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Biodegradation of crude oil in the Aquitaine basin
Biodegradation o f crude oil in the Aquitaine basin JACQUES CONNAN Soci6t6 Nationale Elf Aquitaine (Production), D6partement Laboratoire de G6ologie, Centre Micoulau, 64000 Pau, France and ANNICK RESTLE and PIERRE ALBRECHT Universit6 Louis Pasteur, Institut de Chimie, 1, rue Blaise Pascal, 67000 Strasbourg, France Abstract--Two crude oils from the Aquitaine basin (SW France) have been subjected to in vitro biodegradation with the bacterium Pseudomonas oleovorans for periods ranging from five days to three months. A rapid disappearance of the linear, branched and isoprenoid structures is observed in the alkane fraction. The degree of alteration is highly dependent upon the quality of the initial material but the residual oils, obtained after biodegradation of either immature or moderately mature crudes, display a virtually identical alkane distribution. Such a distribution is also observed in natural asphalts i.e. heavily biodegraded crudes from the Aquitaine tar belt or in crude oils biodegraded in soils from well sites. This alkane distribution displays a high concentration of tri-, tetra- and pentacyclic alkanes. These polycyclic alkanes, organized into several ubiquitous families in the Aquitaine crudes, remain unchanged by bacterial attack either in the laboratory or in nature (soils, boreholes). They therefore represent several classes of compounds that should be of potential interest to correlate unaltered to biodegraded crudes in other basins. Among the families of polycyclics which are ubiquitous in the Aquitaine crudes, we find a novel group of 4 low mol wt steranes (a C2, and a C22 sterane, a C22 and a C23 4 methylsterane), a series of tricyclic terpanes (C19-C26) a series of tetracyclic terpanes (C24-C26) and the ~-fl hopane series (C27-C35).
INTRODUCTION
Water washing and biodegradation of crude oil in reservoirs is now accepted as a widespread phenomenon affecting many pooled occurrences. According to DEMAISON (1977), biodegradation has taken place in most supergiant tar sands or heavy oil deposits from Western Canada (Athabasca), Eastern Venezuela (Orinoco heavy oil belt), Malagasy (Bemolanga deposits), and southeastern USA (Utah tar triangle). The heavy oil accumulations of Western Canada or Eastern Venezuela represent oil reserves comparable to the proved oil fields of the whole Middle East. This information emphasizes the striking importance of the effects of biodegradation to practical petroleum geology. Since the first outstanding paper on the subject, presented by WINTERS and WILLIAMS in 1969, subsequent geochemical studies have been undertaken. A summary of the basic literature, available in 1977, on biodegradation of crude oils, has been published by MmNER et al. (1977) in a review paper dealing with the transformations of petroleum in reservoirs. Since then, more detailed analyses have been performed, especially by using computerized gas chromatographic-mass spectrometric techniques. Among the most common classes of compounds investigated are the polycyclic alkanes, namely the steranes and triterpanes (REED, 1977; RUBINSTEIN et al., 1977; SEIEERT and MOLDOWAN, 1979). These biological markers are recognized as useful tools to correlate unaltered crudes (e.g. VAN DORSSELAERet al., 1978; SEIFERTand MOLDOWAN, 1978). Are they also reliable parameters in correlation problems among biodegraded crudes? REED (1977), RUaINSTEIN et al. (1979) and SEIFERTand MOLDOWAN (1979) have attempted to provide decisive answers to this question. Up to now no conclusive results have been obtained while RUBINSTEINet al. (1977) report steranes and triterpanes as unaffected either in the laboratory experiments or in fossil fuels, whereas REED (1977), SEIFERTand MOLDOWAN (1979) conclude that these families of polycyclics are destroyed by bacteria.
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
PECORADE OIL FIELO
GAS
ANO 6AS FIELD SCALE 0
5
I0
15km
•
OROOEOILS OIOOEGRADEDIN THE LABORATORY
•
CRUDEOIL BIOOEORAOEOIN SOIL
•
NATURALLYBIOOEGRAOEDCRUDES(NATURALASPHALTS)
Torb
Fig. 1. Geographical location of the samples analyzed.
This study does not intend to provide more definite conclusions than those reported by previous researchers. We only want to discuss one case history in which the polycyclic alkanes (tri-, tetra-, pentacyclics) are not obviously biodegraded either in the laboratory or under natural conditions (soils, boreholes). Our results, therefore, agree with those of RUBINSTEIN et al. (1977). The area under study is the Aquitaine basin (SW France). The stratigraphic column covers the time span from upper Cretaceous to Trias. Oil shows occur along the whole stratigraphic column. Two oil accumulations, recognized as economical, are under production: the Lacq oil field (Upper Cretaceous) and the Pecorade oil field (Lower Cretaceous to Jurassic). On the northern rim of the basin, many boreholes have encountered heavy asphaltic crudes which were previously characterized (CONNANand VAN DER WEIDE, 1978) as natural asphalt i.e. severely biodegraded crudes (no n-alkanes, no isoprenoids). The origin of the natural asphalts from the Aquitaine tar belt (Fig. 1) still remains a subject of controversy. Are these asphalts immature or mature biodegraded crudes? A preliminary answer to this question was supplied by means of heating experiments in sealed glass tubes. The thermal behaviour of natural asphalts parallels that of immature unaltered crudes (CONNANand VAN DER WEIDE, 1978). Consequently the tar belt asphalts from the Aquitaine basin may be referred to as immature biodegraded crudes. This geochemical diagnosis was re-examined using biodegradation experiments. Both immature and moderately mature crudes were submitted to bacterial attack in the laboratory by a pure aerobic species: Pseudomonas oleovorans. Crude oil samples, biodegraded in the laboratory, were compared with natural asphalts from the Aquitaine tar belt as well as with Aquitaine crude oils, degraded under natural conditions in soils from a drilling site (PCE 3, Fig. 1) in the Aquitaine basin. EXPERIMENTAL Samples analysed In vitro experiments. In vitro biodegradation experiments were performed on two crudes from the Aquitaine basin: The Lameac I (LMC l) crude (DST 2, 2750-2759 m, Hettangian to Rhaetian) is a sulfur-rich (6.7~o by weight), low gravity (22 ° API) crude which was classified among the immature non biodegraded crudes from the Aquitaine basin (CONNAN and VAN DER WEIDE, 1978).
Biodegradation in the Aquitaine Basin
3
The Pecorade 2 (PCE 2) crude (Production test, 2500 m, Portlandian) is a less viscous crude (29.3' AP1) which still remains sulphur-rich (3.0~o). This crude is considered as moderately mature because the polycyclic alkanes, are still present in significant amounts in this crude. Soils. The PCE 3 drilling site, in the Pecorade oil field, has been selected as reference to investigate the effects of biodegradation of the Aquitaine crude oils under natural conditions in soils. When drilling the PCE 3 well in 1976, an eruption of gas 'and oil was encountered at a depth of 2795 m (Aptian, Lower Cretaceous). A significant amount of oil was spread out over the soil surface in the vicinity of the well. Two years later, we collected two oil-stained soil samples from the PCE 3 oil spill in order to record biodegradation effects in soils. A sample of the original PCE 3 crude was analyzed for comparison purposes. This crude is a rather heavy crude (16°API) which is also sulfur-rich (5.5~). Aquitaine tar belt. Two natural asphalts (Cg 101, 2682-2693 m, Cenomanian and Aire 1, 1910 m, Upper Cretaceous) i.e. severely biodegraded crudes (CONNAN and VAN DER WEIDE, 1978) have been compared to unaltered crudes, found deeper in the same boreholes (Cg I01, DST 5, 2895-2917 m, Neocomian and Aire 1, core 1, 2979 m Jurassic). Each asphalt is considered as the possible biodegraded residue of the associated unaltered crude. The Cg 101 crude resembles the PCE 3 crude: 22.5 ° API, 6.0~ sulfur. The Aire 1 oil (2979 m) looks like an asphalt but does contain a detectable amount of n-alkanes. This asphalt-like crude, poor in saturates (5~o) and enriched in sulfur-bearing structures (7.0~o sulfur), does not show any obvious.signs of biodegradation (occurrence of n-alkanes). The unusual properties of this rather deeply buried crude (3000 m) were thought to result from a combined effect of biodegradation followed by maturation. This explanation, however, is not fully satisfactory; other possibilities such as a low maturity or a restricted biodegradation may not be ruled out. One may agree with MILNER et al. (1977) that "the problem of distinguishing between immaturity and biodegradation is far from being solved". The Aire l asphaltic crude (2979 m), not obviously biodegraded as shown by its n-alkane content, does belong to the puzzling case histories evoked by MILNER et al. 0977). Techniques
A pure species of Pseudomonas oleovorans was selected as microorganism for the in vitro experiments; 250 ml of mineral salt medium (1000ml H 2 0 with 6'.~o,, K2HPO,~; 3'!~',o KH2PO4, 017,~, MgSO4; 1~o (NH4)z SO4 + 2 ml of a solution containing 1000ml H20, 2g FeCI3, 6 H 2 0 + 1.5 ml concentrated HCI; pH = 7), stored in a 1 liter Erlenmeyer, was inoculated by 100 mg (by dry weight) of bacteria and 1-2 g of crude oil. Crude Oil consumption was carried out under aerobic conditions at 25°C with a continuous agitation. Incubation times were as follows: 5, 10, 30 and 90 days. Each experiment was compared to a blank run, performed with the crude oil in the absence of bacteria. Crude oil sample were extracted from the mineral medium with chloroform. The oil-stained soils from PCE 3, the cuttings and the cores from Cg 101 and Aire 1, were soxhlet-extracted with chloroform. The PCE 3 and the Cg 101 crudes were evaporated under vacuum (5 hr, 50°C, 12 mm Hg) in order to eliminate the most volatile compounds. This preliminary treatment provides crude oil residues which are more or less comparable to chloroform extracts. Chloroform extracts or evaporated crudes, desulfurised with activated copper, were subsequently fractionated into asphaltenes, resins, aromatics and saturates. Saturates (total alkanes), purified by TLC on SiO2/Ag N O 3, were analyzed by capillary GC (GLASS WCOT, OV I01, 30 m x 0.25 mm i.d., 100-286°C, 2°C/mm or Polymethylsiloxane, 25 m x 0.3 mn i.d., 120-300°C, 3°C/mm.). Branched and cyclic alkanes, separated by 5 A molecular sieve treatment, were studied by GC and computerized GC MS methods. (GC: SE 30, 25 m × 0.3 mm i.d., 10(~300~C, 3°C/min; MS: LKB 9000 S, PDP 11 E 10). The computerized GC-MS technique involved "Multiple ion cross scan" methods as described by RUBINSTEINet al. (1977). Within the branched and cyclic alkane fraction, particular attention has been paid to cyclic structures. Patterns of mono-, di-, tri-, tetra-, pentacyclic alkanes were compared in all the samples of this study. Characteristic ions of each family of compounds, analyzed by computerized G C - M S are listed in Table 1. A multiple Table 1. Ions analyzed by computerized-GC-MS in the branched and cyclic alkane fraction CHARACTERISTIC IOR$
MONOCYCLiCS
192 1M
210 224 230 252 266 260
|ICYCLICS
1DO 104 20| 222 23|
TRICYCLOC| TERPANES
101
19-26
TERPANES
191
24-27
STEflANES
149
217
21-22 + 27-30
4 - M|THY~STERAN|S
163
231
22-23 + 27-30
250 264 270
13-20
13-20
TET~ACYCLGCS
PENTACYCLOCS HOPANES
I§1
27-35
4
JACQUESCONNAN,ANNICKRESTLEand PIERREALBRECHT
IT ETRACYCLICS~,~, ~24C25
C26
CROSS SCAN OF role 191.TRICYCLICS AND TETRACYCLICS
JSTERANES~I, C211 [4-METHYL-ST~
~
C22 C22 ~
C23
163
MULTIPLE ION CROSS SCAN OF: m/e 149. 163. 217. 231 _C21.C23 RANGE lSTERANES~C27 C28 C2.9
II
~
-
:
~
.
