C34 17a(H),21b(H)-hopanes, and the C29
17a(H),21b(H)-25-norhopane. The C3517a(H),21b(H)-hopane and 18a(H)-oleanane were conserved. Further, the C28±
C34 17a(H),21b(H)-25-norhopanes were degraded and no formation of 25-norhopanes was observed. Degradation
caused preferential removal of the 22R versus the 22S isomer in both the extended hopanes and 25-norhopanes, implying that bacteria remove these compounds in aerobic environments. These data demonstrate 25-norhopane degradation on a time scale similar to that for other biomarkers.#2001 Elsevier Science Ltd. All rights reserved.
Keywords:Biodegradation; 25-Norhopane; Hopane; Crude oil; Venezuela
1. Introduction
Biomarkers are structurally complex components of petroleum derived from biological molecular precursors, such as chlorophyll, sterols, and hopanoids (Peters and Moldowan, 1993). Many biomarkers in crude oil are resistant to biodegradation and are used by petroleum geochemists to assess genetic relationships, thermal maturity and biodegradation. The biomarker pro®le of a crude oil is distinctive and diagnostic, often allowing correlation of an oil to its source rock.
Hopanes are a class of pentacyclic triterpane bio-markers that originate from hopanoids in bacterial membranes (Ourisson et al., 1984; Prince, 1987). Numerous studies show that C30 17a,21b(H)-hopane
and its extended homologs (homohopanes) are biode-graded in the environment and laboratory (Goodwin et al., 1983; Peters and Moldowan, 1991; Chosson et al., 1992; Parker and Acey, 1993; Peters and Moldowan, 1993; Moldowan et al., 1995; Morris et al., 1995). Some reservoired crude oils contain 25-norhopanes, pre-sumably formed by demethylation of the A/B ring at the C-10 position (RullkoÈtter and Wendisch, 1982; Volk-man et al., 1983). The 25-norhopanes were ®rst observed as ``degraded hopanes'' in an oil-impregnated sandstone from the Uinta Basin in Utah (Reed, 1977). Since that report, other investigators have observed 25-norhopanes in heavily degraded reservoir oils (e.g. Sei-fert and Moldowan, 1979; Volkman et al., 1983; SeiSei-fert et al., 1984; Requejo and Halpern, 1989; Moldowan et al., 1995). In most cases, a relative decrease in the
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* Corresponding author. Tel.: 843-762-5533; fax: +1-843-762-5535.
1 Present address: Dept. of Environmental Sciences and
Engineering, 104 Rosenall Hall, University of North Carolina, Chapel Hill, NC 27599-7400, USA.
2 Present address: Exxon Mobil Upstream Research Co., PO
abundance of the hopanes corresponds to increased 25-norhopanes (Requejo and Halpern, 1989; Moldowan and McCarey, 1995). In a later study of reservoir oils from the West Siberia and San Joaquin basins, Peters et al. (1996) concluded that the conversion of hopanes to their corresponding 25-norhopanes was selective, with lower molecular-weight extended hopanes degraded before the high molecular-weight homologs. Further-more, they showed that C-25 demethylation favors the 22Sepimers of the C31and C32 hopanes compared to
22R, while the opposite applies to the C34 and C35
hopanes. Molecular modeling showed that steric dier-ences between the two C-22 epimeric forms explain this stereoselective degradation.
The distribution of 25-norhopanes in petroleum reservoirs is not ubiquitous. Blanc and Connan (1992) contended that 25-norhopanes are already present in petroleum and are ``unmasked'', or concentrated, during biodegradation. In earlier experiments, though, the 25-norhopanes were not a product of kerogen cracking in laboratory pyrolysis studies of Western Australian shales, suggesting that 25-norhopanes occur in sediments as isolated compounds, not as complete series as in biodegraded oils (Noble et al., 1985). Addi-tionally, Peters and Moldowan (1991) normalized the concentrations of various crude oil components in bio-degraded and non-biobio-degraded oils to a conserved C27
diasterane to demonstrate that the concentration eect of biodegradation could not account for the 25-norho-pane concentrations present in the biodegraded oil.
