Changes in Crude Oil Composition during Laboratory Biodegradation

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Changes in Crude Oil Composition during Laboratory Biodegradation: Acids and Oil–Water, Oil–Hydrate Interfacial Properties Kristin Erstad,*,† Ina V. Hvidsten,† Kjell Magne Askvik,‡ and Tanja Barth† Department of Chemistry, UniVersity of Bergen, Alle´gt. 41, 5007 Bergen, Norway, and StatoilHydro ASA, R&D, SandsliVn. 90, 5254 Sandsli, Norway ReceiVed January 15, 2009. ReVised Manuscript ReceiVed May 20, 2009

The process of reservoir biodegradation imposes significant effects on crude oil quality, in terms of chemical composition and physical properties. In the present work the impact of 10 months of anaerobic and aerobic laboratory biodegradation on a crude oil has been studied separately. The processes has been carried out by means of two pure, novel bacterial strains, and the two biodegraded oils have been compared to the original nonbiodegraded crude oil. It is observed that the distribution of hydrocarbons, SARA (saturated hydrocarbons, aromatic hydrocarbons, resins, asphaltenes) fractions, interfacial tension between oil and water, and oil density are affected very differently in the two different systems. Moreover, the anaerobic and aerobic processes have increased the amounts of acids of the oil. High performance liquid chromatography (HPLC) and Fourier transform infrared (FTIR) studies reveal significant differences in the composition of the acid extracts. The effects of the types of acid extracts on gas hydrate/crude oil interfacial properties are also studied. The results suggest that the potential of an oil to form hydrate plugs may possibly be related to the acid species produced during anaerobic biodegradation.

Introduction Microbial degradation of crude oils in the reservoir is a very important postaccumulation process in terms of influence on oil quality and composition. The biodegradation process results in the formation of increasingly heavier oils, leading to higher oil density, increased emulsion stability, and increased content of NSO compounds, including the polar acidic compounds. The higher content of polar compounds in biodegraded crude oils may in principle be caused by (i) an increase in the relative concentration of heavy polar components resulting from removal of the light hydrocarbons by microbial activity or by (ii) products resulting from microbial processes. Acidic compounds in crude oils comprise one subfraction of the NSO compounds that is known to increase in concentration in biodegraded oils.1,2 On the other hand, the organisms responsible for crude oil degradation are also known to utilize carboxylic acids as substrates.3,4 Both short-chain and fatty acids are very often preferred to hydrocarbons as substrates for microbial growth, for example, see refs 5 and 6. Hence, the * To whom correspondence should be addressed. E-mail: kristin.erstad@ kj.uib.no. † University of Bergen. ‡ StatoilHydro ASA. (1) Tomczyk, N. A.; Winans, R. E.; Shinn, J. H.; Robinson, R. C. Energy Fuels 2001, 15, 1498–1504. (2) Barth, T.; Høiland, S.; Fotland, P.; Askvik, K. M.; Pedersen, B. S.; Borgund, A. E. Org. Geochem. 2004, 35, 1513–1525. (3) Magot, M.; Ollivier, B.; Patel, B. K. C. Anton. Leeuw. Int. J. G. 2000, 77, 103–116. (4) Kim, S.; Stanford, L. A.; Rodgers, R. P.; Marshall, A. G.; Walters, C. C.; Qian, K.; Wenger, L. M.; Mankiewicz, P. Org. Geochem. 2005, 36, 1117–1134. (5) Madigan, M. T.; Martinko, J. M.; Dunlap, P. V.; Clark, D. P. Brock Biology of Microorganisms, 12th ed.; Benjamin Cummins: San Francisco, CA, 2008.

composition of the acid fractions of microbially altered oils is expected to be different from those of nonbiodegraded oils. Crude oils contain acids that are surface active, which have been shown to cause antiagglomerating hydrate behavior.7 In petroleum production using multiphase transport systems at low temperatures, gas hydrate formation may present a severe problem in the sense that hydrates may aggregate and plug pipelines and platform equipment. However, in production systems containing crude oil, it is observed that some systems are associated with high risk of hydrates aggregating into large plugs, while in other systems the hydrates form as small dispersed particles that are easily transported in the fluid, even when the pressure and temperature conditions are well within the range of stable hydrate formation. The dispersed hydrates are interpreted as having “oil-wet” surfaces because of the adsorption of surface active petroleum acids. This promotes flocculation rather than agglomeration.7 Forming oil-wet hydrates is, to a high degree, correlated with some degree of previous biodegradation of the oils in the reservoirs,8 in the sense that most nonplugging oils are biodegraded, but not all all biodegraded oils are nonplugging. This observation is initially based on a sample set of nineteen oils from eleven fields (ten in the North Sea, Høiland et al.31 and Borgund et al.8). One way to understand these observations is to consider the microbial processes to be the source of some type of specific compounds that can have a strong and selective affinity for hydrate surfaces. Petroleum degrading microbes are known to (6) Watson, J. S.; Jones, D. M.; Swannell, R. P. J.; van Duin, A. C. T. Org. Geochem. 2002, 33, 1153–1169. (7) Høiland, S.; Borgund, A. E.; Barth, T.; Fotland, P.; Askvik, K. M. Proceedings of the 5th International Conference on Gas Hydrates; Tapir Akademisk Forlag: Trondheim, Norway, 2005. (8) Borgund, A. E.; Erstad, K.; Barth, T. Energy Fuels 2007, 21, 2816– 2826.