.
.
~
.
~
W
259
315 MULTIPLE ION CROSS SCAN OF m/e 217.231. 259. 315 - C27.C30 RANGE C290(~8Hopane ;30 o<~5Hopane
C ._ 27~P Mopane C28
C31 C32 C33 C34 C35 ~ (:30 ~ rJ'l r'L'l r'L'l , ~ i
Hopane
pC30
CROSS SCAN OF m/e 191 - PENTACYCLICS Fig. 2. An example of computerized-GC-MS analysis: the Cg 101 crude oil (DST 5, 2895-2917 m, Barremian). ion cross scan analysis of tricyclics, tetracyclics, steranes (C2~-C23 and C27-C30 range) and pentacyclics (hopane family)is presented in Fig. 2. Identification of stereochemical structures of trieyclics (m/e 191), tetracyclies (m/e 191), C21-C23 steranes and 4-methylsteranes (m/e 149-163-217-231) is still in progress and will be reported later. RESULTS AND DISCUSSION: BIODEGRADATION OF THE AQUITAINE CRUDE OILS IN THE LABORATORY, IN SOILS AND IN THE SUBSURFACE (TAR BELT)
Gross composition Results, compiled in Tables 2 and 3, only refer to chloroform extracts of both original (blank run) and biodegraded crudes. The percentage of crude oil, metabolized by bacteria, was not determinated in the in vitro experiments. The gross composition of the chloroform extracts, although practically identical after 5 days, changes gradually within the 5-90 days time scale. O u r bacterial culture (Pseudomonas oleovorans) appears to be less active than those used by BAILEY et al. (1973) and R U a I N S T ~ et al. (1977), who observed drastic changes in a 3-4 day experiment.
Biodegradation in the Aquitaine Basin
5
Table 2. Gross composition of Pecorade biodegraded crudes (Pseudomonas oleovorans, 5 to 90 days) HI I | ~ Of T I CNL01t0FORgIXTIUWT
I --
'
il
imi
=6
ii Ili [l~:L, •
0
30.4
40.1
21,7
2.11
1.31
23.3
7,H
0.1I
0.21
I.TI
5
30.7
40,0
26.2
3.0
1,30
21.0
I,I3
O.li
0,22
0,7I 0.43
10
17,4
45.0
35.0
I.I
2.U
2,1
0.37
-
-
30
13,0
4I.l
40,8
4.0
|,I5
I.I
0.22
-
--
N
ll.I
34.0
43.0
;.I
3.16
3.2
0.37, I
--
Table 3. Gross composition of Lameac biodegraded crudes (Pseudomonas oleovorans, 5 to 90 days) GROSSCOMPOIITIONOf THE
iJ i i--'-° '
,,-
i! 0.83
I,B
1.40
O.I6
0
0,3
23,0
07.1
10,1
2.53
1.0
5
0,1
23.9
N.2
10,1
2.12
1,0
0.73
I.i0
1.31
0.51
10
6,3
22.9
H.O
11,9
3.H
I,I
0.11
-
-
0.44
f
30
I
4,4
10.5
U.0
7.3
4.47
1.2
0.05
-
-
-
H
]
4.2
19.2
iI,S
7.1
4.52
0.7
0.03
-
-
--
Biodegradation of the LMC 1 and PCE 2 crudes follows a similar pathway in both cases (Fig. 3). However, the extent of biodegradation effects varies from one sample to the other. The PCE 2 crude is still affected after 30 days of bacterial attack, whereas the LMC 1 crude did not show any significant evolution for the same time interval. The biodegradability of a crude depends upon its quality and especially its n-alkane content (HIGGINSand GILBERT,1978). The crude oil with the highest saturate content is the most SATURATES
\ AROMATICS
REIHN| 4. AIPNALTENEI I P C E 2:MODERATELY M A T U R E OIL
O IN V I T R O B I O D E G R A D A T I O N OF A NORTH C A N T A L CRUDE ( A L B E R T A -CANADA ):
k-ILMC 1 : I M M A T U R E O I L •
RESULT OF B A I L E Y ET A L (1973)
N A T U R A L ASPHALTS FROM THE A Q U I T A I N E TAR B E L T
B I O O E O R A D A T I O N EFFECT l0
90 D A Y S OF METABOLISM
Fig. 3. Effect of bacterial degradation on the gross composition of crude oils: results from in vitro experiments.
6
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
readily degradable. In vitro biodegradation experiments with the Aquitaine crude oils confirm these assessments. The medium-grade PCE 2 crude is more extensively consumed than the naphteno-aromatic, sulfur-rich LMC 1 crude. The biodegradation of crudes always involves a stepwise uptake of saturates (CLARET et al., 1977; BAILEY et al., 1973, Fig. 3; HEISE, 1975; DEROO et al., 1974; HIGGINS and GILBERT,1978; our results, Fig. 3). The alkane removal, predominating in the 0-10 day period, entails a relative enrichment of polar compounds (aromatics, resins and asphaltenes, Fig. 3). Later, after l0 days of incubation, aromatics are also attacked as documented by the resin + asphaltenes enrichment. Biodegradation of aromatics is well documented in the recent literature (HIGGINS and GILBERT, 1978; RUBINSTEINet al., 1977). As far as resins and asphaltenes are concerned, susceptibility to biodegradation still remains unclear. BAILEY et al. (1973) and JOBSON et al. (1972) claim that polar compounds are synthesized by bacterial attack of crude oils, whereas RUBINSTEIN et al. (1977) draw opposite conclusions on the basis of their own experiences. In vitro biodegradation experiments on the Aquitaine crude oils, provide an acceptable model to simulate bacterial attack under natural conditions. Fig. 4 summarizes some results obtained on crude oils biodegraded either in reservoirs (Emeraude oil field, Congo, after CLARETet al., 1977; MC 5 oils, Williston basin, Canada, after BAILEY et al., 1973 and EVANS et al., 1971) or in soils (PCE 3 well site, Aquitaine basin). Stepwise biodegradation under natural conditions (Fig. 4) follows an evolution pathway very similar to those recorded in laboratory experiments (Fig. 3). In the particular case of the Aquitaine basin, results of the in vitro experiments agree with data on crude oils altered in soils. In soil, however, phenomena additional to biodegradation may contribute largely to the formation of the residual weathered petroleum. These phenomena comprise solution, evaporation, autoxidation or photooxidation. Alteration of oil requires oxygen which is less available at depth. Lack of oxygen, beneath the soil surface, may limit degradation as suggested by results on the PCE 3 soil samples. The surface sample is indeed more affected than the sample collected at depth. Comparison of gross compositions of the Aquitaine crude oils, biodegraded either in soils (Fig. 4) or in the laboratory, with gross properties of natural asphalts from the Aquitaine tar belt (Fig. 3), may provide some insight into the origin of these heavily biodegraded crudes. In vitro metabolism of the heavy immature LMC 1 crude leads to SATURATES
RESINS ÷ ASPHALTENES
AROMATICS
BIODEGRADATION IN RESERVOIRS
BIODEGRADATIONIN SOIL
[ ] EMERAUDE OIL FIELD (CONGO)
• PCE 3 CRUDE
O WILLISTON BASIN (CANADA)
~
BIODEGRADATION EFFECT
Fig. 4. Effect of bacterial degradation on the gross composition of crude oils: results on naturally biodegraded crudes (soils, boreholes).
Biodegradation in the Aquitaine Basin
7
biodegradation residues with a composition approaching those of natural asphalts. Simulation experiments, performed in this study, confirm previous conclusions: the tar belt asphalts (Cg 101, Aire 1) originate through bacterial degradation of rather immature heavy crudes similar to the LMC 1 oil (CONNAN, 1972; CONNAN and VAN DER WEIDE, 1978). Total alkanes In vitro experiments. The gas chromatograms of the total alkanes from the original crude oils provide some basic information on the origin and the maturity of both samples. The concentrations of biological markers (isoprenoids, steranes and triterpanes) related to maturity, differentiate the samples analyzed. The LMC t crude shows high quantities of both isoprenoids (pr/n-Cl~ = 0.65, phyt/n-C18 = 1.40, Table 3) and polycyclic alkanes (steranes and triterpanes) and may be ascribed as a low maturity crude. The PCE 2 crude displays an obvious depletion (Fig. 5) in these two classes of chemical fossils (refer to pristane/n-C~7 and phytane/n-C~8 ratios, Table 2) which agrees with its more mature character. A clue to the origin of both crudes is provided by the pristane to phytane ratios. Pristane to phytane ratios lower than 1.0 (Tables 2 and 3), along with other characteristic geochemical and sedimentological criteria confirm that both crudes originate from carbonate source rocks (algal-type kerogen) deposited under very reducing conditions (DIDYK et al., 1978). Occurrence of a detectable predominance of even n-alkanes is well in keeping with this assumption (WELTEand WAPLES, 1973). Figure 5 depicts the gradual degradation of the alkane fraction from the LMC 1 and PCE 2 crudes. Significant changes take place in the 5-10 day period, n-Alkanes, i.e. the most readily degradable structures, are completely removed. Simultaneously, homologous series of branched and cyclic compounds (probably iso- and anteiso-alkanes from the GC-MS study) disappear, while isoprenoids are still present. Isoprenoid uptake, however, has already begun as documented by the pristane/phytane decrease (Tables 2 and 3). During the 10-30 day period of incubation, acyclic isoprenoids are apparently removed. The stepwise uptake of the main classes of alkanes, observed herein, reproduces
Z
_o ,< O < e~
o o_z <
u
_z
PCE 2 CRUDE
LNIC 1 CRUDE
Fig. 5. Metabolismof the total alkane fraction of LMC 1 and PCE 2 crude by Pseudomonas oleovorans.Gas chromatographicconditions:OV 101,30 m x 0.25mm i.d., 100-285°C,2°C/mm.