Biodegradation of hopanes commonly occurs without the formation of 25-norhopanes (Peters and Moldowan, 1993 and references therein). 25-Norhopanes have been observed in petroleum reservoirs where the hopanes are demethylated prior to sterane alteration (Brooks et al., 1988). However, if the steranes are degraded prior to the hopanes, then 25-norhopanes are not formed. In an earlier study (Seifert and Moldowan, 1979), no 25-nor-hopane formation was observed in a Texas oil that had been depleted of hopanes. This observation has been duplicated in laboratory studies (Goodwin et al., 1983; Chosson et al., 1992; Morris et al., 1995). At a petro-leum re®nery landfarm, Moldowan et al. (1995) did not observe 25-norhopanes despite evidence of hopane degradation in material that had been deposited at the site almost 10 years earlier. This suggests that in aerobic surface environments hopane degradation either yields products other than the norhopanes or that the 25-norhopanes are subject to similar degradative mechan-isms as the hopanes.
To determine the susceptibility of 25-norhopane to biodegradation, we investigated the fate of both the hopanes and 25-norhopanes in two Venezuelan crude oils (Table 1) using a microbial culture previously shown to degrade the C30 17a,21b-hopane in Bonny
Light crude (Frontera-Suau et al., 1997). Nine
micro-organisms were isolated from this culture, none of which have the ability to degrade C3017a,21b-hopane in
pure culture using crude oil as the sole carbon source (unpublished results). The data presented are from experiments with one of two Venezuelan crude oils, since the extent and pattern of degradation were com-parable for both. Our microbial culture demonstrated simultaneous degradation of the hopanes and 25-nor-hopanes, suggesting that in many aerobic surface envir-onments these biomarkers have similar fates.
2. Experimental
2.1. Venezuelan oils
The two oils used in this study are heavy production oils collected from the Maturin-Temblador basin in Venezuela. These oils contain the C29±C35 17a,21b
-hopanes as well as the C28±C3417a,21b-25-norhopanes.
Table 1 lists various characteristics of the two oils.
2.2. Microbial enrichment culture
The microbial enrichment culture (LC culture) was originally enriched with soil from a creosote-con-taminated site in Fairhope, AL, using a Nigerian Bonny Light crude oil (Table 1) as the sole carbon source (2 mg/ml). The LC culture was maintained with monthly transfers (4% inoculum) into fresh basal medium, BMTM (Hareland et al., 1975), with Bonny Light crude oil. At the time of this experiment, the LC culture had been transferred for 27 consecutive months and had consistently maintained C30 17a,21b-hopane-degrading
activity on Bonny Light crude oil.
Table 1
Characteristics of Venezuelan and Bonny Light crude oils
Characteristics Venezuelan
API gravity 15 12 35
% Saturates
a Oil used in enrichment of the LC culture for this study.
2.3. Time-course culture conditions
During the monthly transfer of the LC culture, a 4% inoculum (109 colony forming units/ml) was used to
inoculate each 50 ml glass tube containing 10 ml of BMTM supplemented with 2 mg/ml of either Venezue-lan oil as the sole carbon source. Triplicate inoculated samples and triplicate uninoculated control samples were set up for each of the following time points: 0, 1, 2, 3 and 5 weeks. Cultures were incubated at 30C in the dark and shaken at 200 rpm.
2.4. Crude oil extraction
At each time point triplicate uninoculated controls and triplicate LC-inoculated samples were sacri®ced for each Venezuelan oil. Oil was extracted from the cultures by shaking three times with 10-ml aliquots of dichloro-methane (Omnisolv HR-GC grade, 99.9%, EM Science, Gibbstown, NJ). Extracts were combined, dried with anhydrous sodium sulfate (J. T. Baker, Phillipsburg, NJ), evaporated under vacuum to reduce volume, and air-dried. Hexane (10-ml GC2grade, 99.9%, Burdick &
Jackson, Muskegon, MI) was then added to each sample to precipitate the asphaltenes prior to gas chro-matographic analysis.
2.5. Gas chromatography (GC)
Deasphaltened samples in hexane were analyzed using a Hewlett-Packard Model 5890 Series IIPlusgas chro-matograph equipped with a ¯ame ionization detector (GC-FID) and an HP-5 column (25 m0.32 mm i.d.0.17 mm). The injector and detector temperatures were 290 and 315C, respectively. The carrier and com-bustion gases were helium and hydrogen, respectively. The temperature program began at 50C for 1 min and proceeded at 5C/min to 310C with a hold at 310C for 20 min.