10.1021/ef900038z CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

Changes in Crude Oil Composition

produce biosurfactants that facilitate access to insoluble hydrocarbons for use as substrates, since the bacteria live in the water phase and not in the oil.9 Different organisms will produce different biosurfactant structures.10 The presence of different biosurfactants resulting from the presence of different organisms performing biodegradation can then explain the fact that not all biodegraded oils show the same properties regarding hydrate plugging or dispersion. In naturally degraded reservoir oils, it is difficult to evaluate the influence of biodegradation as a separate effect, since the original oil composition is often not known, and the type of microorganisms that have caused the changes in composition cannot be determined with any degree of certainty. Controlled laboratory experiments are therefore indicated as test systems. The very slow rate of the microbial processes is a challenge, and long-term experiments are required for even moderate degrees of degradation to be observed. In this work, we characterize the chemical composition of acid extracts and their wetting properties, before and after laboratory biodegradation lasting 10 months, using two different pure strains of hydrocarbon degrading bacteria. After biodegradation, the chemical and physical properties of the bulk oil phases are investigated to determine how the biodegradation processes have altered the acid fraction concentration and composition, and also the bulk oil properties. Finally, the effect of the biodegradation processes on the hydrate plugging tendency mitigation properties of the oils is investigated at conditions comparable to oil pipeline transport systems. Experimental Section Materials. The sample set consists of a start oil and two laboratory biodegraded oils. The start oil is kindly provided by the group of Professor Terje Torsvik, Centre for Integrated Petroleum Research, Norway. The start oil (labeled SO) is a sweet, nonbiodegraded crude oil from the Norwegian continental shelf. This oil is added to an aqueous medium and inoculated separately with two novel bacterial cultures, of which one is anaerobic and one is aerobic. These two samples are labeled Ban (anaerobically biodegraded) and Bae (aerobically biodegraded). The anaerobic bacterium, Desulfotignum toluenicum, has been isolated and described by Ommedal and Torsvik.11 The aerobic bacterium has been isolated and described by Bødtker et al.12 It belongs to the genus Dietzia and is designated strain A14101. The incubation systems consists of 30 mL crude oil, 50 mL bacterial inoculum, and 500 mL aqueous medium phase. For the anaerobic sample, the medium 383a from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), with optimizing conditions described by Ommedal and Torsvik, 2007,11 is used. The aerobic medium is described by Kowalewski et al.13 The incubation is carried out in 1 L flasks, at 30 °C, for a period of approximately 10 months. The two biodegraded oil/water systems are transferred to separation funnels, and the oil phase is collected after the systems are allowed separate for 24 h. Prior to sampling for all analyses described in this paper the oils are, by routine procedure, heated to 60 °C and homogenized. Smaller scale experiments with the same crude oil and bacteria have also been performed with an incubation time of up to eight (9) Peters, K. E.; Walters, C. C.; Moldowan, J. M. Biomarkers and Isotopes in Petroleum Systems and Earth History; The Biomarker Guide, 2nd ed., Vol. 2.; Cambridge University Press: Cambridge, U.K., 2005. (10) Desai, J. D.; Banat, I. M. Microbiol. Mol. Biol. Res. 1997, 61, 47– 64. (11) Ommedal, H.; Torsvik, T. Int. J. Syst. EVol. Microbiol 2007, 57, 2865–2869. (12) Bødtker, G. Microbial response to nitrate treatment in offshore oil fields, Dr. Philos. thesis, University of Bergen, Bergen, Norway, 2009. (13) Kowalewski, E.; Stensen, J. Å.; Gilje, E.; Bødtker, G.; Bjørkvik, B.; Strand, K. A. Presented at the International Symposium of the Society of Core Analysts, 2004.

Energy & Fuels, Vol. 23, 2009 4069 Table 1. Composition of Green Canyon Synthetic Gas Mixture component

mol %

methane ethane propane n-butane iso-butane nitrogen iso-pentane n-pentane

87.243 7.570 3.080 0.792 0.510 0.403 0.202 0.200

weeks. 0.5 mL crude oil are incubated with 33 mL of aqueous phase, giving a lower oil:water ratio than used in the main experiments. Sterile control samples are included in the sample sets and analyzed after 8 weeks. For evaluation of biodegradation, the crude oil from the sterile control is used as the reference to ensure that the observed changes in composition are specifically due to the biodegradation processes. All solvents are of p.a. or HPLC quality. The synthetic natural gas mixture (Green Canyon) is supplied by Yara International ASA. The composition is given in Table 1. Double-distilled, deionized water is used. Acid Extraction. The acids are extracted by use of ion exchange methodology. Very briefly, an activated QAE Sephadex ionexchange resin is transferred to the crude oil, and the mixture is left to stir for 16 h. The acids bond to the Sephadex material whereas the excess oil is filtered off. Toluene and methanol are used to remove the excess oil from the Sephadex. The acids are recovered from the Sephadex by adding formic acid and a solvent mixture of toluene and methanol. The mixture is left to stir for 4 h before the ion exchange mass is removed by filtration. The solvents from the filtrate are removed by a rotary evaporator; the acid extract is redissolved in a small amount of dichloromethane (DCM)/MeOH (93:7 by volume), and the products are quantified (see below). The method has previously been applied to a set of crude oils and has shown good reproducibility.8 Details on this technique can be found in papers of Mediaas et al.14 and Borgund et al.8 Methods for Quantification of Acid Content. GraVimetrically by Electrobalance. Five microliters of the acid extract, dissolved in DCM/MeOH 93:7 (v/v), are deposited on a weighing pan on a Cahn electrobalance (range 0.0001-2 mg). The solvent is allowed to evaporate (exactly 20 min). The weight of the nonvolatile residue is then obtained. The concentration of acids in the crude oil is determined from eq 1