8
JACQUESCONNAN, ANNICK RESTLEand PIERRE ALBRECHT
the biodegradation sequences described by previous authors (JOBSONet al., 1972; BAILEY et al., 1973; RUBTNSTEINet al., 1977; HIGGINS and GILBERT, 1978). Efficient consumption of iso-, anteiso-, isoprenoid alkanes entails a correlative increase in the alkane fraction of those members with four and five rings. This polycyclic alkane enrichment takes place both in the immature and in the more mature crude (Fig. 5). The alkane GC traces of the heavily biodegraded crudes (90 days, Fig. 5) display very similar patterns, especially within the high mol wt range. Biodegradation of the Aquitaine crude oils has severely obscured the specific character of the original crude oil alkanes (Fig. 5). The alkane fingerprint of biodegraded crudes is too unreliable to assess their maturity (Fig. 5). P C E 3 soils. Figure 6 represents the total alkane pattern of the PCE 3 crudes before and after the oil-spill in the PCE 3 soil. Similar events to those recorded in the laboratory affect the crude oil in the PCE 3 soil. C15 + n-alkanes are completely metabolized by bacteria in the surface sample, but they do survive at depth (10 to 20 cm below the soil surface, Fig. 6). Isoprenoid alkanes, a poorer substrate for hydrocarbon-oxidizing bacteria, are preserved at depth but are slightly attacked on at the surface (refer to pristane to phytane ratios, Table 4). Iso- and anteiso-alkanes also appear to be consumed. The
I
PCE 3
~LTERANIESETTRITERPANEE A •
EVAPORATION BIODEGRADATION
DISSOLUTION OXIDATION SOiLSURFACE
""~d L__
HIIII
OIL SPILL
I
10 to 20 cm BELOWSOILSURFACE
ERUPTION OFTHEPCE3
|
WELLAT 2 7|15.20m
~
j
~
REFERENCE
LOWER CRETACEOUS 2 795.20m
Fig. 6. Degradation of crude oils in soils: an example of the alkane alteration in a PCE 3 soil (Aquitaine basin). Same chromatographic conditions as in Fig. 5.
Biodegradation in the Aquitaine Basin Table 4. Analytical data on the PCE 3 crude altered in soils from the PCE 3 well site
.
so,P,R
352
HCE3CORDE [RUPTIONAT 2 736.M m
917
WiTHTaEPCEICRORE SWIFXCE- 2 YEARS
[,
°
I
.[i
17.1 36.0 33.0 11.0
2.1
15
|.75
11.0 32.1 45.0 I1.1
2.9
1.2
1.23
11.8 33.0 40.7 14.7~ 2.0
0,4
1.34
0.54
0,65
0,60
AFTER ERUPTION
910
POE 3 SOIL OIL - STAINED iFITHTHE PCE3CRUOE 10-2OCfll BELOW RNRFAC 2 TEARS AFTER ERUPTION
1.80
3.29
0.68
I
CHLOROFORMEXTRACTOR TOPPER OOOOE-TRE VOLATILEFRACTION, EVAPORATEDOY TOPPING REPRESENTS13 % OF THE WHOLECOUOE(BY WEIGHT)
end-products of the alkane metabolism (Fig. 6) closely resemble the alkane spectra of the Aquitaine crudes, biodegraded in the laboratory (Fig. 5). Transformation of alkanes in a soil by a mixed culture including hydrocarbon degraders of the genera Pseudomonas, proceeds through pathways analogous to the in vitro biodegradation of alkanes by a pure culture of Pseudomonas oleovorans. This assumption was further cross-examined by performing subsequent in vitro experiments. The mixed culture, growing in the oil-stained PCE 3 soil, was isolated. Its oilmetabolizing capability was compared to those of the pure genera of Pseudomonas oleovorans used in our experimental work. Similarities in degradation behaviour are strickingly visible on the alkane distribution patterns of Fig. 7. The in vitro experiments, applied in this study, seem to be fairly acceptable for simulating the combined effects of abiotic and biotic alteration of alkanes in soils. The slower rate of degradation in soils, however, is probably due to the different, less aerobic conditions. A 2 yr degradation in the PCE 3 soil may be reproduced by a 5 day degradation in the laboratory under more aerobic conditions. Branched and cyclic alkanes. Branched and cyclic alkanes have been examined in detail using a capillary-GC and a computerized-GC-MS analysis. Branched and cyclic alkanes of the Aquitaine crude oils, biodegraded under artificial (laboratory, Fig. 5 and 7) or natural (PCE 3 soil, Fig. 6 and 8; Cg 101 and Aire 1, Fig. 9) conditions exhibit very similar fingerprints. Major definite peaks occur within three molecular weight ranges corresponding to temperatures of: 150-200°C (isoprenoids), 200-250°C (peaks A to E, Fig. 8), > 250°C (steranes and triterpanes, Fig. 8). Steranes and triterpanes, detectable in all samples, are concentrated in the branched and cyclic alkane fraction of biodegraded crudes as a consequence of the consumption in other classes of compounds (iso-, anteiso-, etc.). A concentration process seems also to be involved in the magnification of compounds coded A to E. These compounds, well resolved in the PCE 3 biodegraded crude, are recognizable within the homologous series of iso- and anteisoalkanes of the PCE 3 original crude (Fig. 8). They have been tentatively identified by GC-MS (Multiple ion cross scan of m/e 149-163-217-231, Fig. 2) as two steranes (C21, M ÷ = 288; C22, M ÷ = 302) two 4-methylsteranes (C22, M ÷ = 302; C2a, M ÷ = 316) and a C24 (M ÷ = 330) tetracyclic terpane (Fig. 10). These compounds, revealed in the Aquitaine crudes by biodegradation, are not by-products of the bacterial activity. They also
10
JACQUES CONNAN, ANNICK RESTLE a n d PIERRE ALBRECHT
J C
W
j a:
L REFERENCE (FIG.6)
,<
3 MIXEO CULTURE- 5 OAYS
<
<
PSEUDOMQNAS OLEOVORANS . 50AYS
Li
,4
PSEUDOMONAS 0LEOVORANS- 1O DAYS
.:~
Fig. 7. Metabolism of the PCE 3 crude by a Pseudomonas oleovorans and a mixed culture isolated from the PCE 3 soil: gas chromatograms of total alkanes (C15+). Same conditions as in Fig. 5.
do exist in the unaltered crudes (Cg 101, 2895-2917 m, Fig. 9 and PCE 2, Fig. 5) where they are hidden within the homologous series of branched alkanes.
Cyclic alkanes Monocyclics and bicyclics. Polysubstituted monocyclic and bicyclic alkane series (Table 2), magnified in biodegraded crudes, are generally easy to identify. In some unaltered crudes, however, their detection still remains questionable due to their low concentration. M o n o - and bicyclic distributions are not identical in all the samples. In crude
Biodegradation in the Aquitaine Basin
11
PCE 3
(17~, 21~) H-HOPANE t~
$
E OC
u
J~
BA t
t
I
"...S
(17 ~ , 21 ~ ) H-HOPAN| ¢ •
I
-':
BENEATHSOIL SURFACE
10 to 20 cm
j
° c
,.
I
II
•
-I(
REFERENCE
LOWER CRETACEOUS 2 795.20 m
Fig. 8. Degradation of crude oil in soils: an example of the alteration of the branched and cyclic alkane fraction in a PCE 3 soil (Aquitaine basin). Same conditions as in Fig. 5.
oils, biodegraded in the laboratory, both series of compounds have been compared. In the LMC 1 crude, no removal of mono- and bicyclic alkanes was observed after 3 months of bacterial attack by the pure culture of Pseudomonas oleovorans. In natural asphalts from the Aquitaine tar belt (Aire 1, Cg 101, Fig. 9) i.e. more heavily altered crudes, mono- and bicyclic alkanes are obviously affected. This obvious uptake of small cyclic compounds, however should not be exclusively ascribed to biodegradation for it may also be partly due to solubilization by water washing. A progressive loss of monocyclic and to a lesser extent of bicyclic alkane has already been recorded by RUBINSTEIN et al. (1977) in their in vitro biodegradation experiments on the Prudhoe Bay crude. Tri-, tetra- and pentaeyclies. Several series of tri-, tetra- and pentacyclic terpanes were systematically examined in all the samples by computerized-GC-MS analysis (Fig. 2). These series were found to occur either in unaltered or in altered crudes. Their ubiquity in the crude oils from the Aquitaine basin was revealed due to the biodegradation study. The C21-C23 steranes series, for instance, easily detectable in natural asphalts or crudes biodegraded in the laboratory, was also detected later in all the unaltered crudes (Cg 101, 2895-2917 m, Fig. 9 for example). In this particular study, dealing with SW Aquitaine crude oils, one may consider that the tri- and tetracyclic series (Fig. 2) are as ubiquitous as the well-known hopane family (VAN DORSSELAER, 1975; VAN DORSSELAER and ALBRECHT, 1976). This remains, of course, to be proved on a larger number of samples.
12
JACQUES CONNAN, ANNICK RESTLE a n d PIERRE ALBRECHT
t~
o "
I[
o
u
E
°ii
t
Iiiiiiiiiiiiiiiiiiiii!~i~i~\\\7 l
V,~
~
~
o O -o
N
Biodegradation in the Aquitaine Basin
13
C29 C30 ~-j~ Hopane
Hoj ^ o~-~3Hopane I z'2! t. ~tl3~]21
C--
i ~'H7op ane C22 4C22eSt
co~r~r~r~ ,11¢L LI~ I .
