CRUDE OIL BIODEGRADATION AND ENVIRONMENTAL FACTORS AT THE RIUTORT OIL SHALE MINE, SE PYRENEES

123Journal of Petroleum Geology, Vol. 33(2), April 2010, pp 123-140

© 2010 The Authors. Journal compilation © 2010 Scientific Press Ltd

INTRODUCTION

Biodegradation is the alteration and/or removal ofhydrocarbons from oil by microbial action duringmigration or in a reservoir (e.g. Connan, 1984; Larteret al., 2003; Head et al., 2003; Jones et al., 2008), inan oil seepage at the surface (Watson et al., 2000;

1 Dpt. de Geoquímica, Petrologia i Prospecció Geològica,Facultat de Geologia, Universitat de Barcelona. Martí iFranquès, s/n, 08028 Barcelona, Catalonia, Spain.2 Área de Prospección e Investigación Minera, Universidadde Oviedo, 33600 Mieres, Asturias, Spain.3 Dpt. Petrología y Geoquimica, Universidad Compluensede Madrid, 28040 Madrid, Spain. Present address: REPSOL,CTR, 28931 Móstoles, Spain.4 TOTAL, CSTJF, 64000 Pau, France.

* Corresponding author, email:[email protected]

Key words: biodegradation, oil seeps, oil shales, Eocene,Armàncies Formation, Pyrenees.

Liquid oil seeps from organic-rich source rock intervals in the Eocene Armàncies Formation in thewalls of the underground Riutort oil-shale mine in the SE Pyrenees. The mine was excavated at thebeginning of the last century for oil shale extraction. For this study, oil samples were recoveredfrom fractures in the mine walls, and from pools of water on the mine floor. Some oil is present atthe bottom of these pools; oil also floats on the surface of the water in association with emulsionscolonized by microbial mats. The oils have undergone variable degrees of biodegradation.

The physical and chemical environment in the mine was studied in order to establish thecontrols on biodegradation processes. The results show that the degree of biodegradation dependedon factors including the location of the oil (i.e. floating on the top of the water or from the bottomof a pool), and the addition of fresh seepage oil. The biodegradation observed mainly involved theprogressive removal of n-alkanes, isoprenoids and some aromatics. Biodegradation was also assessedin terms of the sulphur content and by quantitative analyses of molecular markers in the aromaticfraction. These approaches indicated that at least 50% of the oil was lost as a result of biodegradation.Isotope studies were also undertaken but isotope signatures did not provide significant data.

Microbiological data were consistent with data collected from chemical analyses. Evidence forthe presence of hydrocarbon-degrading bacteria were obtained from laboratory studies.

CRUDE OIL BIODEGRADATION ANDENVIRONMENTAL FACTORS AT THERIUTORT OIL SHALE MINE, SE PYRENEES

A. Permanyer1*, J. L. R.Gallego2, M. A. Caja3 and D. Dessort 4

Röling et al., 2006), or at an oil spill (Bragg et al.,1994; Atlas and Cerniglia, 1995; Gallego et al.,2006a). Microbial degradation, due mainly to bacterialactivity, causes a progressive loss of chemically-simple structures and hydrogen-rich compounds in asequence which is similar both in a deeply-buriedreservoir and at the surface. The loss of lightcomponents leads to a relative increase in aromaticand polar compounds and therefore in density (Headet al., 2003).

During biodegradation, various classes ofcompounds are degraded simultaneously but atsignificantly different rates (Peters et al., 1996; Eliaset al., 2007). Biodegradation kinetics depend on therange of compounds present in an oil, and the resultingprocesses can be rapid (e.g. the removal of n-alkanes)

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124 Biodegradation at the Riutort oil shale mine, SE Pyrenees

to very slow (e.g. the removal of biomarkers such asdiasteranes, polar compounds, resins and asphaltenes).The sequential depletion and removal of families ofcompounds has been used as a basis forbiodegradation scales such as the “PM scale”proposed by Peters and Moldowan (1993), whichfocussed on moderate to severe levels ofbiodegradation. By contrast, Wenger et al. (2001)proposed a sequential degradation scheme with moreemphasis on initial and moderate levels of alteration.The stepwise sequence of biodegradation can in somecases follow different paths (Chosson et al., 1992)and biodegradation scales based on biomarkers shouldtherefore be used with caution (Peters et al., 2005).

Biodegradation in a reservoir takes place overgeological time-scales and has major economicconsequences (Aitken et al., 2004; Elias et al., 2007).Biodegradation of oil results in a decrease inhydrocarbon content and in API gravity, and anincrease in sulphur content, acidity and viscosity. TheOrinoco and Athabasca “tar sands” in Venezuela andCanada appear to be composed of severelybiodegraded oil (Demaison, 1977).

Biodegradation processes in reservoirs werepreviously ascribed to the action of aerobic bacteria(Chosson et al., 1992; Connan, 1984; Connan andDessort, 1987; Peters and Moldowan, 1993).However, this hypothesis has recently been challenged(e.g. Connan et al., 1997; Magot et al., 2000; Head etal., 2003) as a result of detailed descriptions of oilfieldmicrobiota. These studies showed the occurrence ofviable bacteria at depths of 1500 m or more, and thedominance of strict anaerobes (sulphate-reducing andmethanogenic bacteria) in locations where the oilreservoirs had not reached the “sterilization”

temperature of 80ºC (Wilhelms et al., 2001).Consequently, the rate of oil biodegradation in thesubsurface appears to be limited by the temperaturehistory of the reservoir and by the presence of water,given that most biodegradation occurs at oil-waterinterfaces. This is controlled by the size of the waterleg which controls the delivery of nutrients (Larter etal., 2001; Larter et al., 2003; Röling et al., 2003).