C)

macid × Vsol Vsyr × moil

(1)

where C is the acid concentration in the oil (mg/g), macid is the mass of acids determined from the electrobalance (mg), Vsol and Vsyr are the volumes of the total solution and the syringe, respectively (µL), and moil is the mass of oil used for extraction (g). Total Acid Number (TAN) Potentiometric Titration. A Metrohm autotitrator (model 798 MPT Titrino) equipped with a combination solvotrode for nonaqueous titrations (Metrohm Solvotrode combined LL pH glass electrode, model 6.0229.100) is used to measure the TAN of the SO start oil. The titration is performed using the standardized ASTM664-89 method,15 with modifications according to Barth and Strand.16 For the two samples modified by anaerobic/ aerobic biodegradation TAN is not performed because of limited volumes of sample. TAN is estimated by extrapolation from the amount of extracted acids. (14) Mediaas, H.; Grande, K. V.; Hustad, B. M.; Rasch, A.; Rueslåtten, H. G.; Vindstad, J. E. Soc. Pet. Eng. 2003, 80404. (15) Standard Test Method for Acidic Number of Petroleum Products by Potentiometric Titration. Annual Book of ASTN Standards; American Society for Testing Materials: Philadelphia PA, 1989; Section 5. (16) Barth, T.; Strand, M. H. Presented at the 23rd International Meeting on Organic Geochemistry, 2007; Poster P270-WE.

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High Performance Liquid Chromatography (HPLC). The acid extracts are analyzed by HPLC equipped with an UV-detector, using a cyano-bonded column. Details on the instrumentation and method can be found in Borgund et al.17 The method separates the acid extracts into four well-defined fractions of acid compounds: FA, weakly polar compounds; FB, saturated carboxylic acids; FC, phenols; FD, polyfunctional acids. Some minor adjustments have been made to the method. The chromatograms are divided into fractions as follows: FA, 0-10 min; FB, 10-21 min; FC, 21-34 min; FD, 34-47 min. The samples are run in duplicate, and series at two different concentrations are performed: high concentration (7 mg/mL) to ensure sufficient response for assessment of the phenolic signal range, and low concentration (0.7 mg/mL) to make an overall semiquantitative comparison of chromatograms of the different acid extracts. The samples are dissolved in DCM/MeOH 93:7 (v/v). Fourier Transform Infrared Spectroscopy (FTIR). The FTIR spectra are recorded on a Nicolet Protege 460 FTIR spectrometer with a diamond attenuated total reflection (ATR) Dura sampler cell (from SensIR). A small droplet of the acid extract dissolved in DCM/MeOH 93:7 (v/v) is deposited on the diamond, and the spectra is recorded once the solvent has evaporated. Plugging Tendency. The mitigation potential of the acid extracts on gas hydrate plugging tendency is tested in a qualitative manner, using a high pressure rig equipped with a stirrer that allows visual inspection through a sapphire cell window (additional details and specifications of this equipment can be found in papers by Fadnes18 and Fotland and Askvik19). The equipment allows for monitoring of temperature, pressure, volume, and stirring torque as a function of time. In addition, video recordings can be carried out throughout the whole experiments. The experiments are performed at isobaric conditions at 100 bar, varying the volume by moving a piston inside the cell. The water cut is 50% (v), the water phase is 3.5 wt % brine (NaCl). The volume of the oil samples is 13 to 15 mL (see below for preparation of the oil samples with acid extracts as additives). The step-by-step procedure is as follows: a fixed volume of brine (slight excess) is transferred to the clean and evacuated sapphire cell, subsequently followed by transfer of the oil phase, and finally the supply of the synthetic natural gas phase. To obtain the correct water cut, the volumes are adjusted by discharging the excess of water. Stirring is then started (750 rpm). A system stability check, measuring the bubble point, is done prior to each experiment. The system is then heated to 45 °C to ensure sufficient solubilization of fluids. Cooling is then started to initiate gas hydrate formation. The temperature rates are set as following: start 40-0 °C at 5 °C/ hour. The system is then heated to 10 at 3 °C/hour to allow decomposition the hydrates and then of 0.5 °C/hour until the experiment is terminated. The plugging tendency is assessed by visual inspection with support from the torque data. Preparation of the Acid Extracts for Plugging Tendency Tests. The SO start oil is modified by adding acid extracts from the anaerobically and aerobically biodegraded oils, Ban and Bae, separately. The same acid concentrations are used as determined for these biodegraded oils. In addition, the start oil with increased content of its original acids are tested (same total concentration as the sample modified with Bae acids). Interfacial Tension measurements (IFT). The interfacial tension measurements are performed using the drop-volume (dropweight) method for determination of interfacial tension between two liquids, described by Harkins-Brown equation.20,21 The droplet volume of the oil phase, surrounded by water is measured at ambient temperature, using a 2 mL Gilmont micrometer syringe with