L'24
Penta
J
i
Tri
St
J
o
b
PCE 3 - SOIL S U R F A C E C29
c~ Hopane fl Hopane C30 / L
c3~
C32 C33
ca9
St / C
C23 4 Me St C22
C34il1 l ll'~op~n*
c
4MeSt
I
I O
Penta I O
Tri I r~
I O
Cg 101- ASPHALT "CENOMANIAN 2682 : 2693 m
Fig. 10. Branched and cyclic alkanes of two biodegraded crudes: identification of major compounds. Gas chromatographic conditions: Polymethylsiloxane, 25 m × 0.3 mm i.d., 120-300°C,
3°C/ram.
But one may emphasize, in favour of the ubiquity that samples examined herein came from horizons from the Upper Cretaceous to the Trias. As far as these polycyclic compounds are concerned, the main question that arises concerns their biodegradability. Can these structures be metabolised by bacteria? Referring to the recent literature on the subject, one immediately notices that the question remains open. REED (1977) concludes that low mol wt tricyclic and tetracyclic terpanes are unchanged whereas pentacyclic triterpanes (hopane family) are partially degraded. SEIFERT and MOLDOWAN (1979) report that regular steranes and hopanes are transformed, but that tricyclic terpanes and diasteranes survive. In both cases no in vitro biodegradation experiments were carried out. RUBINSTEINet al. (1977), on the contrary, found that the sterane and the hopane families remain intact after biodegradation in vitro and in nature. Our results, related only to one case history, namely the Aquitaine Basin, confirm the conclusions of RUBINSTEIN et al.: the tri-, tetra-, pentacyclic families examined (Fig. 2) appear to be not significantly affected by bacterial degradation in the laboratory, in soils or under geological conditions in some boreholes from the Aquitaine tar belt. This conclusion may be drawn by comparing tri- tetra- and pentacyclic patterns presented in Figs 11 to 13. Figure 11 represents the tri- and tetracyclic patterns (role 191) of original and in vitro biodegraded crudes. This pattern, different from one crude (LMC 1) to the other (PCE 2) as shown by the C2a tricyclic to C24 tetracyclic ratio, remains unchanged after 90 days of bacterial attack by the Pseudomonas oleovorans (Fig. 11). Figure 12 extends the results of Fig. 11 to other classes of compounds, namely the tricyclic, tetra-
14
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
C24 C25
bETRACYCLICSI~
C26
@ ® @@, . °
LAMEAC
LAMEAC
ORIGINAL
BIODEGRADED
CRUDE-BLANK
RUN-90 DAYS-
CRUDE - PSEUDOMONAS-90 ITETRACYCLICS ],ID *
@® PECORADE-ORIGINAL
PECORADE.BIODEGRADED
C24
DAYS -
C2(
@@ ® CRUDE-BLANK
RUN - 30 DAYS -
CRUDE- PSEUDOMONAS-
90 D A Y S -
Fig. 11. Cross scan of m/e 191 from original and in vitro biodegraded crudes. Tri- and tetracyclic alkanes are not affected by bacterial degradation.
cyclic and pentacyclic alkanes. The striking similarity in the distribution of polycyclic alkanes of both the unaltered (PCE 2) original crude and the biodegraded (PCE 2, 90 days, Pseudomonas oleovorans) crude, shows the non-degradability of these structures in our laboratory experiments. This non-degradability appears also to be the rule in the subsurface as documented by the comparison of the polycyclic distributions of both a natural asphalt (Aire 1, 1910m) and an asphaltic unaltered crude (Aire 1, 2979m) (Fig. 13). Furthermore no evidence for the formation of 4-desmethylhopanes was found in the m/e = 177 fragmentogram, confirming that the hopane skeleton was untouched under both natural and laboratory conditions.
15
Biodegradation in the Aquitaine Basin
:] :] ~] ~] ~]
~]
a. o en~l.
Q
%
'----.e 0
:[
._= r~
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E r~
•
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16
JACQUESCONNAN, ANNICK RESTLEand PIERRE ALBRECHT STERANES m/e 217 - 231
Airu 1 TRICYCLICS m/e 191
Depth (m)
TETRACYC LICS ~ m/e 191
PENTACYCblCS mte 191
P, - C27 " C30
C21 C23
1000
2OOO
:":':':" •
It
:':':"
"
•:.:.: I_0
,; ,~ 2:,. 2~ i, ;9 a
1, ;9 2; 1;., 2', 4_Melt
'2; 21 •
' ;, 2g
' 30
2',
' ='= ' 22
1'~ '
23 o ~ HOPAidE~7
,; ' 29*
30*
30
1liar 31 31 32 12 33 33 34 34 35 3S
30*
~
ASPHALT (UPPER CRETACEOUS-1910 m)
3~-
4m4MaSt 21
29
30
22
23 ot.~ HOPANE27
2|*
32 32 33 33 34 14 39 35
• ASPHALTIC CRUDE (JURASSIC,CA : 2979 m) r---1
TERTIARY
p'--////~
UPPERTO M,DDLE JURASSIC
~
UPPER CIIETACEOUS
~
LOWER JURASSIC
LOWER CRETACEOUS
*
not evaluated : saturated peak
Fig. 13. Tri-, tetra- and pentacyclic alkane distributions of crude oils biodegraded under natural conditions: an example on the Aire 1 well.
CONCLUSIONS
Biodegradation of some Aquitaine crude oils in the laboratory using the Pseudomonas oleovorans strain, provides a suitable model to simulate biodegradation of hydrocarbons under natural conditions. The results of the in vitro biodegradation experiments are in agreement with the results obtained either in soils (oil spill) or underground in boreholes belonging to the Aquitaine tar belt area. Comparison of gross compositions of laboratory biodegraded crudes and of natural asphalts, suggests that the latter are more likely to be immature biodegraded crudes. Degradation mechanisms similar in the laboratory and in nature, occur at various rates. A transformation, observed after a 2-yr period at the soil surface, may be reproduced in the laboratory in a 5-day treatment period under aerobic conditions. Biodegradation of crude oils affects alkanes and to some extent aromatics. The alkane transformation entails a stepwise removal of n-, iso-, anteiso- and isoprenoid alkanes. This gradual metabolism of specific alkane structures obscures the original characteristics of the total alkanes. Therefore, no maturity assessment could be done on the basis of alkanes from drastically biodegraded crudes. Apart from the above-mentioned alkanes, recognized as consumed by bacteria, one may emphasize the survival of tri-, tetra-, and pentacyclic alkanes. These families of polycyclic alkanes have been found to be unattacked by bacteria in the laboratory (Pseudomonas oleovorans or mixed culture), in the PCE 3 soil (mixed culture), in the subsurface (boreholes from the Aquitaine tar belt). These classes of compounds, resistant to bacterial attack in the case of Aquitaine crudeoils, may be regarded as possible useful tools to correlate biodegraded to unaltered crudes.
Biodegradation in the Aquitaine Basin
17
Biodegradation of crude oils in the Aquitaine basin has revealed the ubiquity of several families of tri-, tetra- and pentacyclic alkanes. Among these, we have identified the ~fl hopane family and detected the presence of a series of tricyclic terpanes ranging from C~9 to C26 and of a novel group of C21-C23 steranes (C21-C22 steranes, C22-C23 4-methylsteranes). The elucidation of the exact chemical structures of these compounds is at present under investigation. REFERENCES
BAILEY N. J. L., JOBSONA. M. and ROGERS M. A. (1973) Bacterial degradation of crude oil: comparison of field and experimental data. Chem. Geol. 11, 203-221. CLARET J., TCHIKAYA J. B., TISSOT B., DEROO G. and VAN DORSSELAER A. (1977) Un exemple d'huile biodegrad~e b, basse teneur en soufre: le gisement d'Emeraude (Congo). In Advances in Organic Geochemistry 1975 (eds R. CAMPOSand G. GONI) pp. 509-522. ENADIMSA. Madrid. CONNAN J. 0972) Laboratory simulation and natural diagenesis 1. Thermal evolution of asphalts from the Aquitaine basin (SW France). Bull. Centre Rech. Pau-SNPA 6, 1, 195-214. CONNAN J. and VA~ DER WEIDE B. M. (1978) Thermal evolution of natural asphalts. Chapter 3. In Bitumens, Asphalts and Tar sands (eds G. V. CHILINGARIAN and T. F. YEN) pp. 27-55, Elsevier Scientific Publishing Company. DEMAISON G. T. (1977) Tar sands and supergiant oil fields. AAPG Bull. 61, 11, 1950-1961. DEROO G., TISSOT B., McCROSSAN R. G., DER F. (1974) Geochemistry of heavy oils of Alberta. In Oil Sands Fuel of the Future Memoir 3. Can. Soc. Petro. Geol. 148-167, 184-189. DIDYK B. M., SIMONEIT B. R. T., BRASSELLS. C. and EGLINTON G. (1978) Organic geochemical indicators of palaeoenvironmental conditions of sedimentation. Nature 272, 5650, 216-222. EVANS C. R., ROGERS M. A. and BAILEY N. J. L. (1971) Evolution and alteration of petroleum in Western Canada. Chem. Geol. 8, 147-170. HEISE H. (1975) Geochemistry of Beaufort basin oil. New Ground Can. Petrol. 20, 7, 41-43. HIGGINS I. J. and GILBERT P. D. (1978) The biodegradation of hydrocarbons. Chapter 7. In The Oil Industry and Microbial Ecosystems (eds K. W. A. CHATER and H. J. SOMERVILLE)pp. 80-117. Heyden and Son Ltd. JOBSON A., Cook E. D. and WESTLAKED. W. S. (1972) Microbial utilization of crude oil. Applied Microbiology 23, 6, 108~1089. MILNER C. W. D., ROGERS M. A. and EVANS C. R. (1977) Petroleum transformation in reservoirs. J. Geochim. Explor. 7, 101-153. REED W. E. (1977) Molecular compositions of weathered petroleum and comparison with its possible source. Geochim. Cosmochim. Acta 41,237-247. RUBINSTE1N I., STRAUSZO. P., SPYCKERELLEC., CRAWFORD R. J., WESTLAKED. W. S. (1977) The origin of the oil sands bitumens of Alberta: a chemical and a microbiological simulation study. Geochim. Cosmochim. Acta 41, 1341-1353. SEIEERT W. K. and MOLDOWAN J. M. (1978) Applications of steranes, terpanes and monoaromatics to the maturation, migration and source of crude oils. Geochim. Cosmochim. Acta 42, 77-95. SEIEERT W. K. and MOLDOWAN J. M. (1979) The effect of biodegradation on steranes and terpanes in crude oils. Geochim. Cosmochim. Acta 43, 111-126. VAN DORSSELAERA. (1975) Triterpanes de s6diments. Th6se de Doctorat 6s-Sciences. Universit6 Louis-Pasteur, Strasbourg. VAN DORSSELAER A. and ALBRECHT P. (1976) Marqueurs biologiques: origine, evolution et applications. Bull. Centre Rech. Pau-SNPA 10, i, 193-200. VAN DORSSELAERA., SCHMITTERJ. M., ALBRECHTP., CLARETJ. and CONNAN J. (1978) Use of biological markers in correlation problems. 8th International Congress of organic geochemistry, Moscow (in press). WELTED. M. and WAPLES D. (1973) Uber die Bevorzugung gerad zahlicher n-Alkane in sedimentgesteinen. Naturwissenschaften 60, 516-517. WINTERS J. C. and WILLIAMS J. A. (1969) Microbiological alteration of crude oil in the reservoir. Symp. on petroleum transformations in geologic environments. Am. Chem. Soc. Div. Petroleum Chem. New York, 7-12 September, Preprints 14(4), E 22-E 31.