2.6. Gas chromatography±mass spectrometry (GC±MS)
The method of McDonald and Kennicutt (1992) was used to analyze the samples for biomarkers. In summary, the hydrocarbon fraction of each sample (in dichloro-methane) was added to the top of a chromatographic col-umn packed with 5 g of alumina. The surrogate standards, 5b-cholane,d10-phenanthrene, andd12-chrysene (0.5 ml),
were added as a mixture to the chromatographic column after addition of the sample. The sample/surrogate mixture was eluted from the column with 15-ml dichloromethane, collected in a 20-ml centrifuge tube, and gently evaporated to 500ml with puri®ed nitrogen. The eluant was separated into saturate fractions using high pressure liquid chroma-tography (HPLC) with a Partisil 5 mm PAC Magnum HPLC preparative column and a solvent gradient from
100% hexane to 100% dichloromethane. The saturate fraction was treated with molecular sieve beads prior to analysis to remove then-alkanes. The biomarkers were separated on an HP-1 fused silica capillary column (30 m0.25 mm i.d.). Gas chromatography/mass spectro-metry analyses (m/z=177, m/z=191, m/z=217) were performed using a Hewlett-Packard 5890 II gas chro-matograph interfaced to a Hewlett-Packard 5972 MSD (operated at 70 eV in the selected ion mode, GC/MS/ SIM).
2.7. Biomarker quantitation
Biomarker ratios were calculated using peak areas from them/z=191 andm/z=217 chromatograms. For quantitative analysis, the response factor for the surro-gate standard was calculated by dividing the surrosurro-gate concentration (5.0mg/ml) by the respective peak area. Concentrations for the C30±C35 17a,21b(H)-hopanes
(22Sand 22R), 18a-oleanane, and C2713b,17a
-diaster-ane (20S) were determined by multiplying the respective peak areas (m/z=191, m/z=217) from the mass chro-matograms by the response factor for the surrogate stan-dard. For each of the tricyclic terpanes (C28, C29, or C30),
the peak areas for the 22Rand 22Sepimers were added and divided by the combined value of the 22Rand 22S epimers for the conserved C3517a,21b(H)-homohopane or
by the peak area for the C2713b,17a-diasterane (20S) from
them/z=217 mass chromatograms. Also, the peak area for the C2713b,17a-diasterane (20S) from the m/z=217
mass chromatograms was divided by the combined peak areas for the 22R and 22S epimers of the C35
17a,21b(H)-homohopane. The Ts to Tm ratios were calculated from the peak areas of the m/z=191 chromatograms according to the formula Ts/(Ts+Tm). Peak areas for both C-22 epimers were used in the for-mula%C35 (22R+22S)/(C31±C35) (22R+22S) to
com-pute the homohopane index. The oleanane index was calculated by dividing the peak areas of them/z=191 chromatograms for 18a(H)-oleanane by those of C30
17a,21b(H)-hopane. Calculations for the homohopane and oleanane indices using the absolute concentration values were virtually identical to the values calculated using the peak areas. In the regular sterane to hopane ratio, the C27, C28, and C29 aaa (20R+20S) andabb
(20R+20S) regular sterane areas from the m/z=217 mass chromatograms were divided by the C29±C33
17a(H)-hopane (22R+22S) areas from the m/z=191 mass chromatograms.
3. Results and discussion
for each time point did not dier signi®cantly from the time zero chromatograms shown. The n-alkanes, pristane, and phytane degraded within the ®rst week, with few qualitative changes in the GC-FID pro®les of culture extracts in the following weeks. Mass spectro-metric analysis of other biomarkers in these residues revealed conservation of the steranes, and degradation of the hopanes, 25-norhopanes, and the tricyclic terpanes.