The study of biodegradation in deep oil reservoirsis not straightforward. Obtaining adequate samples isnot easy, and there are inherent difficulties in thelaboratory study of thermophilic and anaerobicbacteria. Complexity can also be added by the mixingof degraded and non-degraded oils, while reactionsin the biodegradation process can be difficult tosimulate in the laboratory (Röling et al., 2006).Aerobic processes are faster, and therefore easier tomodel, for complex mixtures of hydrocarbons thananaerobic biodegradation. At shallow depths andwhere oil seeps are degraded, oxygen is the dominant(but not exclusive) electron acceptor, the circulationof meteoric water is widespread, nutrients areabundant, and, in consequence, oil degradation canbe measured on a human time-scale. On average, oilseeps show a degree of degradation which is directlycontrolled by environmental factors, and which caneasily be related to phenomena such as water washing,mixing and oxidation.

At the Riutort oil shale mine in the SE Pyrenees,liquid oil seeps from the Eocene organic-richArmancies Formation at numerous locations(Permanyer, 2004). Excavation of the mine took placemore than 100 years ago. The slow percolation ofgroundwater through the mine galleries has resultedin pools of water of various sizes. Oil is present in

PYRENEES

N30 Km

THRUST SHEETS

UPPER

LOWER B AS E ME NT AUTOCHTHON

PYRENEES

DUEROBASIN

TAJOBASIN

IBERIANCHAIN

EBROBASIN

BETICCORDILLERA

300 km

IBERIANMASSIF

PYRENEES

0

N

AXIAL ZONE

E B R OB A S I N

CADITHR USTSHEET

NO RT H P Y R E NE AN FAULT

STUDYAREA

Terrades

ANDORRAJaca

IBERIAN PENINSULA

0º43ºN

42ºN

BagàSOUTH

PYRENEAN CENTRAL UNIT

Tremp

EBROBASIN

Fig. 1. Location map of the studied area and structural features of the SE Pyrenees.

125A. Permanyer et al.

some of these pools, and is accompanied by the growthof microbial mats (Permanyer, 2000; Permanyer andDessort, 2004; Permanyer and Caja, 2005; Permanyeret al., 2006; Gallego et al., 2006b). Thebiodegradation of oil can therefore be observed in situ.Biodegradation processes at the mine are mainlyaerobic, and therefore do not apply to the anaerobicconditions in deeply-buried reservoirs. The absenceof light from the mine galleries has excluded thegrowth of photosynthetic organisms.

In this context, this study attempts to: (i) describethe physical and chemical parameters which controlthe mine environment; (ii) determine the degree ofaerobic biodegradation of oils sampled at diverselocations in the mine; and (iii) investigate themicroorganisms involved in the degradationprocesses.

GEOLOGICAL SETTING

The SE portion of the Pyrenees consists of a stack ofsouth-vergent thrust units which include bothsedimentary cover and basement (Fig. 1), and whichoverthrust the Lower Eocene-Oligocene Ebro Basin.Two thrust systems can be distinguished (Muñoz etal., 1986): upper and lower. The Cadí thrust sheet,which is composed of a thick succession of lower-middle Eocene and Paleocene sediments restingdiscordantly on basement, is the most important ofthe lower units in the studied area (Puigdefábregas etal., 1986; Vergés and Martínez, 1988; Clavell et al.,1988) (Fig. 2).

The Eocene Armàncies Formation forms part ofthe Cadí thrust sheet and crops out along a distanceof about 100 km. It is composed of shales, marls,carbonates and locally sandstones up to 600 m thick(Fig. 2). The lower 220 m of the formation has a highorganic matter content. The formation is composedof alternating argillaceous limestones and mudstones(Fig. 3). The cabonate content in the shales variesinversely with the organic carbon content (Permanyeret al., 1988). Organic material is mainly of marineorigin and consists of filamentous algae, dinoflagellatecysts, remains of fish scales and other unrecognisabledebris. Vitrinite and material of terrestrial origin isvery rare or absent. The total organic carbon contentin the shale is between 1.4% and 14.5%.

Rock Eval pyrolysis of shale samples (Permanyer,2004) gave an S2 of 6.4 to 48.2 mg HC /g of rock,with S1 up to 4.7 mg HC /g of rock. These valuesdiminish from shales to marls and are reduced verysharply in limestones in inverse proportion to thecarbonate content. Average values of the HydrogenIndex range between 475 and 650 (maximum up to745), while the Oxygen Index ranges from 2 to 14(Permanyer, 2004; Permanyer et al., in prep.).

GA

RU

MN

IAN

FAC

IES

CA

DI

FmS

AG

NA

RI

FmC

OR

ON

ES

Fm

Low

er M

bU

p. Mb

AR

MÀ

NC

IES

Fm

VALL

FOG

ON

A F

mB

EU

DA

GY

PS

UM

AGE

L U

T E

T I

A N

Low

erU

pper

Mid

dle

Low

er

C U

I S

I A

NIL

ER

DIA

N Upp

erLo

w. &

Mid

.

Upp

.K.-

THA

NE

TIA

N

2000

0

500

1000

1500

m

alluvial fans

shallowcarbonateplatform

prodelta

delta plain

RESTRICTEDPLATFORM

SLOPE

basin

turbidites

marineevaporites

G RMUnits

detachment surface

Fig. 2. Composite stratigraphic column for the CadíThrust Sheet. The Riutort Mine exposures arelocated in the lower part of the ArmànciesFormation.


The thermal maturity of organic material fromArmàncies Formation was indicated by Rock-Eval Tmaxvalues of 438º and 441ºC. Measured reflectivity onrare vitrinite remains is 0.65 % Ro.

Chromatograms of saturated hydrocarbons show apredominance of n-alkanes in the range C17 to C23,indicating an input of algal material. There is aprevalence of even over odd n-alkanes in the rangeC26 to C30. This can be attributed to the carbonate-richnature of the Armàncies Formation sediments(Permanyer et al., in prep.).

Underground galleries at the Riutort mine are cutinto the Armàncies Formation. The mine wasoperational during the first few decades of the 20thcentury, producing approximately 3400 tons of rock(Riba, 1985) for shale oil production. Oil productionwas 90 l of oil per ton of rock heated. Some 8-9 kg ofammonium per ton of rock was also produced.Approximately 80% of rocks at the mine are composedof oil shales. At the present day, oil is expelled alongbedding planes and vertical fractures from organic-richshale intervals intercalated within limestones and marls(Fig. 3). Different degrees of oil degradation (diversecolours and viscosities) can be observed on the ceiling,walls and in the pools of water. Oil is mixed withmicrobial mats in pools (Fig. 4).