INTRODUCTION
Water washing and biodegradation of crude oil in reservoirs is now accepted as a widespread phenomenon affecting many pooled occurrences. According to DEMAISON (1977), biodegradation has taken place in most supergiant tar sands or heavy oil deposits from Western Canada (Athabasca), Eastern Venezuela (Orinoco heavy oil belt), Malagasy (Bemolanga deposits), and southeastern USA (Utah tar triangle). The heavy oil accumulations of Western Canada or Eastern Venezuela represent oil reserves comparable to the proved oil fields of the whole Middle East. This information emphasizes the striking importance of the effects of biodegradation to practical petroleum geology. Since the first outstanding paper on the subject, presented by WINTERS and WILLIAMS in 1969, subsequent geochemical studies have been undertaken. A summary of the basic literature, available in 1977, on biodegradation of crude oils, has been published by MmNER et al. (1977) in a review paper dealing with the transformations of petroleum in reservoirs. Since then, more detailed analyses have been performed, especially by using computerized gas chromatographic-mass spectrometric techniques. Among the most common classes of compounds investigated are the polycyclic alkanes, namely the steranes and triterpanes (REED, 1977; RUBINSTEIN et al., 1977; SEIEERT and MOLDOWAN, 1979). These biological markers are recognized as useful tools to correlate unaltered crudes (e.g. VAN DORSSELAERet al., 1978; SEIFERTand MOLDOWAN, 1978). Are they also reliable parameters in correlation problems among biodegraded crudes? REED (1977), RUaINSTEIN et al. (1979) and SEIFERTand MOLDOWAN (1979) have attempted to provide decisive answers to this question. Up to now no conclusive results have been obtained while RUBINSTEINet al. (1977) report steranes and triterpanes as unaffected either in the laboratory experiments or in fossil fuels, whereas REED (1977), SEIFERTand MOLDOWAN (1979) conclude that these families of polycyclics are destroyed by bacteria.
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
PECORADE OIL FIELO
GAS
ANO 6AS FIELD SCALE 0
5
I0
15km
•
OROOEOILS OIOOEGRADEDIN THE LABORATORY
•
CRUDEOIL BIOOEORAOEOIN SOIL
•
NATURALLYBIOOEGRAOEDCRUDES(NATURALASPHALTS)
Torb
Fig. 1. Geographical location of the samples analyzed.
This study does not intend to provide more definite conclusions than those reported by previous researchers. We only want to discuss one case history in which the polycyclic alkanes (tri-, tetra-, pentacyclics) are not obviously biodegraded either in the laboratory or under natural conditions (soils, boreholes). Our results, therefore, agree with those of RUBINSTEIN et al. (1977). The area under study is the Aquitaine basin (SW France). The stratigraphic column covers the time span from upper Cretaceous to Trias. Oil shows occur along the whole stratigraphic column. Two oil accumulations, recognized as economical, are under production: the Lacq oil field (Upper Cretaceous) and the Pecorade oil field (Lower Cretaceous to Jurassic). On the northern rim of the basin, many boreholes have encountered heavy asphaltic crudes which were previously characterized (CONNANand VAN DER WEIDE, 1978) as natural asphalt i.e. severely biodegraded crudes (no n-alkanes, no isoprenoids). The origin of the natural asphalts from the Aquitaine tar belt (Fig. 1) still remains a subject of controversy. Are these asphalts immature or mature biodegraded crudes? A preliminary answer to this question was supplied by means of heating experiments in sealed glass tubes. The thermal behaviour of natural asphalts parallels that of immature unaltered crudes (CONNANand VAN DER WEIDE, 1978). Consequently the tar belt asphalts from the Aquitaine basin may be referred to as immature biodegraded crudes. This geochemical diagnosis was re-examined using biodegradation experiments. Both immature and moderately mature crudes were submitted to bacterial attack in the laboratory by a pure aerobic species: Pseudomonas oleovorans. Crude oil samples, biodegraded in the laboratory, were compared with natural asphalts from the Aquitaine tar belt as well as with Aquitaine crude oils, degraded under natural conditions in soils from a drilling site (PCE 3, Fig. 1) in the Aquitaine basin. EXPERIMENTAL Samples analysed In vitro experiments. In vitro biodegradation experiments were performed on two crudes from the Aquitaine basin: The Lameac I (LMC l) crude (DST 2, 2750-2759 m, Hettangian to Rhaetian) is a sulfur-rich (6.7~o by weight), low gravity (22 ° API) crude which was classified among the immature non biodegraded crudes from the Aquitaine basin (CONNAN and VAN DER WEIDE, 1978).
Biodegradation in the Aquitaine Basin
3
The Pecorade 2 (PCE 2) crude (Production test, 2500 m, Portlandian) is a less viscous crude (29.3' AP1) which still remains sulphur-rich (3.0~o). This crude is considered as moderately mature because the polycyclic alkanes, are still present in significant amounts in this crude. Soils. The PCE 3 drilling site, in the Pecorade oil field, has been selected as reference to investigate the effects of biodegradation of the Aquitaine crude oils under natural conditions in soils. When drilling the PCE 3 well in 1976, an eruption of gas 'and oil was encountered at a depth of 2795 m (Aptian, Lower Cretaceous). A significant amount of oil was spread out over the soil surface in the vicinity of the well. Two years later, we collected two oil-stained soil samples from the PCE 3 oil spill in order to record biodegradation effects in soils. A sample of the original PCE 3 crude was analyzed for comparison purposes. This crude is a rather heavy crude (16°API) which is also sulfur-rich (5.5~). Aquitaine tar belt. Two natural asphalts (Cg 101, 2682-2693 m, Cenomanian and Aire 1, 1910 m, Upper Cretaceous) i.e. severely biodegraded crudes (CONNAN and VAN DER WEIDE, 1978) have been compared to unaltered crudes, found deeper in the same boreholes (Cg I01, DST 5, 2895-2917 m, Neocomian and Aire 1, core 1, 2979 m Jurassic). Each asphalt is considered as the possible biodegraded residue of the associated unaltered crude. The Cg 101 crude resembles the PCE 3 crude: 22.5 ° API, 6.0~ sulfur. The Aire 1 oil (2979 m) looks like an asphalt but does contain a detectable amount of n-alkanes. This asphalt-like crude, poor in saturates (5~o) and enriched in sulfur-bearing structures (7.0~o sulfur), does not show any obvious.signs of biodegradation (occurrence of n-alkanes). The unusual properties of this rather deeply buried crude (3000 m) were thought to result from a combined effect of biodegradation followed by maturation. This explanation, however, is not fully satisfactory; other possibilities such as a low maturity or a restricted biodegradation may not be ruled out. One may agree with MILNER et al. (1977) that "the problem of distinguishing between immaturity and biodegradation is far from being solved". The Aire l asphaltic crude (2979 m), not obviously biodegraded as shown by its n-alkane content, does belong to the puzzling case histories evoked by MILNER et al. 0977). Techniques
A pure species of Pseudomonas oleovorans was selected as microorganism for the in vitro experiments; 250 ml of mineral salt medium (1000ml H 2 0 with 6'.~o,, K2HPO,~; 3'!~',o KH2PO4, 017,~, MgSO4; 1~o (NH4)z SO4 + 2 ml of a solution containing 1000ml H20, 2g FeCI3, 6 H 2 0 + 1.5 ml concentrated HCI; pH = 7), stored in a 1 liter Erlenmeyer, was inoculated by 100 mg (by dry weight) of bacteria and 1-2 g of crude oil. Crude Oil consumption was carried out under aerobic conditions at 25°C with a continuous agitation. Incubation times were as follows: 5, 10, 30 and 90 days. Each experiment was compared to a blank run, performed with the crude oil in the absence of bacteria. Crude oil sample were extracted from the mineral medium with chloroform. The oil-stained soils from PCE 3, the cuttings and the cores from Cg 101 and Aire 1, were soxhlet-extracted with chloroform. The PCE 3 and the Cg 101 crudes were evaporated under vacuum (5 hr, 50°C, 12 mm Hg) in order to eliminate the most volatile compounds. This preliminary treatment provides crude oil residues which are more or less comparable to chloroform extracts. Chloroform extracts or evaporated crudes, desulfurised with activated copper, were subsequently fractionated into asphaltenes, resins, aromatics and saturates. Saturates (total alkanes), purified by TLC on SiO2/Ag N O 3, were analyzed by capillary GC (GLASS WCOT, OV I01, 30 m x 0.25 mm i.d., 100-286°C, 2°C/mm or Polymethylsiloxane, 25 m x 0.3 mn i.d., 120-300°C, 3°C/mm.). Branched and cyclic alkanes, separated by 5 A molecular sieve treatment, were studied by GC and computerized GC MS methods. (GC: SE 30, 25 m × 0.3 mm i.d., 10(~300~C, 3°C/min; MS: LKB 9000 S, PDP 11 E 10). The computerized GC-MS technique involved "Multiple ion cross scan" methods as described by RUBINSTEINet al. (1977). Within the branched and cyclic alkane fraction, particular attention has been paid to cyclic structures. Patterns of mono-, di-, tri-, tetra-, pentacyclic alkanes were compared in all the samples of this study. Characteristic ions of each family of compounds, analyzed by computerized G C - M S are listed in Table 1. A multiple Table 1. Ions analyzed by computerized-GC-MS in the branched and cyclic alkane fraction CHARACTERISTIC IOR$
MONOCYCLiCS
192 1M
210 224 230 252 266 260
|ICYCLICS
1DO 104 20| 222 23|
TRICYCLOC| TERPANES
101
19-26
TERPANES
191
24-27
STEflANES
149
217
21-22 + 27-30
4 - M|THY~STERAN|S
163
231
22-23 + 27-30
250 264 270
13-20
13-20
TET~ACYCLGCS
PENTACYCLOCS HOPANES
I§1
27-35
4
JACQUESCONNAN,ANNICKRESTLEand PIERREALBRECHT
IT ETRACYCLICS~,~, ~24C25
C26
CROSS SCAN OF role 191.TRICYCLICS AND TETRACYCLICS
JSTERANES~I, C211 [4-METHYL-ST~
~
C22 C22 ~
C23
163
MULTIPLE ION CROSS SCAN OF: m/e 149. 163. 217. 231 _C21.C23 RANGE lSTERANES~C27 C28 C2.9
II
~
-
:
~
.