As a class, the tricyclic terpanes are quite recalcitrant. Their degradation typically occurs well after hopane
removal, generally at the same time as the diasteranes (Reed, 1977; Seifert and Moldowan, 1979). In Fig. 2, however, m/z=191 chromatograms reveal extensive degradation of both theRandSepimers of the C28
tri-cyclic terpane after week 3. A recent study, based on molecular volumes and surface areas, considers the sec-ond-eluting peak (inferred to be the 22Repimer) for the C26±C29tricyclic terpanes more readily biodegraded by
a proposed C-10 demethylation process similar to that hypothesized for the hopanes (Peters et al., 1996; Peters,
2000). A study of the tricyclic terpanes in heavy Venezuelan reservoir oils showed preferential removal of the second-eluting tricyclic terpane peak (Alberdi et al., 2000). In our laboratory study, however, no epimer speci®city was observed for the degradation of the C28
tri-cyclic terpane. After 5 weeks, the ratio of the C28tricyclic
terpane to C3517a,21b-homohopane had decreased 32%
from time zero (Table 2). When the C28tricyclic terpane
is compared to the C27 13b,17a-diasterane (20S), the
ratio over the same time decreased 82%. However, analysis of the C28tricyclic terpane epimers revealed no
discernable preference (data not shown). The C29 and
C30 tricyclic terpanes in Venezuelan 14103, however,
remained relatively conserved over the course of the experiment when compared to C3517a,21b-homohopane
and C2713b,17a-diasterane (20S) (Fig. 2, Table 2).
18a-Oleanane was more resistant to degradation than other compounds, yielding a 10-fold increase in the
oleanane index between the controls and inoculated sam-ples at week 5 (Table 2). Degradation of three norhopane species, 17a(H)-28,30-bisnorhopane, 18a (H)-22,29,30-trisnorneohopane (Ts), and 17a (H)-22,29,30-trisnorho-pane (Tm), was observed on them/z=191 chromatogram (Fig. 2). Although both Ts and Tm were degraded in Venezuelan oil (Fig. 2), a slight increase in the Ts/Tm ratio was observed (Table 2), implying that Tm is more readily biodegraded than Ts.
By week 5, C3017a,21b-hopane and C31±C3417a,21b
-homohopanes were reduced relative to C35 hopane at
time zero (Fig. 2). Signi®cant increases in both the oleanane index and C35 17a,21b-homohopane:C30
17a,21b-hopane ratio reveal more extensive degradation of C3017a,21b-hopane compared to C35 homohopane
or oleanane (Table 2). Preferential degradation of theR isomer over theS isomer was observed in the C31±C34
17a,21b-homohopane degradation (Fig. 2). However, the C35-homohopane index, commonly used for
nonbiodegraded oils to indicate the redox potential of the source sediments, increased in the Venezuelan sam-ples from 16% in controls at week 5 to 41% in week 5 inoculated samples (Table 2). Additionally, the ratio of the recalcitrant C2713b,17a-diasterane (20S) to the C35
17a,21b-homohopane did not signi®cantly vary over the course of the experiment (Table 2). Similar trends in hopane degradation were observed in the quantitative data (Table 3). These data further illustrate the relative conservation of C35 17a,21b-homohopane during the
experiment.
C3517a,21b-Homohopane conservation was observed
in tar-sand samples from the Pt. Arena (Monterey) Formation by Requejo and Halpern (1989). Moldowan et al. (1995) later demonstrated degradation of the C30
through C34 hopanes with preservation of the C35
hopane in samples from a petroleum landfarm site. Peters et al. (1996) observed conservation of higher molecular-weight hopanes with preferential degradation
Table 2
Biomarker ratios calculated for Venezuelan oil samples at time zero and after 5 weeks of biodegradation with the LC culturea
C28TT/
a All values are the averages of triplicate samples with the standard deviations of those values. Controls are uninoculated cultures
maintained under the same conditions as the inoculated samples.
b Calculated from m/z=191 mass chromatogram peak areas of the C
28 through C30tricyclic terpanes (TT) (22R+22S), C35
17a,21b(H)-homohopane (C35H) (22R+22S), and C3017a,21b(H)-hopane (C30H). c Calculated from m/z=191 mass chromatogram peak areas of the C
28through C30 tricyclic terpanes (TT) (22R+22S), C35
17a,21b(H)-homohopane (C35H) (22R+22S), and C3017a,21b(H)-hopane (C30H) along with the peak areas for the C2713b,17a
-diasterane (20S) (C27D) from them/z=217 mass chromatograms. d Calculated fromm/z=191 mass chromatogram peak areas of the C
27 17a(H)-22,29,30-trisnorhopane (Tm) and C2718a
(H)-22,29,30-trisnorneohopane (Ts).
e %C
35(22R+22S)/(C31±C35) (22R+22S) homohopanes as determined from peak areas fromm/z=191 chromatograms.