MATERIALS AND METHODS

SamplingThe Riutort mine includes several differentunderground galleries (Figs 5A and 5B). One particulargallery, the “study” gallery, located approximately 70m from the main entrance, was selected as a sampling

area because of the good preservation of oil there.Control points for water quality were established.Two types of samples were collected for analysis:oils and microbial aggregates.

Pools of water were present on the gallery floor.The physical and chemical characteristics of waterwere determined in 13 pools (samples W1 to W13;Fig. 5B). Drip water (sample W0) was collecteddirectly from a carbonate stalactite in a plastic bottle.

Oil sampling points were selected in accordancewith the nature of the oil and its location with respectto the pools of water. Samples were:

M1: a composite sample of oil droplets drainingfrom the rock;

M3: a composite sample of “black” oil floatingon a pool of water;

M5: a composite sample of oil from the bottomof a pool;

M6 and M7: oil mixed with microbial aggregates;M8: fresh oil from a fracture; andM11: a composite sample of “brownish” oil

floating on a pool (Fig. 4).All the oil samples were collected in glass vials

and kept at 4ºC until analysis.A striking feature of the water pools was the

growth of microbial mats on the oil floating on thesurface (Fig. 4). After sampling, the biologicalaggregates were preserved in sterile bottles at roomtemperature prior to microbiological characterization.

Water AnalysesTemperature, pH, oxidation–reduction potential (Eh),electrical conductivity and dissolved oxygen in thewater were measured in situ using hand-held

Fig. 3. Left: field photograph of the Armàncies Formation exposed near the Riutort mine; Right: view of thestudy gallery (see Fig. 5).


AA

CC

B

D

B

DD

Oil

Oil

Oil

Oil + Water

Microbial mat

Microbial mat5 cm

25 cm

20 cm

20 cm

Mineentry

Studygallery

A

B

N

10 m

BWater-pool sample

1

2

3

45

6

7b8a

8b9

101112

7a

0

Water-pools

Drip water (stalactite)

13

Gallery door

> 10ºAPI

< 10ºAPI

Oil

Water

Bacterial matOil

Clays C

Fig. 5. (A) Schematic map of the Riutort Mine showing the location of the study gallery (modified from Riba,1985). (B) Detailed map of the study gallery showing the position of the water samples. (C) Schematic cross-section of a pool of water. Pools may be 20 cm to 1 m long and 30 cm wide; depth is about 20cm.

Fig. 4. (A). Photograph of the mine wall showing seeping oil; (B and D). Photographs of water pools with blackand brownish oil and microbial mats; (C). Photograph of the mine ceiling with stalactites and seeping oil.


instrumentation systems (Hanna Instruments). Inaddition, portable gas testers (Dräger Accuroaspiration system) were utilized to determine gascontents in the mine (CO2, CO, H2S).

Oil AnalysesTo quantify the degree of biodegradation,hydrocarbons were fractionated into saturates andaromatics. Gas chromatography was carried out onsaturated fractions using a Delta Chrom Series 9980apparatus with a flame ionization detector. Separationwas achieved on a fused silica capillary column (J&WPONA) (50 m * 0.2 mm * 0.5 μm). Chemstation AgilentTechnologies 1990–2000 software was used. The GCoperating conditions were as follows: temperature wasmaintained at 35ºC for 15 min, then increased from35 to 320ºC at a rate of 2 ºC/min, and maintained at320ºC for 30 min. Saturated fractions were alsoanalysed by gas chromatography–mass spectrometry(GC–MS) using a Thermo Electron MD 800 highresolution mass spectrometer coupled to a ThermoElectron 8060 gas chromatograph. The GC/MS useda non-polar J&W DB-5 column (60 m * 0.25 mm *0.1 μm). Operating conditions were as follows:temperature was maintained at 40ºC for 1 min, thenincreased from 40 to 300ºC a rate of 2ºC/min, andmaintained at 300ºC for 60 min. The mass spectrometerwas operated in electron impact mode with anionization voltage of 40 eV and a source temperatureof 200ºC. Data were recorded by selected ionmonitoring mode of eight ions. Terpane, sterane anddiasterane ratios were calculated from the resultingm/z 177, m/z 191, m/z 217, m/z 218 and m/z 259 massfragmentograms. Data were processed with Masslab1.4 software.

The aromatic hydrocarbons were analysed usingan HP 6890/5973 GC/MS/FID instrument and d10-phenanthrene, d8-dibenzothiophene and d12-chrysene

as internal standards. GC conditions were 60m x 0.25mm DB5 (J&W) column, 0.1 μm phase thickness. Inaddition, a 5 m deactivated precolumn was installedbetween the injection point and the analytical columnto improve the chromatographic resolution. Thesample was injected in the cold on-column injectorto avoid discrimination of the heavy compounds. Afterinjection, the injector temperature was raised to 300°Cto sweep the heavier compounds from the injector.The temperature programme of the oven rangedbetween 40°C and 300°C at a rate of 2°C/min, then66 min at 300°C.

The column end was connected (using a glass Y-connector) to the MS and to a Flame IonisationDetector (FID), and the mass spectrometer wasswitched in full scan mode (50-550 amu/sec.). Thisacquisition mode is necessary to detect all aromaticcompounds. After acquisition was completed,automatic data processing was carried out using aproprietary protocol for geochemical compounds(Dessort and Connan, 1995)

Carbon isotope measurements of saturate andaromatic hydrocarbons were performed on a ThermoFinnigan Series 1112 elemental analyser coupled toa Finnigan Mat Delta C mass spectrometer. Thereference materials were Graphite (USGS 24),Saccharose (IAEA-CH6), Polyethylene (IAEACH7)and Oil (NBS-22).

The sulphur content was determined by elementalanalyses, using a Carlo Erba EA 1108 CHNS-Oinstrument.