.
.
~
.
~
W
259
315 MULTIPLE ION CROSS SCAN OF m/e 217.231. 259. 315 - C27.C30 RANGE C290(~8Hopane ;30 o<~5Hopane
C ._ 27~P Mopane C28
C31 C32 C33 C34 C35 ~ (:30 ~ rJ'l r'L'l r'L'l , ~ i
Hopane
pC30
CROSS SCAN OF m/e 191 - PENTACYCLICS Fig. 2. An example of computerized-GC-MS analysis: the Cg 101 crude oil (DST 5, 2895-2917 m, Barremian). ion cross scan analysis of tricyclics, tetracyclics, steranes (C2~-C23 and C27-C30 range) and pentacyclics (hopane family)is presented in Fig. 2. Identification of stereochemical structures of trieyclics (m/e 191), tetracyclies (m/e 191), C21-C23 steranes and 4-methylsteranes (m/e 149-163-217-231) is still in progress and will be reported later. RESULTS AND DISCUSSION: BIODEGRADATION OF THE AQUITAINE CRUDE OILS IN THE LABORATORY, IN SOILS AND IN THE SUBSURFACE (TAR BELT)
Gross composition Results, compiled in Tables 2 and 3, only refer to chloroform extracts of both original (blank run) and biodegraded crudes. The percentage of crude oil, metabolized by bacteria, was not determinated in the in vitro experiments. The gross composition of the chloroform extracts, although practically identical after 5 days, changes gradually within the 5-90 days time scale. O u r bacterial culture (Pseudomonas oleovorans) appears to be less active than those used by BAILEY et al. (1973) and R U a I N S T ~ et al. (1977), who observed drastic changes in a 3-4 day experiment.
Biodegradation in the Aquitaine Basin
5
Table 2. Gross composition of Pecorade biodegraded crudes (Pseudomonas oleovorans, 5 to 90 days) HI I | ~ Of T I CNL01t0FORgIXTIUWT
I --
'
il
imi
=6
ii Ili [l~:L, •
0
30.4
40.1
21,7
2.11
1.31
23.3
7,H
0.1I
0.21
I.TI
5
30.7
40,0
26.2
3.0
1,30
21.0
I,I3
O.li
0,22
0,7I 0.43
10
17,4
45.0
35.0
I.I
2.U
2,1
0.37
-
-
30
13,0
4I.l
40,8
4.0
|,I5
I.I
0.22
-
--
N
ll.I
34.0
43.0
;.I
3.16
3.2
0.37, I
--
Table 3. Gross composition of Lameac biodegraded crudes (Pseudomonas oleovorans, 5 to 90 days) GROSSCOMPOIITIONOf THE
iJ i i--'-° '
,,-
i! 0.83
I,B
1.40
O.I6
0
0,3
23,0
07.1
10,1
2.53
1.0
5
0,1
23.9
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10,1
2.12
1,0
0.73
I.i0
1.31
0.51
10
6,3
22.9
H.O
11,9
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0.11
-
-
0.44
f
30
I
4,4
10.5
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7.3
4.47
1.2
0.05
-
-
-
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4.2
19.2
iI,S
7.1
4.52
0.7
0.03
-
-
--
Biodegradation of the LMC 1 and PCE 2 crudes follows a similar pathway in both cases (Fig. 3). However, the extent of biodegradation effects varies from one sample to the other. The PCE 2 crude is still affected after 30 days of bacterial attack, whereas the LMC 1 crude did not show any significant evolution for the same time interval. The biodegradability of a crude depends upon its quality and especially its n-alkane content (HIGGINSand GILBERT,1978). The crude oil with the highest saturate content is the most SATURATES
\ AROMATICS
REIHN| 4. AIPNALTENEI I P C E 2:MODERATELY M A T U R E OIL
O IN V I T R O B I O D E G R A D A T I O N OF A NORTH C A N T A L CRUDE ( A L B E R T A -CANADA ):
k-ILMC 1 : I M M A T U R E O I L •
RESULT OF B A I L E Y ET A L (1973)
N A T U R A L ASPHALTS FROM THE A Q U I T A I N E TAR B E L T
B I O O E O R A D A T I O N EFFECT l0
90 D A Y S OF METABOLISM
Fig. 3. Effect of bacterial degradation on the gross composition of crude oils: results from in vitro experiments.
6
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
readily degradable. In vitro biodegradation experiments with the Aquitaine crude oils confirm these assessments. The medium-grade PCE 2 crude is more extensively consumed than the naphteno-aromatic, sulfur-rich LMC 1 crude. The biodegradation of crudes always involves a stepwise uptake of saturates (CLARET et al., 1977; BAILEY et al., 1973, Fig. 3; HEISE, 1975; DEROO et al., 1974; HIGGINS and GILBERT,1978; our results, Fig. 3). The alkane removal, predominating in the 0-10 day period, entails a relative enrichment of polar compounds (aromatics, resins and asphaltenes, Fig. 3). Later, after l0 days of incubation, aromatics are also attacked as documented by the resin + asphaltenes enrichment. Biodegradation of aromatics is well documented in the recent literature (HIGGINS and GILBERT, 1978; RUBINSTEINet al., 1977). As far as resins and asphaltenes are concerned, susceptibility to biodegradation still remains unclear. BAILEY et al. (1973) and JOBSON et al. (1972) claim that polar compounds are synthesized by bacterial attack of crude oils, whereas RUBINSTEIN et al. (1977) draw opposite conclusions on the basis of their own experiences. In vitro biodegradation experiments on the Aquitaine crude oils, provide an acceptable model to simulate bacterial attack under natural conditions. Fig. 4 summarizes some results obtained on crude oils biodegraded either in reservoirs (Emeraude oil field, Congo, after CLARETet al., 1977; MC 5 oils, Williston basin, Canada, after BAILEY et al., 1973 and EVANS et al., 1971) or in soils (PCE 3 well site, Aquitaine basin). Stepwise biodegradation under natural conditions (Fig. 4) follows an evolution pathway very similar to those recorded in laboratory experiments (Fig. 3). In the particular case of the Aquitaine basin, results of the in vitro experiments agree with data on crude oils altered in soils. In soil, however, phenomena additional to biodegradation may contribute largely to the formation of the residual weathered petroleum. These phenomena comprise solution, evaporation, autoxidation or photooxidation. Alteration of oil requires oxygen which is less available at depth. Lack of oxygen, beneath the soil surface, may limit degradation as suggested by results on the PCE 3 soil samples. The surface sample is indeed more affected than the sample collected at depth. Comparison of gross compositions of the Aquitaine crude oils, biodegraded either in soils (Fig. 4) or in the laboratory, with gross properties of natural asphalts from the Aquitaine tar belt (Fig. 3), may provide some insight into the origin of these heavily biodegraded crudes. In vitro metabolism of the heavy immature LMC 1 crude leads to SATURATES
RESINS ÷ ASPHALTENES
AROMATICS
BIODEGRADATION IN RESERVOIRS
BIODEGRADATIONIN SOIL
[ ] EMERAUDE OIL FIELD (CONGO)
• PCE 3 CRUDE
O WILLISTON BASIN (CANADA)
~
BIODEGRADATION EFFECT
Fig. 4. Effect of bacterial degradation on the gross composition of crude oils: results on naturally biodegraded crudes (soils, boreholes).
Biodegradation in the Aquitaine Basin
7
biodegradation residues with a composition approaching those of natural asphalts. Simulation experiments, performed in this study, confirm previous conclusions: the tar belt asphalts (Cg 101, Aire 1) originate through bacterial degradation of rather immature heavy crudes similar to the LMC 1 oil (CONNAN, 1972; CONNAN and VAN DER WEIDE, 1978). Total alkanes In vitro experiments. The gas chromatograms of the total alkanes from the original crude oils provide some basic information on the origin and the maturity of both samples. The concentrations of biological markers (isoprenoids, steranes and triterpanes) related to maturity, differentiate the samples analyzed. The LMC t crude shows high quantities of both isoprenoids (pr/n-Cl~ = 0.65, phyt/n-C18 = 1.40, Table 3) and polycyclic alkanes (steranes and triterpanes) and may be ascribed as a low maturity crude. The PCE 2 crude displays an obvious depletion (Fig. 5) in these two classes of chemical fossils (refer to pristane/n-C~7 and phytane/n-C~8 ratios, Table 2) which agrees with its more mature character. A clue to the origin of both crudes is provided by the pristane to phytane ratios. Pristane to phytane ratios lower than 1.0 (Tables 2 and 3), along with other characteristic geochemical and sedimentological criteria confirm that both crudes originate from carbonate source rocks (algal-type kerogen) deposited under very reducing conditions (DIDYK et al., 1978). Occurrence of a detectable predominance of even n-alkanes is well in keeping with this assumption (WELTEand WAPLES, 1973). Figure 5 depicts the gradual degradation of the alkane fraction from the LMC 1 and PCE 2 crudes. Significant changes take place in the 5-10 day period, n-Alkanes, i.e. the most readily degradable structures, are completely removed. Simultaneously, homologous series of branched and cyclic compounds (probably iso- and anteiso-alkanes from the GC-MS study) disappear, while isoprenoids are still present. Isoprenoid uptake, however, has already begun as documented by the pristane/phytane decrease (Tables 2 and 3). During the 10-30 day period of incubation, acyclic isoprenoids are apparently removed. The stepwise uptake of the main classes of alkanes, observed herein, reproduces
Z
_o ,< O < e~
o o_z <
u
_z
PCE 2 CRUDE
LNIC 1 CRUDE
Fig. 5. Metabolismof the total alkane fraction of LMC 1 and PCE 2 crude by Pseudomonas oleovorans.Gas chromatographicconditions:OV 101,30 m x 0.25mm i.d., 100-285°C,2°C/mm.