Calcula-tions of the homohopane index using the absolute concentration data in Table 3 provided similar values.
f In this ratio, the C
27, C28, and C29aaa(20R+20S) andabb(20R+20S) regular sterane areas from them/z=217 mass
chroma-tograms and the C29±C3317a(H)-hopanes (22R+22S) from them/z=191 mass chromatograms were used. g Calculated fromm/z=191 mass chromatogram peak areas for 18a(H)-oleanane and C
3017a,21b(H)-hopane. Calculations of the
of the 22Repimer. Subsequent molecular modeling of the R and S epimers of the C34±C35 homohopanes
revealed that theSepimer conformation may sterically protect the C-25 methyl group from microbial attack (Peters et al., 1996). Several microbially unique enrich-ment cultures in our laboratory from geographically distinct soils show hopane-degrading activity with con-servation of the C35hopane (unpublished data).
Conservation of the C35hopane in the above studies
contrasts to that of Goodwin et al. (1983), who reported degradation of the higher molecular-weight hopanes after 11.5 months (C35>C34>C33>C32>C31>C30).
The Goodwin et al. (1983) study also observed degra-dation of theRepimer over theSepimer, observations similar to those in the previous examples. In their study, a mineral salts medium was amended with crude oil as the source of inoculum and carbon. 25-Norhopanes were not detected despite hopane degradation. Chosson et al. (1992) observed alteration of the steranes in the saturate fraction of West Rozel oil. Their hopane degradation by pure cultures of Nocardia, Arthrobacter, and Myco-bacterium species, though not as pronounced as the sterane degradation, showed the same C35>C34>C33>
C32>C31>C30 hopane degradation sequence as
observed by Goodwin et al. (1983). The mineral medium used by Chosson et al. (1992) incorporated glycerol and yeast extract, potentially preferred carbon sources that could decrease the catabolic diversity of the micro-organisms and might explain the altered hopane degra-dation sequence in that study. To support this, Mueller et al. (1990) described the biotransformation of
poly-cyclic aromatic hydrocarbons (PAHs) byP. paucimobilis EPA505. In a complex medium that included yeast extract and glucose, resting P. paucimobilis EPA505 cells were capable of growing on only 5 of the 17 PAHs tested. However, ¯uoranthene-grown resting Pseudomo-nas paucimobilis EPA505 cells were capable of trans-forming 11 of the 17 PAHS tested, suggesting that the presence of more labile carbon sources abrogates the transformation of more recalcitrant compounds.
The additional carbon sources included in the Chosson et al. (1992) study may have also stimulated the co-meta-bolism of compounds in the West Rozel crude oil. A study by Kachholz and Rehm (1978) described n-alkane co-metabolism, or incomplete mineralization, by ®ve species ofBacillusduring growth on glucose, peptone, and yeast extract. Ooyama and Foster (1965) described co-metabo-lism of cyclopentane, cyclohexane, cycloheptane, and cyclooctane to their corresponding cycloketones when grown in a medium with propane as the growth substrate. More complex saturated molecules, such as the hopanes, may also be transformed by a co-metabolic activity, as has been suggested by (1) demethylation to form norhopanes, or (2)b-oxidation of the alkyl side chain. To support the latter, microbial conversion of 3a,5a-cyclosterols to 17-ketosteroids has been observed (Martin, 1977).