inverted needle of radius 0.415 mm. Approximately 90% of a droplet is extorted from the syringe. The remaining part of the droplet is very carefully pressed out until the droplet releases itself from the tip after about one minute pause for equilibrium. An average of 10 droplets in a series is measured, and five series of each oil sample are performed. Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID). The instrument is a Iatroscan MK-5 TLC/FID Analyzer with a TU 400 start controller. The crude oils are diluted in DCM/ MeOH 93:7 (v/v) to a concentration of 15 mg/mL. Two µL aliquots of the solutions are transferred to chromatographic rods (Chromarods SIII of silica, from Newman-Howells) by use of a sample spotter (Semiautomatic Sample Spotter Model SES 3202/IS-02, Analyses Systems SES). The rods are then placed in development tanks and the chromatographic separation is performed with the following eluents: hexane (30 min), toluene (10 min) and DCM: MeOH 93:7 (v/v) (3 min). The rods are dried at 60 °C for 90 s before they are scanned through the hydrogen flame in the flame ionization detector (FID) in the Iatroscan instrument. The percent of SARA fractions (saturated hydrocarbons, aromatic hydrocarbons and resins + asphaltenes) is then calculated. Two parallel determinations are made for the degraded samples, which give less than 2% difference between the percent values of the fractions. A welldefined reference oil, the Norwegian Standard Oil (NSO-1), is measured along with the samples and the quality of the data is assessed by the acceptance criteria given by Weiss et al.22 The standard oil is measured four times, and the highest standard deviation observed is 1.3% for the aromatic fraction. Whole Oil Gas Chromatography (WOGC). The oils are analyzed on a ThermoFinnigan Trace GC equipped with a FID. The stationary phase is a HP-PONA dimethylpolysiloxane column (50 m × 0.20 mm × 0.5 µm) from Agilent technologies. The mobile phase is helium. The temperature program is as follows (as described by Skaare23): initial temperature 30 °C held for 15 min, ramp rate 1.5 °C/min to 60 °C, then at 4 °C/min to the final temperature of 320 °C and held for for 35 min. The injector temperature is 300 °C while the FID is kept at 350 °C. The assignment of chromatographic peaks and quality assessment are based on the Norwegian Standard Oil (NSO-1).22 Note that this standard method applies to “stabilized” fluid (i.e., untopped and at ambient surface conditions). In this work, by routine procedure handling of crude oils in our laboratory, the samples are heated to 50 °C prior to sample outtake. This is in order to ensure a homogeneous composition, preventing wax precipitations at room temperature. In addition, this procedure helps facilitating injections of samples that normally are too viscous to be introduced by a syringe. 0.5 µL of warm, homogenized crude oil is introduced manually into the GC system through a syringe, using split injection. Density Measurements. An Anton Paar densitometer DMA 60 equipped with an Anton Paar DMA 602 HT measuring cell is used to determine the densities of liquids. All measurements are performed at ambient temperature.

(17) Borgund, A. E.; Erstad, K.; Barth, T. J. Chromatogr., A 2007, 1149, 189–196. (18) Fadnes, F. H. Fluid Phase Equilib. 1996, 117, 186–192. (19) Fotland, P.; Askvik, K. M. J. Colloid Interface Sci. 2008, 321, 130– 141. (20) Harkins, W. D.; Brown, F. E. J. Am. Chem. Soc. 1919, 41, 499– 524. (21) Adamson, A. W. Physical Chemistry of Surfaces, 5th ed.; John Wiley & Sons: New York, 1990.

(22) Weiss, H. M.; Wilhelms, A.; Mills, N.; Scotchmer, J.; Hall, P. B.; Lind, K.; Brekke, T. NIGOGAsThe Norwegian Industry Guide to Organic Geochemical Analyses [Online]. Edition 4.0; Published by Norsk Hydro, Statoil, Geolab Nor, SINTEF Petroleum Research and the Norwegian Petroleum Directorate, 102 pp, http://www.npd.no/engelsk/nigoga/default. htm [accessed 28 August 2008]. (23) Skaare, B. B. Effects of initial anaerobic biodegradation on crude oil and formation water composition. Ph.D. thesis, University of Bergen., Bergen, Norway, 2007.

Results and Discussion Observations. Figure 1 illustrates some observations done when opening the sample flasks after 10 months of laboratory biodegradation. The Ban anaerobic biodegraded oil/water/bacteria system seems to have been producing hydrogen sulphide (H2S analysis is not performed but the characteristic odor of the gas is observed when opening the sealed flask). This is attributed


Figure 1. Features of the biodegraded samples. Left: Solid precipitations in the water phase of the Ban anaerobic system. Right: Bacterial activity in the oil/water interface of the Bae aerobic system.

to the sulfate-reducing abilities of this bacterium.11 In addition, a solid material has accumulated in the water phase, see Figure 1. This can possible be a salt formed from metal cations present in the aqueous medium and the sulphide anion, but this has not been confirmed. In the Bae system of aerobic biodegraded oil, the bacterial activity in the oil/water interface is very apparent, observed as a bacterial “mat” (not observed for the Ban system). Bulk Oil Properties. Bulk crude oil composition data collected from Iatroscan-, density-, and IFT-measurements are presented in Table 2. The results show that the changes in the oil composition and properties are different for the two cultures. In the Bae sample a slight increase in the relative amount of polar compounds (i.e., resins + asphaltenes) compared to the start oil SO is observed, as expected. However, this is not observed for the Ban sample. Also, for the Ban the content of aromatics has decreased relative to the amount of saturated hydrocarbons. This may be related to the specific bacteria Desulfotignum toluenicum which has shown to be able to selectively utilize monoaromatic compounds.11 Though the changes are not very large, they are outside the range of variation of the method, and thus can be used to illustrate the difference between the two systems. The density has increased slightly in both the biodegraded samples, which can be related to the removal of light hydrocarbons and/ or production of new and heavier components during biodegradation. The IFT measurements show that IFT is significantly reduced only for the Bae sample. This observation is in accordance with the increase in the content of polar compounds for the Bae oil, which may be attributed to the production of new surface-active component (biosurfactants). This particular bacterium has previously been demonstrated to be highly active in the interface of crude oil/water/bacteria systems, as shown by the dramatical drop in IFT measured by use of a laser-light scattering technique.13,24 (24) Kowalewski, E.; Rueslåtten, I.; Gilje, E.; Sunde, E.; Bødtker, G.; Lillebø, B. L. P.; Torsvik, T.; Stensen, J. Å.; Bjørkvik, B.; Strand, K. A. Presented at the European Symposium on Improved Oil Recovery, 2005.