Microbial analysesDetection of aerobic microorganisms (bacteria, yeastand fungi) was carried out by spreading 0.1 ml ofoil-microbial aggregates on plates with three media:GAE medium to grow culturable bacteria (Braña etal., 1986); GPIA medium to detect yeasts (40 g/l

Sample Type pH Eh (mV) T (ºC) O2 (mg/l) Conductivity (mS/cm) Comments0 Drip water 7.3 -270 - 3.3 3 No oil 1 Pool water 7.8 -350 11.7 <0.5 3.8 Oil present2 Pool water 8.4 -275 11.7 1.3 3.7 Oil present3 Pool water 7.9 -309 11.4 <0.5 3.4 Oil present4 Pool water 7.7 -345 11.3 <0.5 3.4 No oil 5 Pool water 7.7 -225 - 4 3.1 No oil 6a Pool water 7.6 -136 - <0.5 2 No oil 6b Pool water 7.6 -115 - -0.5 2 Oil present7a Pool water 8 -117 11.4 2.4 1.9 No oil 7b Pool water 7.6 -73 11.4 2.4 1.9 No oil 8 Pool water 7.6 -72 - 1.4 1.9 No oil 9 Pool water 7.7 -226 11.2 <0.5 1.8 Oil present

10 Pool water 7.5 -281 - <0.5 1.9 Oil present11 Pool water 7.7 -154 - 4 1.7 No oil 12 Pool water 7.6 -172 - 4 1.7 No oil 13 Pool water 7.6 -171.6 - 4 1.7 No oil

Table 1. Summary of the physical and chemical properties of the water samples from the Riutort mine.


glucose, 0.5 g/l micopeptone, 0.5 g/l yeast extract and20 g/l agar); and a selective medium to grow fungi(Sabouraud, Conda) supplied with 0.5 g/lchloramphenicol to prevent bacterial growth. Plates wereincubated at 30ºC. Dominant microorganisms were re-isolated and identified: sequence analysis of theribosomal 16S gene (16S rDNA) was performed afterDNA extraction (Bac 100 Kit, Chemagen), PCRproducts were purified by GFXTM PCR DNA and GelBand Purification (Amersham Biosciences), andsequences were obtained in an ABI PrismTM 3100Genetic Analyzer (Perkins Elmer). Identification wasmade using the Gapped BLAST database (Altschul etal., 1997).

RESULTS AND DISCUSSION

Physical and chemical environment andconsequences for biodegradationThe gallery studied had a relative humidity of 100%and a constant temperature of 12ºC (± 0.5ºC),irrespective of the time of the year.

The presence of light and oxygen can favour theoxidation of some petroleum constituents, especiallyaromatics and saturates (Charié-Duhaut et al., 2000).

However, as the studied gallery was in constant totaldarkness, neither biodegradation due tophotosynthetic organisms nor photo-degradation ofaromatics was expected.

Fresh drip-water from stalactites had a pH of7.3. This was lower than the pH in pools of waterwhich was elevated by the presence of carbonates(Table 1). The drip-water had a content of dissolvedoxygen (3.3 mg/l) which was lower than thatexpected in percolating waters (6 to 8 mg/l). Thiscould be due to oxygen consumption while the waterwas within the rock. Given the low permeability ofthe sedimentary rocks in the Armàncies Formationand given that the galleries are approximately 50 to75 m from the surface, the residence time ofrainwater in the rocks may be lengthy. Oxygendissolved in the water may therefore have beenpartially consumed by interactions with the countryrocks.

Pool-waters had pH values of 7.6 to 8.4, almostconstant temperatures of 11.2 to 11.7ºC, electricalconductivities of 1.7 to 3.8 mS/cm, and redoxpotentials ranging from -350 to -72 mV (there wasa moderate negative correlation with dissolvedoxygen levels: Table 1). Levels of dissolved oxygen

Peaknumber

Molecularformula Weight Compound

1 C19H34 262 Tricyclic terpane 2 C20H36 276 Tricyclic terpane 3 C21H38 290 Tricyclic terpane 4 C23H42 304 Tricyclic terpane 5 C24H44 318 Tricyclic terpane 6 C25H46 346 Tricyclic terpane 7 C24H42 330 Tetracyclic terpane 8 C26H48 360 Tricyclic terpane (22R, 22S)9 C28H52 388 Tricyclic terpane (22R)10 C28H52 388 Tricyclic terpane (22S)11 C29H52 402 Tricyclic terpane (22R)12 C29H52 402 Tricyclic terpane (22S)13 C27H46 370 18α(H)-22,29,30-Trisnorneohopane (Ts) 14 C27H46 370 17α(H)-22,29,30-Trisnorhopane (Tm) 15 C29H50 398 17α(H)-21β(H)-30-Norhopane 16 C29H50 398 18α(H)-Norneohopane (29Ts) 17 C30H52 412 Diahopane18 C30H52 400 17α(H),21β(H)-Hopane19 C30H52 412 17β(H),21α(H)-Moretane 20 C31H54 426 17α(H),21β(H)-Homohopane (22S) 21 C31H54 426 17α(H),21β(H)-Homohopane (22R)22 C30H52 412 Gammacerane 23 C32H56 440 17α(H),21β(H)-Homohopane (22S)24 C32H56 440 17α(H),21β(H)-Homohopane (22R)25 C33H58 454 17α(H),21β(H)-Homohopane (22S)26 C33H58 454 17α(H),21β(H)-Homohopane (22R)27 C34H60 468 17α(H),21β(H)-Homohopane (22S)28 C34H60 468 17α(H),21β(H)-Homohopane (22R)29 C35H62 482 17α(H),21β(H)-Homohopane (22S)30 C34H60 482 17α(H),21β(H)-Homohopane (22R)

Table 2. Terpane (m/z191) identification ofpeaks in Fig. 6.


were controlled by the presence or absence ofmicrobial mats which were associated with either oilfloating on the surface of, or at the bottom of, thepools (Fig. 3). Pools in which oil and microbial matswere not present are relatively enriched in dissolvedoxygen (up to 4 mg/l); whereas pools with oil hadoxygen contents less than 1.5 mg/l. These values werestill greater than the levels of oxygen (<0.5 mg/l) inpools located at the bottom of the gallery (Table 1).Oxygen consumption was due to aerobic microbialactivity in pools containing oil, and this wasparticularly significant in the area at the bottom ofthe gallery.