8
JACQUESCONNAN, ANNICK RESTLEand PIERRE ALBRECHT
the biodegradation sequences described by previous authors (JOBSONet al., 1972; BAILEY et al., 1973; RUBTNSTEINet al., 1977; HIGGINS and GILBERT, 1978). Efficient consumption of iso-, anteiso-, isoprenoid alkanes entails a correlative increase in the alkane fraction of those members with four and five rings. This polycyclic alkane enrichment takes place both in the immature and in the more mature crude (Fig. 5). The alkane GC traces of the heavily biodegraded crudes (90 days, Fig. 5) display very similar patterns, especially within the high mol wt range. Biodegradation of the Aquitaine crude oils has severely obscured the specific character of the original crude oil alkanes (Fig. 5). The alkane fingerprint of biodegraded crudes is too unreliable to assess their maturity (Fig. 5). P C E 3 soils. Figure 6 represents the total alkane pattern of the PCE 3 crudes before and after the oil-spill in the PCE 3 soil. Similar events to those recorded in the laboratory affect the crude oil in the PCE 3 soil. C15 + n-alkanes are completely metabolized by bacteria in the surface sample, but they do survive at depth (10 to 20 cm below the soil surface, Fig. 6). Isoprenoid alkanes, a poorer substrate for hydrocarbon-oxidizing bacteria, are preserved at depth but are slightly attacked on at the surface (refer to pristane to phytane ratios, Table 4). Iso- and anteiso-alkanes also appear to be consumed. The
I
PCE 3
~LTERANIESETTRITERPANEE A •
EVAPORATION BIODEGRADATION
DISSOLUTION OXIDATION SOiLSURFACE
""~d L__
HIIII
OIL SPILL
I
10 to 20 cm BELOWSOILSURFACE
ERUPTION OFTHEPCE3
|
WELLAT 2 7|15.20m
~
j
~
REFERENCE
LOWER CRETACEOUS 2 795.20m
Fig. 6. Degradation of crude oils in soils: an example of the alkane alteration in a PCE 3 soil (Aquitaine basin). Same chromatographic conditions as in Fig. 5.
Biodegradation in the Aquitaine Basin Table 4. Analytical data on the PCE 3 crude altered in soils from the PCE 3 well site
.
so,P,R
352
HCE3CORDE [RUPTIONAT 2 736.M m
917
WiTHTaEPCEICRORE SWIFXCE- 2 YEARS
[,
°
I
.[i
17.1 36.0 33.0 11.0
2.1
15
|.75
11.0 32.1 45.0 I1.1
2.9
1.2
1.23
11.8 33.0 40.7 14.7~ 2.0
0,4
1.34
0.54
0,65
0,60
AFTER ERUPTION
910
POE 3 SOIL OIL - STAINED iFITHTHE PCE3CRUOE 10-2OCfll BELOW RNRFAC 2 TEARS AFTER ERUPTION
1.80
3.29
0.68
I
CHLOROFORMEXTRACTOR TOPPER OOOOE-TRE VOLATILEFRACTION, EVAPORATEDOY TOPPING REPRESENTS13 % OF THE WHOLECOUOE(BY WEIGHT)
end-products of the alkane metabolism (Fig. 6) closely resemble the alkane spectra of the Aquitaine crudes, biodegraded in the laboratory (Fig. 5). Transformation of alkanes in a soil by a mixed culture including hydrocarbon degraders of the genera Pseudomonas, proceeds through pathways analogous to the in vitro biodegradation of alkanes by a pure culture of Pseudomonas oleovorans. This assumption was further cross-examined by performing subsequent in vitro experiments. The mixed culture, growing in the oil-stained PCE 3 soil, was isolated. Its oilmetabolizing capability was compared to those of the pure genera of Pseudomonas oleovorans used in our experimental work. Similarities in degradation behaviour are strickingly visible on the alkane distribution patterns of Fig. 7. The in vitro experiments, applied in this study, seem to be fairly acceptable for simulating the combined effects of abiotic and biotic alteration of alkanes in soils. The slower rate of degradation in soils, however, is probably due to the different, less aerobic conditions. A 2 yr degradation in the PCE 3 soil may be reproduced by a 5 day degradation in the laboratory under more aerobic conditions. Branched and cyclic alkanes. Branched and cyclic alkanes have been examined in detail using a capillary-GC and a computerized-GC-MS analysis. Branched and cyclic alkanes of the Aquitaine crude oils, biodegraded under artificial (laboratory, Fig. 5 and 7) or natural (PCE 3 soil, Fig. 6 and 8; Cg 101 and Aire 1, Fig. 9) conditions exhibit very similar fingerprints. Major definite peaks occur within three molecular weight ranges corresponding to temperatures of: 150-200°C (isoprenoids), 200-250°C (peaks A to E, Fig. 8), > 250°C (steranes and triterpanes, Fig. 8). Steranes and triterpanes, detectable in all samples, are concentrated in the branched and cyclic alkane fraction of biodegraded crudes as a consequence of the consumption in other classes of compounds (iso-, anteiso-, etc.). A concentration process seems also to be involved in the magnification of compounds coded A to E. These compounds, well resolved in the PCE 3 biodegraded crude, are recognizable within the homologous series of iso- and anteisoalkanes of the PCE 3 original crude (Fig. 8). They have been tentatively identified by GC-MS (Multiple ion cross scan of m/e 149-163-217-231, Fig. 2) as two steranes (C21, M ÷ = 288; C22, M ÷ = 302) two 4-methylsteranes (C22, M ÷ = 302; C2a, M ÷ = 316) and a C24 (M ÷ = 330) tetracyclic terpane (Fig. 10). These compounds, revealed in the Aquitaine crudes by biodegradation, are not by-products of the bacterial activity. They also
10
JACQUES CONNAN, ANNICK RESTLE a n d PIERRE ALBRECHT
J C
W
j a:
L REFERENCE (FIG.6)
,<
3 MIXEO CULTURE- 5 OAYS
<
<
PSEUDOMQNAS OLEOVORANS . 50AYS
Li
,4
PSEUDOMONAS 0LEOVORANS- 1O DAYS
.:~
Fig. 7. Metabolism of the PCE 3 crude by a Pseudomonas oleovorans and a mixed culture isolated from the PCE 3 soil: gas chromatograms of total alkanes (C15+). Same conditions as in Fig. 5.
do exist in the unaltered crudes (Cg 101, 2895-2917 m, Fig. 9 and PCE 2, Fig. 5) where they are hidden within the homologous series of branched alkanes.
Cyclic alkanes Monocyclics and bicyclics. Polysubstituted monocyclic and bicyclic alkane series (Table 2), magnified in biodegraded crudes, are generally easy to identify. In some unaltered crudes, however, their detection still remains questionable due to their low concentration. M o n o - and bicyclic distributions are not identical in all the samples. In crude
Biodegradation in the Aquitaine Basin
11
PCE 3
(17~, 21~) H-HOPANE t~
$
E OC
u
J~
BA t
t
I
"...S
(17 ~ , 21 ~ ) H-HOPAN| ¢ •
I
-':
BENEATHSOIL SURFACE
10 to 20 cm
j
° c
,.
I
II
•
-I(
REFERENCE
LOWER CRETACEOUS 2 795.20 m
Fig. 8. Degradation of crude oil in soils: an example of the alteration of the branched and cyclic alkane fraction in a PCE 3 soil (Aquitaine basin). Same conditions as in Fig. 5.
oils, biodegraded in the laboratory, both series of compounds have been compared. In the LMC 1 crude, no removal of mono- and bicyclic alkanes was observed after 3 months of bacterial attack by the pure culture of Pseudomonas oleovorans. In natural asphalts from the Aquitaine tar belt (Aire 1, Cg 101, Fig. 9) i.e. more heavily altered crudes, mono- and bicyclic alkanes are obviously affected. This obvious uptake of small cyclic compounds, however should not be exclusively ascribed to biodegradation for it may also be partly due to solubilization by water washing. A progressive loss of monocyclic and to a lesser extent of bicyclic alkane has already been recorded by RUBINSTEIN et al. (1977) in their in vitro biodegradation experiments on the Prudhoe Bay crude. Tri-, tetra- and pentaeyclies. Several series of tri-, tetra- and pentacyclic terpanes were systematically examined in all the samples by computerized-GC-MS analysis (Fig. 2). These series were found to occur either in unaltered or in altered crudes. Their ubiquity in the crude oils from the Aquitaine basin was revealed due to the biodegradation study. The C21-C23 steranes series, for instance, easily detectable in natural asphalts or crudes biodegraded in the laboratory, was also detected later in all the unaltered crudes (Cg 101, 2895-2917 m, Fig. 9 for example). In this particular study, dealing with SW Aquitaine crude oils, one may consider that the tri- and tetracyclic series (Fig. 2) are as ubiquitous as the well-known hopane family (VAN DORSSELAER, 1975; VAN DORSSELAER and ALBRECHT, 1976). This remains, of course, to be proved on a larger number of samples.
12
JACQUES CONNAN, ANNICK RESTLE a n d PIERRE ALBRECHT
t~
o "
I[
o
u
E
°ii
t
Iiiiiiiiiiiiiiiiiiiii!~i~i~\\\7 l
V,~
~
~
o O -o
N
Biodegradation in the Aquitaine Basin
13
C29 C30 ~-j~ Hopane
Hoj ^ o~-~3Hopane I z'2! t. ~tl3~]21
C--
i ~'H7op ane C22 4C22eSt
co~r~r~r~ ,11¢L LI~ I .
L'24
Penta
J
i
Tri
St
J
o
b
PCE 3 - SOIL S U R F A C E C29
c~ Hopane fl Hopane C30 / L
c3~
C32 C33
ca9
St / C
C23 4 Me St C22
C34il1 l ll'~op~n*
c
4MeSt
I
I O
Penta I O
Tri I r~
I O
Cg 101- ASPHALT "CENOMANIAN 2682 : 2693 m
Fig. 10. Branched and cyclic alkanes of two biodegraded crudes: identification of major compounds. Gas chromatographic conditions: Polymethylsiloxane, 25 m × 0.3 mm i.d., 120-300°C,
3°C/ram.