Energy & Fuels, Vol. 23, 2009 4071

Degradative loss, described by Elias et al.,25 is used to assess the effects of biodegradation in the anaerobic and aerobic experiments. DegradatiVe loss calculated for Ban and Bae is shown in Figure 2. It is very apparent that the monoaromatic compounds (benzene, toluene, ethylbenzene, and m-, p-, and o-xylenes) are being extensively depleted in both processes. Since the sterile controls from the eight-week incubation experiments are used as the reference oil, abiotic processes like partitioning of aromatic compounds into the water phase are already accounted for. The observed differences in composition can thus be assigned specifically to biodegradation. As mentioned, the Desulfotignum toluenicum culture used for anaerobic biodegradation possesses the unique property of being able to grow solely on toluene (hence its name).11 As can be seen, nearly 90% of toluene is removed during anaerobic biodegradation (note that toluene coelutes with the compound 2,2,3tm-C5 (2,3,3trimethylpentane), but it is assumed that the amount of toluene surpasses the amounts of 2,2,3tm-C5 by far). Interestingly, the results shown here strongly imply that the bacteria also are able to utilize other monoaromatic compounds when present in crude oil: benzene, ethylbenzene, and xylenes are all clearly depleted. According to Ommedal et al.,11 the monoaromates n-C12, ethylbenzene, m-, p-, or o-xylenes are not utilized when given as separate compounds. This is also the case for n-alkanes in the range n-C7-n-C9. A similar effect is seen for the aerobic bacterium Dietzia sp. A14101, which is not able to utilize monoaromatic compounds given individually.12 However, studies on crude oil degradation of this bacteria have shown that toluene and xylene are completely consumed after 120 days, which can explain the findings in this work. In addition to the obvious high degree of depletion of monoaromatic compounds, a general assessment of the degradative losses from Figure 2 show that the high molecular weight compounds are least affected by the biodegradation processes. This observation is in agreement with the lighter, more volatile components being known to be attacked first in the biodegradation process.26 Overall the aerobic process seems to have proceeded more rapidly than the anaerobic process, especially for the compounds in the lowest molecular weight range. As shown, the depletions of the two lightest end members i-C5 and n-C5 are more than 85% for Bae. This observation is in accordance with aerobic processes being known to be faster than those carried out under anaerobic conditions. These findings strongly support that microbial alteration of the oil indeed has taken place. These changes caused by biodegradation would otherwise be difficult to detect if assessed solely by classical biodegradation parameters; for example, the pr/n-C17 and ph/n-C18 ratios26-29 are unaffected for both Ban and Bae. With regards to the Thompson parameters describing aromaticity, benzene/n-hexane and toluene/n-heptane30 are expected to increase for biodegraded oils. For Ban and Bae, these ratios are in fact decreasing. This (25) Elias, R.; Vieth, A.; Riva, A.; Horsfield, B.; Wilkes, H. Org. Geochem. 2007, 38, 2111–2130. (26) Wenger, L. M.; Davis, C. L.; Isaksen, G. H. Soc. Pet. Eng. 2001, 71450. (27) Peters, K. E.; Moldowan, J. M. The Biomarker Guide. Interpreting Molecular Fossils in Petrolem and Ancient Sediments; Prentice Hall: Englewood Cliffs, NJ, 1993. (28) Seifert, W. K.; Moldowan, J. M. Geochim. Cosmochim. Acta 1979, 43, 111–126. (29) Waples, D. Geochemistry in Petroleum Exploration; Reidel Publishing Company: Boston, 1985. (30) Thompson, K. F. M. Geochim. Cosmochim. Acta 1983, 47, 303– 316. (31) H, E.; Barth, T.; Fadnes, F. J. Colloid Interface Sci. 2005, 287, 217–225.

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Table 2. Bulk Oil Property Data, from Iatroscan-, Density-, and IFT-Measurements oil

% saturated hydrocarbons

% aromatic hydrocarbons

% resins + asphaltenes

density (g/mL)

IFT (mN/m)

SO Ban Bae

53.1 ( 0.7 57 ( 3 52 ( 1

38.1 ( 0.8 34 ( 4 37.2 ( 0.6

8.8 ( 0.6 8.5 ( 0.2 10.5 ( 0.8

0.84258 ( 0.00005 0.8604 ( 0.0005 0.8562 ( 0.0001

23 ( 1 23.6 ( 0.5 17.0 ( 0.4

can be attributed to the specific physiological properties of these two bacteria cultures. The degradative loss method requires that the biodegraded oils are compared to a related oil that is less affected by

biodegradation. Hence, the constituents of all the resolvable GCpeaks in the Ban and Bae biodegraded oil are calculated as relative concentrations with regard to the same compounds found in the sterile incubated sample of the SO nonbiodegraded start oil.