Microbial activity is usually accompanied by CO2production. An atmosphere containing 500 ppm ofCO2 was detected in the proximity of pool number 1(Fig. 5B), and the level of CO2 decreased toward theexit (close to pool number 13) where only 250 ppmof CO2 were measured. H2S (2 ppm) was detected afterremoval of oily mud at the bottom of a number ofpools, indicating that anaerobic processes hadoccurred there.

Saturated hydrocarbonsCharacteristics of fresh oilThe composition of the oil samples was affected by:(i) biodegradation at varying stages; (ii) the partialevaporation of the lighter compounds (~ C15-); (iii)variations in the composition of the source material;and (iv) the replacement of the biodegraded oil byfresh oil.

Sample M8 (from a fracture opened duringsampling) was chosen as a reference sample. Thissample was regarded as “fresh” oil because it showedthe highest content of saturated hydrocarbons,especially n-alkanes. A gas chromatogram of thesample showed a significant contribution of n-alkaneswith minor UCM (Unresolved Complex Mixture)(Fig. 6A). The plot was characterised by apredominance of n-alkanes between n-C17 and n-C22with minor dominance of n-C21, and with no odd/evenprevalence. The isoprenoid ratio Pr/Ph was >1.

Pentacyclic terpanes were characterized by lowtrisnorneohopane / trisnorhopane ratios (Ts/Tm = ca.0.8) and by the predominance of the C30 hopane.

Sample M-8 M-1 M-3 M-5 M-11 M-7 M-6

SAT HC mg/g 5557 4713 2546 3699 3198 5000 2900 ARO HC mg/g 2028 2258 2424 1654 4601 3400 2600 NSO mg/g 2415 3028 5029 4748 2301 1600 4500 n-alkanes % 19,6 13,1 8,8 4,9 0,8 0,6 0,4 Iso-cyclo alkanes % 18,1 15,8 13,9 14,5 7,3 1,3 1,3 UCM % 62,3 71,1 77,4 80,7 91,9 98,1 98,3 Pr/Ph 1,2 1,0 1,0 1,0 0,7 0,7 0,5 Pr/nC17 0,7 0,9 1,0 1,7 2,1 2,2 1,6 Ph/nC18 0,5 0,8 0,9 1,5 4,1 2,4 1,9 C27 Steranes (%) 39 39 38 39 51 42 20 C28 Steranes (%) 28 30 33 33 19 10 29 C29 Steranes (%) 33 31 29 28 29 48 51 Diasteranes / Steranes 0,2 0,2 0,1 0,2 0,5 0,4 0,2 Diasteranes index 0,6 0,6 0,4 0,5 1,7 1,7 0,7 C21 / C23 tricyclics 0,8 0,6 0,6 0,6 0,7 0,5 0,5 C26 /C25 tricyclics 0,6 0,6 0,6 0,6 0,5 0,6 0,6 Tricyclics / Hopanes 0,2 0,3 0,4 0,5 0,3 0,5 0,2 Ts/Tm 0,8 0,8 0,9 0,9 0,6 0,9 0,9 Eq. Ro % 0,7 0,7 0,7 0,7 0,7 0,70 0,7 Oleanane index 0,1 0,1 0,1 0,1 0,1 0,2 0,2 Gammacerane. index 0,1 0,1 0,1 0,1 0,1 0,4 0,3 Homohopane index 0,1 0,1 0,1 0,1 0,1 0,1 0,1 Sulphur % 0,49 0,59 0,66 0,82 1,00 nd nd δ13C SAT ‰ -25,1 -25,3 -25,6 -25,4 -22,7 -24,40 -25,30 δ13C ARO ‰ -23,6 -23,6 -23,4 -23,6 -21,1 -23,20 -23,60

UCM: Unresolved Complex Mixture (% of GC area) Pr: Pristane; Ph: Phytane Steranes (%): 5α(H),14α(H),17α(H)-20R-Steranes Diasteranes Index = 13β,17α Diacholestane 20S+20R / ααα Cholestane 20S + 20R C27 RoEq: vitrinite reflectance equivalent = 0.574+(0.148*Ts/Tm), van Graas (1989) Tricyclics/Hopanes: Σ C19-C30 Tric / Σ C29-C35 Hop Ts/Tm: 18α(H)-22,29,30-Trisnorneohopane/17α(H)-22,29,30-trisnorhopane Oleanane index = Oleanane/C30 Hopane Gammacerane index: Gammacerane/αβC30 hopane Homohopane Index= (C35Hop/ΣC31Hop-C35Hop)

Table 3. Geochemical characteristics of studied oil samples from the Riutort mine.


Homohopanes were present in descending order fromC31 to C35 without any dominance, with a homohopaneindex of 0.08. The relative presence of C24 tetracyclicterpanes was notable. Abundant C24 tetracyclicterpanes were interpreted to indicate carbonate andevaporitic depositional environments (Palacas et al.,1984; Connan et al., 1986; Connan and Dessort, 1987;Clark and Philp, 1989). Terpanes were characterisedby the presence of tricyclics (tricyclic/hopane ratio =0.18) with respect to source rock extracts of the

A

B

C

Steranesm/z 217

Terpanesm/z 191

Saturated HCTIC

PrPh

n-C2

1

n-C 2

4

n-C1

7 n-C1

8

n-C1

3

n-C3

0

1 23

45

13

867

9, 10

11, 1

2

1417 19

16

20, 2

1

15

18

23, 2

425

, 26

27, 2

8

29, 3

0

22

C27 Diasteranes

C27 Steranes

C29 Steranes

C28 Steranes

Retention time

Inte

nsity

Fig. 6. Chromatograms of sample M8 (“fresh oil”).(A). Gas chromatogram of saturated hydrocarbonfraction (Pr: pristane, Ph: phytane, nC17: normalalkanes). (B and C). Mass chromatograms showingthe distribution of steranes (m/z 217) and terpanes(m/z 191). See Table 2 for peak identification.

