Organic Geochemistry 32 (2001) 105±114

www.elsevier.nl/locate/orggeochem

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,*
a
Department of Microbiology and Immunology, Medical University of South Carolina, 221 Fort Johnson Rd.,
Charleston, SC 29412, USA
b
B & B Laboratories, 1902 Pinon Dr., College Station, TX 77845, USA
c
Mobil Technology Company, PO Box 650232, Dallas, TX 75265, USA
d
Marine 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 C35 17a(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 Hopanes are a class of pentacyclic triterpane bio-

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.
* Corresponding author. Tel.: +1-843-762-5533; fax: +1-
843-762-5535.
1 sandstone from the Uinta Basin in Utah (Reed, 1977).
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
Box 2189, Houston, TX 77252-2189, USA.
E-mail address: morrisp@musc.edu (P.J. Morris).
0146-6380/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00147-9
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
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; Seifert
et al., 1984; Requejo and Halpern, 1989; Moldowan et
al., 1995). In most cases, a relative decrease in the
106 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114
abundance of the hopanes corresponds to increased 25-
norhopanes (Requejo and Halpern, 1989; Moldowan
and McCa€rey, 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
22S epimers of the C31 and C32 hopanes compared to
22R, while the opposite applies to the C34 and C35
hopanes. Molecular modeling showed that steric di€er-
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-biodegraded oils to a conserved C27
diasterane to demonstrate that the concentration e€ect
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 25-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 C30 17a,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±C34 17a,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 Venezuelan Bonny
oil (no. 14050) oil (no. 14103) Light crudea
API gravity 15 12 35
% Saturates 33 26 58
(HPLC)
% Aromatics 39 43 31
(HPLC)
% Polars 28 31 11
(HPLC)
% Asphaltenes 8 9 2
(isolated)
a
Oil used in enrichment of the LC culture for this study.
Asphaltenes were removed prior to component analysis. Com-
ponent analysis calculations for the saturate, aromatic, and
polar fractions were based on the normalized oil mass remain-
ing after asphaltene removal.
 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114
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 30 C 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 GC2 grade, 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 II Plus gas 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 315 C, respectively. The carrier and com-
bustion gases were helium and hydrogen, respectively.
The temperature program began at 50 C for 1 min and
proceeded at 5 C/min to 310 C with a hold at 310 C 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, and d12-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 500 ml 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
107
100% hexane to 100% dichloromethane. The saturate
fraction was treated with molecular sieve beads prior to
analysis to remove the n-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 the m/z=191 and m/z=217 chromatograms. For
quantitative analysis, the response factor for the surro-
gate standard was calculated by dividing the surrogate
concentration (5.0 mg/ml) by the respective peak area.
Concentrations for the C30±C35 17a,21b(H)-hopanes
(22S and 22R), 18a-oleanane, and C27 13b,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 22R and 22S epimers were added
and divided by the combined value of the 22R and 22S
epimers for the conserved C35 17a,21b(H)-homohopane or
by the peak area for the C27 13b,17a-diasterane (20S) from
the m/z=217 mass chromatograms. Also, the peak area
for the C27 13b,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 the m/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) and abb
(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
Fig. 1 shows the degradation of the n-alkanes and
acyclic isoprenoids in Venezuelan oil 14103 based on
GC-FID analysis over 5 weeks. Uninoculated controls
108 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114

Fig. 1. GC±FID chromatograms of Venezuelan oil 14103 extracted from microbial cultures at times 0, 1, 3 and 5 weeks.

for each time point did not di€er 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 the R and S epimers 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 22R epimer) for the
C26±C29 tricyclic terpanes more readily biodegraded by
a proposed C-10 demethylation process similar to that
hypothesized for the hopanes (Peters et al., 1996; Peters,
 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114 109

Fig. 2. (A) GC±MS chromatograms (m/z=191) of Venezuelan oil 14103 crude oil extracts at times 0, 1, 3, and 5 weeks. (B) GC±MS
chromatograms (m/z=177) of Venezuelan oil 14103 crude oil extracts at times 0, 1, 3 and 5 weeks.

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 C28 tricyclic
terpane to C35 17a,21b-homohopane had decreased 32%
from time zero (Table 2). When the C28 tricyclic terpane
is compared to the C27 13b,17a-diasterane (20S), the
ratio over the same time decreased 82%. However,
analysis of the C28 tricyclic 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 C35 17a,21b-homohopane
and C27 13b,17a-diasterane (20S) (Fig. 2, Table 2).
18a-Oleanane was more resistant to degradation than
other compounds, yielding a 10-fold increase in the
110 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114

Table 2
Biomarker ratios calculated for Venezuelan oil samples at time zero and after 5 weeks of biodegradation with the LC culturea

C28TT/ C29TT/ C30TT/ C35H/ C28TT/ C29TT/ C30TT/ C27D/ Ts/ Homohopane Regular Oleanane
C35Hb C35Hb C35Hb C30Hb C27Dc C27Dc C27Dc C35Hc Tmd index (%)e steranes/ indexg
hopanef