Figure 2. Degradative loss after laboratory biodegradation for compounds in Ban and Bae. For notations, see Weiss et al.22 The error bars show the difference between two parallel GC analyses.


Figure 3. Yields of acid from acid extractions. SO: Nonbiodegraded start oil. Ban: Anaerobic biodegraded oil. Bae: Aerobic biodegraded oil.

The application of the method is slightly modified, adjusted to the properties of these particular samples; after examination of the chromatograms, the aliphatic hydrocarbon n-pentadodecane (n-C25) is chosen as an internal reference compound for normalization. n-C25 is the compound least affected by both the anaerobic- and aerobic biodegradation processes in these experiments. In an ideal approach, the chosen internal reference should be totally unaffected by biodegradation. This is not obtainable within this range of compounds, and hence some negative values are observed in Figure 2. There is also some deviation between the analytical parallels (shown as error bars). Despite these limitations, the degradative loss approach is able to document incipient biodegradation levels with more clarity and detail than alternative methods. Acid Content in the Oils. Acid Extraction Yields. Figure 3 shows the acid content measured in the three samples. A significant increase in acid concentration is observed in the two biodegraded samples, although not to the same level as in the natural biodegraded oils previously investigated by Borgund et al.8 This can be the result of insufficient time of laboratory biodegradation compared to the natural biodegradation processes that take place in the reservoirs throughout a geological time scale. In addition, the extent of biodegradation taking place may be related to the ratio between the surface area and the volume of oil being too small during the incubation period (as discussed in the previous section), that is, using smaller volumes of oil will most likely lead to a relatively larger alteration in oil composition and increase in acid concentration. Total Acid Number. The acid content in the start oil SO, measured by titration, is determined to 0.06 mg KOH/g oil. A low TAN value like this is typically representative for sweet, nonbiodegraded oils that originate from the Norwegian Continental shelf.2,8,31 For the two laboratory biodegraded samples, Ban and Bae, a measure of TAN is estimated by use of a correlation curve (Figure 4). This curve is based on a relationship between extraction yields and TAN that is observed for a set of oils previously characterized with regards to acid content.8 A very strong correlation (R2 ) 0.9923) is observed between these two quantities. Several other authors have also reported this correlation for crude oils.32,33 Using this plot, it can be seen that the two laboratory biodegraded samples are located closer to the nonbiodegraded oils (S-oils) than to the naturally biodegraded ones (B-oils), again implying that the duration of (32) Meredith, W.; Kelland, S. J.; Jones, D. M. Org. Geochem. 2000, 31, 1059–1073. (33) Saab, J.; Mokbel, I.; Razzouk, A. C.; Ainous, N.; Zydowicz, N.; Jose, J. Energy Fuels 2005, 19, 525–531.


Figure 4. Relationship between acid extraction yields and total acid numbers based on data from Borgund et al.8 and the oil SO. The points on the curve representing the laboratory biodegraded oils Ban and Bae are extrapolated. The biodegradation level of the natural biodegraded crude oils from Borgund et al.8 is assessed to be from light to very heavy biodegraded on the Peter and Moldowan scale.27

Figure 5. Distribution of compound classes in the acid extract (from low concentration runs: 0.7 mg/mL).

the laboratory biodegradaton (10 months) has not been sufficient for extensive biodegradation to take place. Assessing the actual extent of biodegradation for these samples is not simple. When compared with TAN values with those from Wenger et al.,26 all the oils classify as being nonbiodegraded (TAN less than 0.2 mg KOH/g oil). However, when the the biodegradation from the alteration in hydrocarbons (Figure 2) is assessed, they could be classified as slightly biodegraded: major alterations of hydrocarbons in the range from C1 to C15 are observed for both the biodegraded samples. HPLC of Acid Extracts. Figure 5 shows distribution of acid compound classes, based on the quantification of peak areas from the low concentration runs at 0.7 mg/mL (no cutoff peaks in the chromatograms). A large part of the Bae acid extract consist of polyfunctional compounds, which can be attibuted to the production of biosurfactant species (also discussed earlier for the IFT measurements). Biosurfactants are known to consist of highly polyfunctional, complex structures.34 It is apparent from Figure 5 that the composition of the Ban acid extract differs very much from that of SO and Bae. Ban contains a lower relative amount of phenolic compounds, and it has a large relative amount of weakly polar compounds. It has been shown that large relative amounts of phenolic compounds in acid extracts may correlate with formation of water wet hydrates and higher plugging tendency.8 It is interesting to compare Ban with the acid distribution observed for an especially hydrate active acid subfraction previously isolated from naturally biodegraded oils (34) Lang, S. Curr. Opin. Colloid Interface Sci. 2002, 7, 12–20.

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Figure 7. FTIR spectra of acid extracts. %R: percent reflectance.