M11

M5

M3

M1

M8

M7

M6

Inte

nsi

tyIn

tensi

ty

nC17nC18

PhPr

nC17

Retention time

Fig. 7. Gas chromatograms of saturated fractions ofstudied samples. The progressive reduction in n-alkanes, iso- and cyclo-alkane contents and theincrease in the UCM (Unresolved Complex Mixture)with biodegradation are apparent.


Armàncies Formation (0.14; Permanyer, 2004) (Fig.6 B and Tables 2 and 3).

Regular steranes showed a similar contribution ofC27 and C29 compounds, with a C27-C28-C29 distributionconsistent with a marine origin. A discrete contributionof diasteranes was noted with respect to the lowcontent of these compounds in the source rock samples(Permanyer, 2004) (Fig. 6 C).

The maturity calculated using the Ts/Tm ratio wasRoEq=0.69% close to the values of source-rockextracts (0.68%), where RoEq, vitrinite reflectanceequivalent, = 0.574 (0.148 * Ts/Tm (Van Graas, 1989).

Biodegraded oil samplesWhen compared to sample M8, the other samplesshowed varying degrees of depletion of n-alkanes, arelative increase in isoprenoids concentration (pristaneand phytane), and an increase in the UCM hump(Table 3). According to these criteria (Figs. 7, 8 and9), sample M1 (oil droplets) is less biodegraded thanM3 (“black” oil floating on water) and M5 (“highdensity” oil at the bottom of a pool). Sample M11(“brownish” oil floating on water) shows a higher level

M-8 M-1 M-3 M-5 M-11 M-7 M-60

10

20

30

40

50

60

70

80

90

100

SAMPLES

n-alkanesIso-cyclo alkanes

UCM

M8

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M5M11

M6M7

500

550

600

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700

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850

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0 50 100 150 200

mg/

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CM

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of degradation than M3, which suggests a longerresidence time in the pool. Samples M6 and M7 (oilmixed with microbial aggregates) present the highestdegrees of biodegradation. However, a dilution effectand/or the existence of anaerobic conditions at thebottom of the pools may slow down the degradationprocess.

The distribution of the C27-C28-C29 regular steranesis similar in samples M8, M1, M3 and M5 (Fig. 10).However, the distribution varies in the case of the morebiodegraded samples (M11, M7 and M6). Observeddeviations are difficult to explain. These variationsmay be related to the heterogeneity of the source rockor to changes in the composition of the microbial floraresponsible for the degradation. The hopanes wereapparently not altered by biodegradation, as indicatedby the terpane ratios (Table 3). The absence of anycorrelation between, for example, the quantity ofUCM and the variations in molecular ratios (forinstance, Ts/Tm) may indicate negligiblebiodegradation of the hopane series, although possibleheterogeneity of the source rock should again be takeninto account.

Fig. 8. Plot showing the depletionof n-alkanes and iso-cycloalkanes, and the increase in UCMas the degree of biodegradationincreases from sample M8 tosample M7 and M6.

Fig. 9. Graph showing the variations ofn-alkanes with respect to UnresolvedComplex Mixture (UCM) in sampleswith different degrees ofbiodegradation.


Finally, the homohopane series had a similardistribution in all the samples, regardless of the degreeof biodegradation (Fig. 11).

Based on these data, biodegradation was at level3-4 on the PM (Peters and Moldowan, 1993) scale.However, the PM scale is applied to anaerobicbiodegradation in deep reservoirs, whereas theprocesses taking place at the Riutort Mine are mainlyaerobic. Sequences and rates of the selective removalof petroleum compounds by biodegradation may differfor aerobic and anaerobic biodegradation (Yamane etal., 1997).

Aromatic hydrocarbonsThe components of the aromatic fraction in theunaltered sample (M8) were investigated, and achromatogram is presented in Fig. 12. Compoundsidentified in the aromatic fraction are typical of“normal” mature crude with an entire series includinglinear and branched alkylbenzenes, naphthalene,phenanthrene and chrysene. The predominance of aderivative of tricyclic compounds (1,2,8trimethylphenanthrene) was apparent. The unresolvedcomplex mixture (UCM) of branched aromaticcompounds under the resolved peak envelope is large.The ratio of Pr/Ph (1.25) to dibenzothiophene/phenanthrene (1.16) suggests a marine shale originfor the oil, in agreement with Hugues et al. (1995),and consistent with the characteristics of the sourcerocks (Permanyer, 2004; Permanyer et al, in prep.).

Analysis of the aromatic fraction (Fig. 13) showsthat alteration is consistent with biodegradation ofsaturates except for the long-chain n-alkylbenzeneseries, which had disappeared in all the samples.Selective depletion in alkylbenzenes and alkyltoluenes

has been recorded by Holba et al. (2004), andinterpreted to indicate an early stage of anaerobicmicrobial degradation (Barman Skaare et al., 2007).Some molecular series and compounds disappear (e.g.the phenanthrene series) or are temporarilyconcentrated (triaromatic steroids; 1,2,8trimethylphenanthrene) as biodegradation proceeds.In sample M11, the 1,2,8 trimethylphenantrene(TMP), which is less refractory than triaromaticsteroids, had been consumed.

In this context, quantitative analyses appear to bemore adequate than molecular ratios to describe theevolution of biodegradation (Dessort and Connan,1995; Elias et al., 2007), particularly because thevarious molecular series are biodegraded at differentrates (Wenger and Isaken, 2002). A relativequantification of aromatic molecular markers (Fig. 14)shows the following:

(i) A dilution of the biodegraded oil because of aninput of non-biodegraded oil (samples M3 and M11).Determining the rate of biodegradation is difficultwhere there is a renewal of the fluids, as the effects offresh oil recharge are not easy to estimate. Removalof oil from the source rock appears to be directlyrelated to hydrostatic pressure and this depends onthe recharge due to rainfall. After periods of heavyrainfall, the amount of oil increases visibly.

(ii) The relative stability of these compounds,assuming that the absence of biodegradation ofrefractory compounds should increase theirconcentration in the oil.