But one may emphasize, in favour of the ubiquity that samples examined herein came from horizons from the Upper Cretaceous to the Trias. As far as these polycyclic compounds are concerned, the main question that arises concerns their biodegradability. Can these structures be metabolised by bacteria? Referring to the recent literature on the subject, one immediately notices that the question remains open. REED (1977) concludes that low mol wt tricyclic and tetracyclic terpanes are unchanged whereas pentacyclic triterpanes (hopane family) are partially degraded. SEIFERT and MOLDOWAN (1979) report that regular steranes and hopanes are transformed, but that tricyclic terpanes and diasteranes survive. In both cases no in vitro biodegradation experiments were carried out. RUBINSTEINet al. (1977), on the contrary, found that the sterane and the hopane families remain intact after biodegradation in vitro and in nature. Our results, related only to one case history, namely the Aquitaine Basin, confirm the conclusions of RUBINSTEIN et al.: the tri-, tetra-, pentacyclic families examined (Fig. 2) appear to be not significantly affected by bacterial degradation in the laboratory, in soils or under geological conditions in some boreholes from the Aquitaine tar belt. This conclusion may be drawn by comparing tri- tetra- and pentacyclic patterns presented in Figs 11 to 13. Figure 11 represents the tri- and tetracyclic patterns (role 191) of original and in vitro biodegraded crudes. This pattern, different from one crude (LMC 1) to the other (PCE 2) as shown by the C2a tricyclic to C24 tetracyclic ratio, remains unchanged after 90 days of bacterial attack by the Pseudomonas oleovorans (Fig. 11). Figure 12 extends the results of Fig. 11 to other classes of compounds, namely the tricyclic, tetra-
14
JACQUES CONNAN, ANNICK RESTLE and PIERRE ALBRECHT
C24 C25
bETRACYCLICSI~
C26
@ ® @@, . °
LAMEAC
LAMEAC
ORIGINAL
BIODEGRADED
CRUDE-BLANK
RUN-90 DAYS-
CRUDE - PSEUDOMONAS-90 ITETRACYCLICS ],ID *
@® PECORADE-ORIGINAL
PECORADE.BIODEGRADED
C24
DAYS -
C2(
@@ ® CRUDE-BLANK
RUN - 30 DAYS -
CRUDE- PSEUDOMONAS-
90 D A Y S -
Fig. 11. Cross scan of m/e 191 from original and in vitro biodegraded crudes. Tri- and tetracyclic alkanes are not affected by bacterial degradation.
cyclic and pentacyclic alkanes. The striking similarity in the distribution of polycyclic alkanes of both the unaltered (PCE 2) original crude and the biodegraded (PCE 2, 90 days, Pseudomonas oleovorans) crude, shows the non-degradability of these structures in our laboratory experiments. This non-degradability appears also to be the rule in the subsurface as documented by the comparison of the polycyclic distributions of both a natural asphalt (Aire 1, 1910m) and an asphaltic unaltered crude (Aire 1, 2979m) (Fig. 13). Furthermore no evidence for the formation of 4-desmethylhopanes was found in the m/e = 177 fragmentogram, confirming that the hopane skeleton was untouched under both natural and laboratory conditions.
15
Biodegradation in the Aquitaine Basin
:] :] ~] ~] ~]
~]
a. o en~l.
Q
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'----.e 0
:[
._= r~
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.-~.~
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16
JACQUESCONNAN, ANNICK RESTLEand PIERRE ALBRECHT STERANES m/e 217 - 231
Airu 1 TRICYCLICS m/e 191
Depth (m)
TETRACYC LICS ~ m/e 191
PENTACYCblCS mte 191
P, - C27 " C30
C21 C23
1000
2OOO
:":':':" •
It
:':':"
"
•:.:.: I_0
,; ,~ 2:,. 2~ i, ;9 a
1, ;9 2; 1;., 2', 4_Melt
'2; 21 •
' ;, 2g
' 30
2',
' ='= ' 22
1'~ '
23 o ~ HOPAidE~7
,; ' 29*
30*
30
1liar 31 31 32 12 33 33 34 34 35 3S
30*
~
ASPHALT (UPPER CRETACEOUS-1910 m)
3~-
4m4MaSt 21
29
30
22
23 ot.~ HOPANE27
2|*
32 32 33 33 34 14 39 35
• ASPHALTIC CRUDE (JURASSIC,CA : 2979 m) r---1
TERTIARY
p'--////~
UPPERTO M,DDLE JURASSIC
~
UPPER CIIETACEOUS
~
LOWER JURASSIC
LOWER CRETACEOUS
*
not evaluated : saturated peak
Fig. 13. Tri-, tetra- and pentacyclic alkane distributions of crude oils biodegraded under natural conditions: an example on the Aire 1 well.
CONCLUSIONS
Biodegradation of some Aquitaine crude oils in the laboratory using the Pseudomonas oleovorans strain, provides a suitable model to simulate biodegradation of hydrocarbons under natural conditions. The results of the in vitro biodegradation experiments are in agreement with the results obtained either in soils (oil spill) or underground in boreholes belonging to the Aquitaine tar belt area. Comparison of gross compositions of laboratory biodegraded crudes and of natural asphalts, suggests that the latter are more likely to be immature biodegraded crudes. Degradation mechanisms similar in the laboratory and in nature, occur at various rates. A transformation, observed after a 2-yr period at the soil surface, may be reproduced in the laboratory in a 5-day treatment period under aerobic conditions. Biodegradation of crude oils affects alkanes and to some extent aromatics. The alkane transformation entails a stepwise removal of n-, iso-, anteiso- and isoprenoid alkanes. This gradual metabolism of specific alkane structures obscures the original characteristics of the total alkanes. Therefore, no maturity assessment could be done on the basis of alkanes from drastically biodegraded crudes. Apart from the above-mentioned alkanes, recognized as consumed by bacteria, one may emphasize the survival of tri-, tetra-, and pentacyclic alkanes. These families of polycyclic alkanes have been found to be unattacked by bacteria in the laboratory (Pseudomonas oleovorans or mixed culture), in the PCE 3 soil (mixed culture), in the subsurface (boreholes from the Aquitaine tar belt). These classes of compounds, resistant to bacterial attack in the case of Aquitaine crudeoils, may be regarded as possible useful tools to correlate biodegraded to unaltered crudes.
Biodegradation in the Aquitaine Basin
17
Biodegradation of crude oils in the Aquitaine basin has revealed the ubiquity of several families of tri-, tetra- and pentacyclic alkanes. Among these, we have identified the ~fl hopane family and detected the presence of a series of tricyclic terpanes ranging from C~9 to C26 and of a novel group of C21-C23 steranes (C21-C22 steranes, C22-C23 4-methylsteranes). The elucidation of the exact chemical structures of these compounds is at present under investigation. REFERENCES
BAILEY N. J. L., JOBSONA. M. and ROGERS M. A. (1973) Bacterial degradation of crude oil: comparison of field and experimental data. Chem. Geol. 11, 203-221. CLARET J., TCHIKAYA J. B., TISSOT B., DEROO G. and VAN DORSSELAER A. (1977) Un exemple d'huile biodegrad~e b, basse teneur en soufre: le gisement d'Emeraude (Congo). In Advances in Organic Geochemistry 1975 (eds R. CAMPOSand G. GONI) pp. 509-522. ENADIMSA. Madrid. CONNAN J. 0972) Laboratory simulation and natural diagenesis 1. Thermal evolution of asphalts from the Aquitaine basin (SW France). Bull. Centre Rech. Pau-SNPA 6, 1, 195-214. CONNAN J. and VA~ DER WEIDE B. M. (1978) Thermal evolution of natural asphalts. Chapter 3. In Bitumens, Asphalts and Tar sands (eds G. V. CHILINGARIAN and T. F. YEN) pp. 27-55, Elsevier Scientific Publishing Company. DEMAISON G. T. (1977) Tar sands and supergiant oil fields. AAPG Bull. 61, 11, 1950-1961. DEROO G., TISSOT B., McCROSSAN R. G., DER F. (1974) Geochemistry of heavy oils of Alberta. In Oil Sands Fuel of the Future Memoir 3. Can. Soc. Petro. Geol. 148-167, 184-189. DIDYK B. M., SIMONEIT B. R. T., BRASSELLS. C. and EGLINTON G. (1978) Organic geochemical indicators of palaeoenvironmental conditions of sedimentation. Nature 272, 5650, 216-222. EVANS C. R., ROGERS M. A. and BAILEY N. J. L. (1971) Evolution and alteration of petroleum in Western Canada. Chem. Geol. 8, 147-170. HEISE H. (1975) Geochemistry of Beaufort basin oil. New Ground Can. Petrol. 20, 7, 41-43. HIGGINS I. J. and GILBERT P. D. (1978) The biodegradation of hydrocarbons. Chapter 7. In The Oil Industry and Microbial Ecosystems (eds K. W. A. CHATER and H. J. SOMERVILLE)pp. 80-117. Heyden and Son Ltd. JOBSON A., Cook E. D. and WESTLAKED. W. S. (1972) Microbial utilization of crude oil. Applied Microbiology 23, 6, 108~1089. MILNER C. W. D., ROGERS M. A. and EVANS C. R. (1977) Petroleum transformation in reservoirs. J. Geochim. Explor. 7, 101-153. REED W. E. (1977) Molecular compositions of weathered petroleum and comparison with its possible source. Geochim. Cosmochim. Acta 41,237-247. RUBINSTE1N I., STRAUSZO. P., SPYCKERELLEC., CRAWFORD R. J., WESTLAKED. W. S. (1977) The origin of the oil sands bitumens of Alberta: a chemical and a microbiological simulation study. Geochim. Cosmochim. Acta 41, 1341-1353. SEIEERT W. K. and MOLDOWAN J. M. (1978) Applications of steranes, terpanes and monoaromatics to the maturation, migration and source of crude oils. Geochim. Cosmochim. Acta 42, 77-95. SEIEERT W. K. and MOLDOWAN J. M. (1979) The effect of biodegradation on steranes and terpanes in crude oils. Geochim. Cosmochim. Acta 43, 111-126. VAN DORSSELAERA. (1975) Triterpanes de s6diments. Th6se de Doctorat 6s-Sciences. Universit6 Louis-Pasteur, Strasbourg. VAN DORSSELAER A. and ALBRECHT P. (1976) Marqueurs biologiques: origine, evolution et applications. Bull. Centre Rech. Pau-SNPA 10, i, 193-200. VAN DORSSELAERA., SCHMITTERJ. M., ALBRECHTP., CLARETJ. and CONNAN J. (1978) Use of biological markers in correlation problems. 8th International Congress of organic geochemistry, Moscow (in press). WELTED. M. and WAPLES D. (1973) Uber die Bevorzugung gerad zahlicher n-Alkane in sedimentgesteinen. Naturwissenschaften 60, 516-517. WINTERS J. C. and WILLIAMS J. A. (1969) Microbiological alteration of crude oil in the reservoir. Symp. on petroleum transformations in geologic environments. Am. Chem. Soc. Div. Petroleum Chem. New York, 7-12 September, Preprints 14(4), E 22-E 31.