Time zero
Control 0.90.1 1.10.1 1.30.0 0.60.0 4.40.3 5.20.4 6.40.5 0.20.0 0.20.0 17.90.2 1.20.0 0.10.0
Sample 1.000.1 1.10.1 1.30.1 0.50.1 4.10.4 4.80.3 5.60.3 0.20.0 0.30.0 16.20.6 1.10.1 0.20.1
Week 1
Control 0.90.1 1.10.1 1.30.2 0.50.0 4.00.2 4.90.1 5.80.3 0.20.0 0.20.0 17.11.2 1.20.0 0.10.0
Sample 0.70.1 1.20.2 1.20.1 0.50.0 3.10.2 5.10.7 5.40.2 0.20.0 0.30.0 17.50.8 1.30.1 0.20.0
Week 2
Control 0.90.1 1.20.0 1.30.1 0.50.0 4.00.1 5.10.2 5.70.5 0.20.0 0.20.0 16.20.5 1.20.0 0.10.0
Sample 0.60.3 1.10.1 1.40.1 0.80.4 2.20.9 4.00.2 4.80.1 0.20.0 0.30.0 21.93.8 2.10.7 0.40.2
Week 3
Control 1.00.0 1.30.1 1.50.1 0.50.0 4.10.3 5.00.7 6.01.0 0.30.1 0.20.0 15.90.4 1.20.0 0.20.0
Sample 0.40.2 1.10.1 1.60.1 1.10.4 1.40.6 3.90.6 5.50.5 0.30.0 0.30.0 32.54.2 4.40.8 0.50.2
Week 5
Control 1.20.0 1.40.1 1.70.1 0.40.0 4.00.4 4.80.5 5.50.5 0.20.0 0.20.0 15.50.3 1.20.0 0.10.0
Sample 0.30.2 1.30.2 1.80.1 2.11.2 0.70.5 3.40.3 4.60.3 0.20.0 0.30.1 41.47.1 7.82.9 1.50.8
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 C28 through C30 tricyclic terpanes (TT) (22R+22S), C35
17a,21b(H)-homohopane (C35H) (22R+22S), and C30 17a,21b(H)-hopane (C30H).
c
Calculated from m/z=191 mass chromatogram peak areas of the C28 through C30 tricyclic terpanes (TT) (22R+22S), C35
17a,21b(H)-homohopane (C35H) (22R+22S), and C30 17a,21b(H)-hopane (C30H) along with the peak areas for the C27 13b,17a-
diasterane (20S) (C27D) from the m/z=217 mass chromatograms.
d
Calculated from m/z=191 mass chromatogram peak areas of the C27 17a(H)-22,29,30-trisnorhopane (Tm) and C27 18a(H)-
22,29,30-trisnorneohopane (Ts).
e
%C35 (22R+22S)/(C31±C35) (22R+22S) homohopanes as determined from peak areas from m/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 C27, C28, and C29 aaa (20R+20S) and abb (20R+20S) regular sterane areas from the m/z=217 mass chroma-
tograms and the C29±C33 17a(H)-hopanes (22R+22S ) from the m/z=191 mass chromatograms were used.
g
Calculated from m/z=191 mass chromatogram peak areas for 18a(H)-oleanane and C30 17a,21b(H)-hopane. Calculations of the
oleanane index using the absolute concentration data in Table 3 provided similar values.

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 the m/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, C30 17a,21b-hopane and C31±C34 17a,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 C30 17a,21b-hopane compared to C35 homohopane
or oleanane (Table 2). Preferential degradation of the R
isomer over the S 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 C27 13b,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.
C35 17a,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
 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114 111

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 (5.0 mg/ml) by the respective peak area. Concentrations for the
C30±C35 17a,21b(H)-hopanes (22S and 22R), 18a-oleanane, and C27 13b,17a-diasterane (20S) were determined by multiplying the
respective peak areas (m/z=191, m/z=217) used to calculate the biomarker ratios in Table 2 by the response factor for the surrogate
standard.

of the 22R epimer. Subsequent molecular modeling of
the R and S epimers of the C34±C35 homohopanes
revealed that the S epimer 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 C35 hopane (unpublished data).
Conservation of the C35 hopane 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 the R epimer over the S epimer, 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) by P. 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
of Bacillus during 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
112 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114
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±C35 extended 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 the m/z=177
chromatograms were not observed until weeks 3 and 5
(Fig. 2). At week 5, the C28 and C29 17a, 21b-25-norho-
panes (Fig. 2) were reduced, as were the R epimers of
the C30±C34 17a, 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 C30 through C34 with conservation of the
C35 hopanes 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 C35 hopane 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 C27 diasteranes in a
fossil fuel soil extract (Morris et al., 1995). In the pres-
ent study, however, microorganisms from the same soil
did not a€ect the steranes or diasteranes (data not
shown, Table 2). These di€ering 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-
hopane, hypothesizing that a ``scorpion'' con®guration
of the hopane molecule aliphatic side chain shields the
C-25 methyl group from microbial attack. Another
mechanism must protect 18a(H)-oleanane, however,
which lacks the side chain in the homohopanes. Simi-
larly, the 17a(H),21b(H)-30-norhopane was conserved
 F.D. Bost et al. / Organic Geochemistry 32 (2001) 105±114
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 a€ect 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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