Figure 6. UV-spectra of the phenolic fractions from Ban and Bae.

with low plugging tendency (i.e., B4a, B4c, and B2b from Figure 4). A very similar pattern is observed, with a high relative amount of weakly polar compounds.35,36 On the other hand, the high content of polyfunctional compounds found in Bae acid extract resembles very much the acid distribution found in a subfraction of a biodegraded, high plugging tendency oil (B1c from Figure 4). The spread in data, measured by standard deviation from the average value of the duplicate runs, vary between 0.03 to 1%. The UV-detector is not universal and thus cannot provide an absolute quantitification of all compounds present, and the response of the saturated carboxylic acid peak is expected to be weak as these compounds have low UV absorbance. Hence, the approach is semiquantitative with the purpose of detecting differences within these three acid extracts. Figure 6 shows UV-spectra extracted from the range of phenolic compounds in the chromatograms (only Ban and Bae shown). The SO and Bae acid extracts have a maximum absorbance at 218 nm with a secondary absorption shoulder with maxima around 258-273 nm. This pattern is typical for phenols.37 However, this secondary absorbance is not very distinct for Ban, again implying that the phenolic fraction has a different composition than those from SO and Bae. FTIR of Acid Extracts. A comparison of the FTIR spectra from acid extract isolated from the SO, Ban and Bae samples is shown in Figure 7. In general, the absorption bands from saturated aliphatic hydrocarbons (group of peaks around 2800-3000 cm-1 and two signals in the fingerprint area at approximately 1461 and 1377 cm-1), aromatic ring hydrocarbons (around 1600 cm-1), and the carboxylic acid carbonyl group (around 1706 cm-1) are dominating all three spectra (band assignments are made according to Coates38). In addition, a broad, less well-defined absorbance band is observed in the range 3700-2500 cm-1, overlapping the C-H stretching region (35) Erstad, K.; Høiland, S.; Barth, T.; Fotland, P. Proceedings of the 6th International Conference on Gas Hydrates; University of British Columbia: Vancouver, British Columbia, Canada, 2008; https://circle.ubc.ca/ handle/2429/1022. (36) Erstad, K.; Høiland, S.; Fotland, P.; Barth, T. Energy Fuels 2009, 23, 2213–2219. (37) Talrose, V.; Yermakov, A. N.; Usov, A. A.; Goncharova, A. A.; Leskin, A. N.; Messineva, N. A.; Trusova, N. V.; Efimkina, M. V. UV/ Visible Spectra. In NIST Chemistry WebBook, NIST Standard Reference Database Number 69; Linstrom, P. J.; Mallard, W. G., Eds; National Institute of Standards and Technology: Gaithersburg, MD, 2005; p 20899, http:// webbook.nist.gov. (38) Coates, J. Interpretation of Infrared Spectra, A Practical Approach. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; pp 10815-10837.

Figure 8. Samples used for wetting property tests. SO start oil: the original nonbiodegraded oil with natural content of acids. SO + Ban acids and SO + Bae acids: Start oil modified with acids from Ban and Bae, respectively (additives in the same concentrations as determined for these oils, see Figure 3). SO + SO acids: Start oil modified with extra addition of its own acids, to the same total acid concentration as the SO + Bae sample.

around 3000 cm-1. This band is assigned O-H stretch from carboxylic acids or possible phenolic compounds and is only distinct for the SO and Bae acid extract spectra. This observation supports the findings from HPLC, discussed in the previous section, again indicating that the Ban acid extract contains relatively less phenolic compounds than SO and Bae. The intensity distribution of the spectral bands vary significantly among the samples. Another observation is that the aromatic CdC-C signal is increasing relative to the CdO signal, indicating increased aromaticity of the two acid extracts from the biodegraded oils. This could possible be a result of carboxylation of aromatic hydrocarbons (the results from Table 2 do not imply any increase in aromatic hydrocarbons) or it could reflect the preferential degradation of fatty acids, which are known to be preferred substrates for degradation.5 Wetting Properties. The hydrate plugging behavior of four oil samples is presented in this section. Three of the oil samples are modified by addition of acids, as described in Figure 8. The “SO + SO acids” sample is the start oil SO with increased concentration of its natural acids. This is tested with the purpose of comparing the wetting effect of acid concentration versus the effect of type of acids (the other modified samples consist of the start oil SO added biodegraded acids from Ban and Bae). Figure 9 shows hydrate formation and dissociation curves from two of the experimental runs, comparing the start oil with the SO + Ban acids modified oil. Point A on the curves represents the onset of hydrate formation. Formation and growth of hydrates continue until point B is reached on the curve. B represents the point where the test cycle reaches its lowest temperature. From this point, heating of the system causes


Figure 9. Volume versus temperature development curves from two of the plugging tendency experimental runs. Upper: Start oil SO. Lower: SO modified with Ban acids.

dissociation of the hydrates. The three samples SO start oil, SO + Bae acids and SO + SO acids all show similar behavior, illustrated in the upper part of Figure 9. Conversely, the SO + Ban sample displays a somehow different behavior, as shown in the lower part of Figure 9. It is observed that the hydrates start to form at a significantly higher temperature in this particular oil sample compared to the start oil (and the other samples). Figure 10 shows the torque versus time, and volume versus time measurements for the same two samples. The torque provides information on the changes in the viscosity of the fluids. Thus, a sudden increase in torque can be related to formation of hydrate lumps/depositions and plugging. For the SO start oil, the point of hydrate formation (sudden drop in volume) corresponds well with maximum torque, meaning that the plugging takes place almost simultaneously with volume drop (this scenario is also observed for the SO + Bae acids and the SO + SO acids, data now shown). For the SO + Ban acids the torque increase less abruptly, but in a stepwise manner, from the onset of hydrate formation until it reaches a maximum value much later than the SO start oil. This corresponds to a point close after point B on the curve in Figure 9 (after the melting of hydrates has been initiated). The volume drop is also less steep than the for the corresponding SO start oil volume curve. This implies that the formation and building up of hydrates to a plug has happened more slowly for this system. A reasonable interpretation can be that the hydrates in this system are more oil-wet, residing to a larger extent in the oil phase, and with less access to water for growth and building up plugs compared to any of the other samples. Figure 11 shows still images of the growing hydrates for all four experiments, recorded between the onset of crystal formation and growth between points A and B. In all the systems plugging occurs, but it seems that the plugging is somehow less


Figure 10. Torque- and volume development as a function of time for the samples SO start oil (upper) and SO + Ban acids (lower).