(iii) A relative decrease in the phenanthrene series,providing evidence that these compounds undergoearly biodegradation in aerobic conditions.

(iv) An increase followed by a decrease in the

M-8M-1

M-3M-5

M-11

M-6

M-7

C27

C28

C29

C31 C 32 C 33 C 34 C 350

5

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35

40

Homohopanes (22S + 22R)

M-8

M-1

M-3

M-5

M-11

M-7

M-6

% o

f Tot

al C

31 to

C35

Hop

anes

Fig. 10. Ternary plot showing the distribution ofregular steranes (C27-C28-C29) in the studied oilsamples.

Fig. 11. Graph showing the distribution of C31-C35homohopanes, which have similar abundances in allthe studied oil samples. This distribution shows thatthese homohopanes are not affected by thebiodegradation process.


40 60 80 100 120 140 160

**

*

** UCM = Unresolved Complex Mixture P = Phenanthrene MP = Methyl Phenanthrene S = Internal Standard

UCM

*

P

MP

SS

S

** ***

Abun

danc

e

= contamination

UCM

**

Time (mn) →

MP

DMP

TMPP

1,2,8trimethylphenanthrene

M-8

M-1

M-3

M-5

M-11

m/z 178+192+206+220+231

Triaromatic steroids

Fig. 12. Gas chromatogram of the total fraction of aromatic hydrocarbons from sample M8, which isconsidered not to be affected by degradation. The unresolved complex mixture (UCM) of branched aromaticcompounds under the resolved peak envelope is large.

Fig. 13. Aromatic hydrocarbons: reconstructed chromatograms using selected mass fragmentograms. Fromtop to bottom, the samples are ranked according to the degree of biodegradation for saturatedhydrocarbons. Biodegradation affects some aromatic compounds such as the phenanthrene series or aretemporarily concentrated (triaromatic steroids; 1,2,8 trimethylphenanthrene) as biodegradation develops.(P: phenanthrene; MP: methylphenanthrene; DM: dimethylphenanthrene, TMP: trimethylphenanthrene).


concentration of 1,2,8 TMP reflecting its moderatesensitivity to biodegradation. In sample M5, theconcentration of 1,2,8 TMP increases up to 150% withrespect to the less biodegraded sample M1. Thisindicates that the C15+ aromatic fraction has lost at leastone-third of its mass at this stage of biodegradation.The behaviour of this compound is typical of productsthat are moderately refractory to biodegradation suchas hopanes and 8,14 secohopanes (Restlé, 1983). Theconcentration of these products reaches a maximumbefore biodegradation.

The UCM, specifically in the aromatic fraction, isknown to be quite refractory to biodegradation(Dessort and Connan, 1995). Our data showed 24%of UCM biodegradation in sample M5 and 23% forsample M11 with respect to the reference sample; thisimplies a medium sensitivity to biodegradation.

IsotopesIsotope analyses were performed on all samplesstudied at the Riutort Mine. Fig. 15 presents isotopicsignatures for saturated and aromatic hydrocarbons,and the relationship between the degradation of n-alkanes and the variations in isotopic values ofsaturated hydrocarbons. Except for sample M11,values for the saturated fraction were between -24.4‰and -25.6‰, while aromatics ranged between -23.2‰and -23.6‰. The difference between the mostbiodegraded sample (M6) and fresh oil (M8) was δ13C= -0.2‰ for saturated and aromatic hydrocarbons.Except for sample M11, no major variations wereobserved in the isotopic signature of saturatedhydrocarbons regarding the loss of n-alkanes withbiodegradation. Sample M11 showed a heavier δ13Csignature than the other samples. This could be linkedto an increasing degree of biodegradation, to

0

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M-8 M-1 M-3 M-5 M-11

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Relative quantity of molecular markers

0.01

0.10

1

10

Phenanthrene series

Triaromatic steranes

1,2,8 TMP /Triaromatic steranes

1,2,8 TMP /Phenanthreneseries

M-1 M-3 M-5 M-11

Increase in biodegradation in accordance with n-alkane content

Selected molecular ratios

Dilution withfresh oil

M-8

/

mg/g

Fig. 14. Aromatic hydrocarbons. A (above). Plot showing the relative quantity of molecular markers in thestudied samples, reflecting a combination of biodegradation and dilution effects; B (below). Plot showingselected molecular ratios in the studied samples, indicating the different behaviour of the aromatics duringbiodegraation depending on their chemical structure (see text for more details).


variations in the source rock, or to local differencesin the microbial flora. Of these, local variations in thesource rock were the most likely. In addition, thissample shows an “anomalous” amount of aromatichydrocarbons.

Stahl (1980) pointed out that aerobicbiodegradation of crude oil in laboratory experimentshad only minor effects on the carbon isotopesignatures of the saturated and aromatic fractions.Experiments on crude oil biodegradation underaerobic conditions with a marine bacterial communityhave showed the stability of isotopic compositions ofthe C16-C30 n-alkanes (Mazeas et al., 2002). Morerecently, Sun et al. (2005) showed that biodegradationhas little effect on the carbon isotope composition ofa whole oil, and that there is no significant carbon-isotope fractionation in the high molecular weight n-alkanes during slight to moderate biodegradation.However, these authors reported a general increaseof up to 4‰ in the δ13C values of low to mediummolecular weight n-alkanes during intensebiodegradation. Enrichments in δ13C of 2% for highmolecular weight n-alkanes during biodegradation has

been also reported by Vieth et al. (2006) and Wilkeset al. (2008), occurring only at very highbiodegradation levels (above 80%). In our study,samples M7 and M6, which are considerably moredepleted in saturated hydrocarbons than M11, showedthe same isotopic signature as the “fresh oil” (M8)and the other less biodegraded samples. In fact, thestudied samples are made up of high molecular weightcompounds (C15+ saturates and C11+ aromatics) and onthe basis of the above considerations, significantisotopic shifts should not be expected.