In addition to co-metabolic alteration, the saturated ring structure of cycloalkanes may also be directly attacked. The general mechanism for the degradation of cyclohexane involves the oxidative conversion to cyclo-hexanol, followed by conversion to cyclic ketones. Hydrolysis leads to further enzymatic attack and
Table 3
Biomarker ratios and absolute concentrations of selected compounds in Venezuelan oil at time zero and week 5a
Time zero Week 5
Control Sample Control Sample
C30H 290.929.5 291.165.4 270.336.8 49.020.4
C31H, 22S 172.814.1 162.919.7 151.219.7 26.211.3
C31H, 22R 126.117.4 120.816.1 109.110.8 19.49.1
C32H, 22S 123.913.7 113.09.7 103.110.9 20.29.0
C32H, 22R 80.310.6 71.37.2 65.18.5 9.04.6
C33H, 22S 87.97.7 77.69.3 74.313.5 14.91.4
C33H, 22R 49.14.9 46.14.6 38.63.7 7.82.5
C34H, 22S 57.25.2 52.06.2 45.95.8 21.26.1
C34H, 22R 35.93.7 32.95.2 26.85.0 7.12.6
C35H, 22S 98.813.6 82.78.1 71.98.8 55.92.0
C35H, 22R 61.05.2 47.78.5 40.56.4 30.95.5
Oleanane 39.15.6 73.449.6 31.76.4 66.930.5
C27D, 20S 32.15.7 31.03.9 31.92.2 31.71.0
a All values (ng/ml) are the averages of triplicate samples with the standard deviations of those values. Controls are uninoculated
cultures maintained under the same conditions as the inoculated samples. The response factor for the surrogate standard (see Experimental) was calculated by dividing the surrogate concentration (5.0mg/ml) by the respective peak area. Concentrations for the C30±C3517a,21b(H)-hopanes (22Sand 22R), 18a-oleanane, and C2713b,17a-diasterane (20S) were determined by multiplying the
aromatization of the ring followed by ring cleavage by dioxygenases (Perry, 1977; Trower et al., 1985, 1989). Degradation of dehydroabietic acid by Flavobacterium resinovorum and Alcaligenes eutrophus is mediated through an attack on the saturated rings similar to that described above for cyclohexane (Biellman et al., 1973; Liss et al., 1997). Although potential pathways were not explored, the complete removal of isopimaric and dehydroabietic acids from the cultures of two Pseudo-monas-like isolates implies cleavage of the saturated rings in the molecule (Wilson et al., 1996). These mechanisms for degradation of smaller saturate mol-ecules provide a framework for understanding microbial attack of more complex cyclic biomarkers.
The LC enrichment culture was used to ascertain whether a culture that is capable of degrading C30
17a,21b-hopane is also capable of degrading the 25-norhopanes. The Venezuelan crude oils contain the C29
17a,21b-hopane, C30 17a,21b-hopane, and the entire
suite of C31±C35extended hopanes (Fig. 2). Of special
interest to this study, however, was the presence of the C28±C34 17a,21b-25-norhopanes (Fig. 2), the putative
demethylation products of the hopanes. Our data show that hopane and 25-norhopane display similar suscept-ibility to degradation. Substantial changes in them/z=177 chromatograms were not observed until weeks 3 and 5 (Fig. 2). At week 5, the C28and C2917a, 21b
-25-norho-panes (Fig. 2) were reduced, as were theR epimers of the C30±C3417a, 21b-25-norhopanes. At no time during
this experiment was an increase in abundance of the 17a,21b-25-norhopanes observed relative to C35
hopane. In a separate experiment, we examined degra-ded Bonny Light crude oil residues daily for the possible formation of 25-norhopanes from hopane by the LC culture (data not shown). This daily analysis revealed no formation of 25-norhopanes, despite C30±C34 hopane
degradation. Accumulation of 17a,21b-25-norhopanes would be expected if 25-norhopanes were the demethyl-ation products of the hopanes. Thus, given the similar susceptibility of hopane and 25-norhopane to degrada-tion, either no 17a,21b-25-norhopanes were formed from hopane transformation or the 25-norhopanes were transiently formed and rapidly degraded.
Certain evidence suggests that hopane degradation is not always accompanied by the production of 25-nor-hopanes. Goodwin et al. (1983) saw no 25-norhopanes in their laboratory-degraded Kent oil samples despite hopane degradation. In laboratory studies of West Rozel crude oil biodegradation by 73 aerobic bacteria, 25-norhopanes were not detected despite demonstrated hopane loss (Chosson et al., 1992). No 25-norhopane formation was observed in samples of heavily bio-degraded oil seeps from Greece although the hopanes were degraded (Seifert et al., 1984). In a study of a landfarm, Moldowan et al. (1995) demonstrated degra-dation of the C30through C34with conservation of the
C35hopanes in samples from the south end of the site.