Figure 11. Still images from plugging tendency test, during the stage of hydrate formation and growth (between point A (onset hydrate formation) and point B (end of hydrate formation and growth)).

aggressive in all the systems with the acid additives, including the SO + SO acid sample. This indicates that acid concentration also has an impact on wettability. The evaluation of plugging tendency in these experiments is based mainly on visual inspection with support from torque data, thus great precautions must be taken when interpreting the results. The duration of hydrate growth between point A and B is indicated in Figure 11. In all the samples with added acids, the

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plugging happens more slowly than in the unmodified oil SO. As discussed, the SO + Ban acids sample stands out being the sample where hydrates form most slowly. The visual observations made for the SO + Ban acid sample corresponds well with the volume- and torque data: no large, crystalline hydrate lumps are observed between point A and B. The unusual behavior of Ban acids may be related to its compositional features discussed in earlier sections. As discussed in the HPLC section, the composition of the Ban acids stands out from the other two acid extracts in having a higher relative amount of weakly polar compounds and a relatively lower content of phenolic compounds. It is also interesting that of the three acid modified samples, this particular sample have the lowest concentration (see Figure 8). Yet, it displays the highest inhibiting effects with regards to the kinetics of hydrate plugging. This is reflected in time of the growth between point A and B, where the SO + Ban acids system uses almost twice as long time to reach point B as the SO + SO acid system, and 8× longer than the nonmodified crude oil SO. Thus, as a very conservative approach, it seems that the acids to some degree affect the wetting properties of the hydrate surface toward a more oil-wet state but not to the same extent that has been observed in previous work, where acid extracts derived from natural biodegraded oils were tested.7 In that work, a complete transformation from plugging to dispersed hydrates was observed when adding these acids to a high plugging tendency oil. A higher acid concentration was used (6500 ppm, which corresponds to a TAN of 1.1 mg KOH/g oil determined for the nonplugging oil the acids were isolated from). As discussed, for the samples Ban and Bae the duration of biodegradation for the oils seems to have been insufficient to produce the required amount or concentration of acids that can transform the oil from being a high plugging tendency oil to a low plugging tendency oil. Summary In the course of the ten month laboratory incubation with the two pure cultures, only a slight degree of biodegradation has taken place, as seen using crude oil analysis and characterization. Still, we observe that the anaerobic and aerobic biodegradation processes have changed the crude oil properties, especially reflected in the acid content and the acid extract composition. Generally the biodegradation has led to increased density, increased acid content, and depletion of certain types of hydrocarbon compounds (corresponding to the preliminary knowledge on the specific physiological properties of the bacteria used in the experiments). Biodegradation has led to an increase in the amount of aromatic acidic species and a decrease

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in the light aromatic hydrocarbons. The changes in composition are different for the two cultures. Bae acid extracts contain the largest amount of acids. This acid extract contains a large quantity of polyfunctional compounds that we believe are biosurfactants produced during aerobic biodegradation. The production of species that is surface active in the oil/water interface is shown in the reduction of the interfacial tension of the Bae crude oil. The anaerobic biodegradation process does not increase the acid concentration to the same extent as the aerobic process. However, the acid extract from Ban has a significantly different composition of acid compound classes, as shown by HPLC and FTIR. Ban acid extract has a high content of weakly polar compounds and low content of phenolic compounds, which may be related to low plugging tendency. As opposed to Bae acids, the Ban acid species do not cause any change in surface activity in the oil/water interface as measured by IFT. However they display significantly larger effects in the oil/hydrate interface. Thus, even though aerobic process produce largest amounts of polyfunctional compounds that may be biosurfactants, the anaerobic process seem to have been producing acids that contain the most hydrate surface active components. This observation supports the idea that different microbial organisms and processes can exert different impacts on the plugging tendency in biodegraded oils. We therefore raise the question whether plugging potential of oils to a large extent can be related to the anaerobic biodegradation processes. None of the acid extracts are able to form systems of completely dispersed hydrate particles. We attribute this to both the quantities and the types of acids not being sufficient to make a sufficiently significant impact on hydrate morphology. However, alterations toward more oil-wet hydrate conditions are indicated in all plugging tendency tests where the SO oil has been spiked with acid additives. For the unmodified SO oil, the plugging takes place very fast, whereas in all the acidmodified samples the hydrate plugs form slower. The SO + Ban acids sample imposes the largest effect of all the modified samples. Acknowledgment. StatoilHydro ASA and the Research Council of Norway are greatly acknowledged for funding this work, StatoilHydro ASA also for the permission to publish results. We are also thankful to the organic geochemistry unit at StatoilHydro ASA for providing laboratory facilities and assistance for TLCFID measurements. We are indebted to Terje Torsvik, Gunhild Bødtker and Hege Ommedal for providing these novel bacteria cultures and oil samples for us to explore in this work, and for all assistance and valuable input and discussions. Anne-Birgit Skåtøy is greatly acknowledged for performing TAN-analysis. EF900038Z

Changes in Crude Oil Composition during Laboratory Biodegradation

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