Sulphur contentThe level of biodegradation can also be quantified interms of the sulphur content. This is because anegligible amount of organic sulphur is incorporatedinto an oil during biodegradation, and sulphur-bearingcompounds are particularly refractory (Dessort andConnan, 1995). Our results indicate that the sulphurcontent increases with biodegradation from 0.51% to1% (Fig. 16).

Resins and asphaltenes can also be assessed toquantifying biodegradation, as these compounds are

-32

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Non marine

Marine

δ13C Saturated HC (‰)

δ13 C

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ic H

C (‰

)

M-11

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% n

-alk

anes

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δ13C saturated HC

δ13 C

Sat

urat

ed H

C (‰

)

M-8 M-1 M-3 M-5 M-110

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30

40

50

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0,50

0,60

0,70

0,80

0,90

1,00

% n-alkanes% sulphur

% s

ulph

ur

% n

-alk

anes

Fig. 16. Relative sulphur content increases withbiodegradation. Fig. shows percent variations ofsulphur and n-alkanes according to biodegradation.

Fig. 15. Left: “Sofer plot” (Sofer, 1984) showing isotopic values for saturates and aromatic hydrocarbons. Notethe mis-match of sample M11. Right: Relationship within biodegradation related to n-alkanes and δ13C isotopicvalues of saturated hydrocarbons.


also refractory. However they may contain polarcompounds (lipids) from microbial cell walls, leadingto the erroneous estimation of the amount of polarspresent.

MicrobiologyMicrobial cultures showed the presence of two speciesof fungus and a large variety of bacteria in the samples(Fig. 17). Samples from oily pools of water showedthe growth of bacteria in concentrations up to 3 x 107

CFU/ml (Colony Forming Units per ml). The mostabundant strains in the cultures were selected foridentification (Table 4). Most of the strains belongedto the Phylum Proteobacteria (predominantly fromthe Class α), which have previously been describedas hydrocarbon degraders. Some of the strains appearable to produce surface-active compounds(biosurfactants or bioemulsifiers: see Fig. 7) whichfacilitate the formation of oil emulsions andsubsequent degradation (Gallego et al., 2007).

Other analyses including selective cultures andmicroscopic observations (Gallego et al., 2006b)suggested that cooperation by mixed cultures

(consortia) led to degradation of both aliphatic andaromatic compounds, as previously reported in naturalenvironments (Mishra et al., 2001; Larter et al., 2003).These diverse microbial populations can beresponsible for the degradation of different types ofhydrocarbon. Different metabolic pathways arerequired, for instance, to process aromatic rings thanmerely to oxidise n-alkanes.

CONCLUSIONS

Seepage oils at the underground Riutort oil shale minein the SE Pyrenees are sourced from the EoceneArmancies Formation. The oils have undergonesignificant biodegradation due to the presence of alarge number of hydrocarbon degraders (mainlybacteria). A specific aerobic microbial community hasdeveloped at the mine, and relies on the metabolism ofhydrocarbons in the absence of other sources of carbon.Biodegradation occurs in the presence of oxygen andwater, at an almost constant temperature (ca.12ºC) andat neutral or slightly alkaline pH. The biodegradation ismainly caused by aerobic bacterial activity.

Fig. 17. Phase contrast microscopicimages (100x) of oil seep sample before(a) and after (b) selective enrichmentexperiment (successive liquid cultivationsof the microbiota isolated from thestudied samples in conditions in which theoil from the mine was the sole carbonsource). Emulsion effect and bacteria withbacillary morphology attached to the oil-water inter-phases can be observed.

Genus/species (a) Comments

Oleomonas sagaranensis (AJ784808)Species previously described as hydrocarbon-degraders

in oil fields (Kanamori et al., 2002)

Bacillus sp (AY748912) Brevundimonas sp (DQ337577) Genera previously described as hydrocarbon-degraders Microbacterium sp. (AB234055)

Pandoraea sp. (DQ167022)

Promicromonospora sp (DQ008600) Dyella yeojuensis (DQ181549) Dyella ginsengisoli (AB245367)

Genera non-previously described as hydrocarbon-degraders

Table 4. Identification of the predominant bacterial strains obtained from the oil seep samples. Gram refersto differential staining of cell membranes. (a) Genus/species which gave higher identity percentage in the 16SRNA sequence (EMBL/Genbank access number in parentheses).


Various different approaches were used in orderto quantify the affects of biodegradation. A study ofsaturated hydrocarbons suggested that biodegradationdid not exceed 3-4 on the Peters and Moldowan (1993)scale. Quantification of the sulphur content, as wellas of molecular markers in aromatic fractions(triaromatic steroids, 1,2,8 trimethylphenanthrene)indicate that 50% of oil had been lost bybiodegradation, to which must be added light oil lostby evaporation. No particular conclusions could beobtained from isotopic analyses. Microbialdegradation remained moderate and was primarilylimited to the alteration of n-alkanes, isoprenoids andsome aromatics (such as naphthalene, phenanthreneand alkybenzene series).

Although the existence of anaerobic processescannot be ruled out, most of the degradation isattributable to aerobic biodegradation. A number ofthese hydrocarbon-degrading micro-organisms wereisolated in aerobic cultures, and some of themproduced bioemulsions similar to those observed inthe mine.

ACKNOWLEDGEMENTS

The authors thank Mrs. M. Ribera, Mayor ofGuardiola de Berguedà, for permission to work in theRiutort mine. Special thanks are due to Montse Sibilaand Jordi Illa for help with sampling and for analyticalsupport. This work was funded by Project CGL2006-01861 of the Spanish Ministry of Science andTechnology and the DURSI of the CatalonianGovernment (2005SGR00890 «Grup de GeologiaSedimentària»). We also gratefully acknowledge theEnvironmental Geochemistry and Biotechnologygroup headed by Professor Jesús Sánchez at theUniversity of Oviedo for assistance in microbialdeterminations. Journal review by H. I. Petersen andan anonymous referee and comments on a previousversion by B. Barman Skaare are acknowledged withthanks.

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CRUDE OIL BIODEGRADATION AND ENVIRONMENTAL FACTORS AT THE RIUTORT OIL SHALE MINE, SE PYRENEES

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