Despite the degradation of the hopanes, 25-norhopanes were not detected. These observations are similar to the degradation sequence observed for our Venezuelan oil, suggesting that conservation of C35hopane and lack of
25-norhopane formation are a common degradation pathway in aerobic environments.
Hopane degradation in petroleum reservoirs has been observed under the following conditions: (1) the hopanes are demethylated to 25-norhopanes prior to sterane alteration, or (2) the steranes are degraded before the hopanes and the hopanes do not degrade to 25-norhopanes (Brooks et al., 1988). Interestingly, the sterane pro®les in our biodegraded Venezuelan oils were not altered during the experiment despite hopane and 25-norhopane degradation (data not shown, Table 2). In addition, no 25-norhopanes formed. The ratio of regular steranes to C29±C33 hopanes increased
seven-fold by week 5 (Table 2). Hopane losses before sterane degradation have been observed previously (Peters and Moldowan, 1991). Further, a previous study using microorganisms enriched from the same soil as the LC culture showed degradation of the C27diasteranes in a
fossil fuel soil extract (Morris et al., 1995). In the pres-ent study, however, microorganisms from the same soil did not aect the steranes or diasteranes (data not shown, Table 2). These diering patterns of biomarker degradation in the Venezuelan oils and in the examples cited above indicate that the composition of the carbon source exerts control on microbial consortia.
Oxygenated intermediates are expected as the result of aerobic alteration of hopanes rather than a direct progression to the saturated 25-norhopanes (Higgins and Gilbert, 1978). Aerobic biodegradation should produce functionalized structures (hydroxyl moieties, carboxylic acid groups, ketones, etc.) at the position being altered (Blanc and Connan, 1992). de Lemos Sco®eld (1990) described a C-10 hopanoic acid as a possible inter-mediate of hopane catabolism. The occurrence of the C-10 hopanoic acid was not monitored for this study; however, future studies with the LC enrichment culture will address the presence of hopanoic acids (Bost et al., 2000). To the best of our knowledge, though, these intermediate compounds have not been detected in laboratory microbial degradation experiments.
Other compounds in oil, such as 18a(H)-oleanane, also contain a methyl group attached to C-10, yet are conserved during degradation of the Venezuelan oil. Peters et al. (1996) convincingly used molecular mechanics to explain the conservation of C35 17a,21b
in the Kent oil used in the Goodwin et al. (1983) study. This 17a(H),21b(H)-30-norhopane contains the same C-10 methyl group present in the hopane and homohopane molecules, yet remains relatively unchanged despite hopane degradation.
The data presented here suggest that hopane and 25-norhopane degradation share a common mechanism, given the similar onset of degradation seen for both classes of compounds. The data also dispute the notion that 25-norhopane is a recalcitrant endpoint to hopane degradation during our de®ned laboratory investiga-tions. Given the similar pro®les of hopane degradation in Venezuelan oil to previous observations (Requejo and Halpern, 1989; Moldowan et al., 1995), this degradation pattern may be representative of what occurs in aerobic surface environments. Tritz et al. (1999) determined that the only product of tritium-labeled hopane oxidation by a cholesterol-induced Arthrobacter simplex was hop-17(21)-ene. Additional studies under aerobic and anaerobic conditions are aimed at determining the mechanisms of degradation of a C30 17a(H),21b
(H)-hopane concentrate from Bonny Light crude oil by a complex microbial consortium (Bost et al., 2000). Understanding the mechanism(s) and pathway(s) of hopane and 25-norhopane transformation will provide a more universal understanding of the possible in-reser-voir biodegradation phenomena that aect the biomarker pro®les of crude oils.
Acknowledgements
This work was supported by the Donors of the Pet-roleum Research Fund, administered by the American Chemical Society. This research was sponsored in part by the U.S. Department of Energy's cooperative agree-ment # DE-FCO2-98CH10902 to the Medical Uni-versity of South Carolina's Environmental Biosciences Program. The authors would also like to recognize the helpful reviews of the manuscript provided by Professor Robert Alexander and an anonymous reviewer.
Associate EditorÐL. Ellis
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