Energy & Fuels 2006, 20, 2079-2082
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Nutrient Supply during Subsurface Oil BiodegradationsAvailability of Petroleum Nitrogen as a Nutrient Source for Subsurface Microbial Activity T. B. P. Oldenburg,*,† S. R. Larter,†,‡ and H. Huang† PRG, Petroleum ReserVoir Group, Department of Geology and Geophysics, UniVersity of Calgary, Calgary, Alberta, Canada, and NRG, School of CiVil Engineering and Geosciences, UniVersity of Newcastle, Newcastle NE1 7RU, United Kingdom ReceiVed April 5, 2006. ReVised Manuscript ReceiVed June 15, 2006
Biodegradation of petroleum is an important alteration process with major negative economic consequences for oil production and refining operations. Most of the world’s crude oil in reservoirs is biodegraded. Although it has been suggested in recent years that biodegradation of petroleum in reservoirs proceeds anaerobically, little is still known about the processes and the controlling factors involved in subsurface hydrocarbon and non-hydrocarbon degradation. As the supply of hydrocarbons is unlikely to be limiting in oil reservoirs, essential nutrients such as nitrogen or phosphorus might control the rate or degree of biological degradation. Here we examine the possibility of crude oil itself providing nitrogen as a nutrient in the form of nitrogen compounds for microbial use. We use excellent natural biodegradation sequences, which cover the whole range from low to severe degrees of biodegradation. We suggest that small amounts of petroleum nitrogen can be used during biodegradation at high degrees of biodegradation [>level 4 of Peters and Moldowan (The Biomarker Guide; Prentice Hall: Englewood Cliffs, NJ, 1993), PM level > 4], but at lower degrees of biodegradation, nitrogen as an essential nutrient has to be supplied from other sources, most likely from ammonium ion, which is abundant in oilfield waters, sourced from clay and other mineral buffers.
Introduction The ability of microorganisms to survive or grow in oilfields depends on the physical characteristics and chemical composition of the petroleum reservoir, with temperature as the main limiting factor. Even though microorganisms are known to survive at high temperatures (.100 °C),2-4 Philippi5 noted that in situ oil biodegradation was never observed in oil reservoirs whose temperatures exceeded 82 °C. In addition, Wilhelms et al.6 reported that oils in reservoirs that, though currently cooler than 80 °C, have been heated to temperatures higher than 80 °C are also not biodegraded, which was suggested to be the result of sterilization of these petroleum reservoirs by heating during deep burial, inactivating hydrocarbon-degrading organisms that occur in petroleum reservoirs. The availability of electron donors and acceptors governs the metabolic activities of microorganisms in oil reservoirs. As oilfields are generally isolated from surface waters, their redox potentials are very low and some electron acceptors are most generally absent, in particular, oxygen and nitrate. Reservoir waters generally contain sulfate, even if at very low concentrations, and bicarbonate,
factors which have led to the assumption that the major metabolic processes occurring in such ecosystems are sulfate reduction, methanogenesis, acetogenesis, and fermentation.7-9 It appears that methanogenesis is the common terminal process in most low sulfate petroleum reservoirs.10-12 The potential electron donors include hydrogen and numerous organic molecules, including hydrocarbons. Resins and asphaltenes are important fractions of crude oil containing thousands of complex heterocompounds whose composition has been partly elucidated, and they are an abundant potential source of electron donors for anaerobic metabolism.7 It has been suggested that bioavailability of petroleum compounds is a major factor controlling biodegradation in reservoirs.13 This seems unlikely as the sequential nature of the biodegradation process, in which n-alkanes of a large carbon number range, which are the least water soluble and chemically reactive components of a crude oil at any carbon number, degrade first. This suggests that compounds are degraded in a sequence largely driven by common compound abundance in petroleum more than by reactivity or physical properties. The process is most likely controlled by evolutionary response to petroleum composition
* To whom correspondence should be addressed. E-mail: toldenbu@ ucalgary.ca. † University of Calgary. ‡ University of Newcastle. (1) Peters, K. E.; Moldowan, J. M. The Biomarker Guide; Prentice Hall: Englewood Cliffs, NJ, 1993. (2) Stetter, K. O.; Huber, R.; Blo¨chl, E.; Kurr, M.; Eden, R. D.; Fielder, M.; Cash, H.; Vance, I. Nature 1993, 365, 743-745. (3) Blo¨chl, E.; Rachel, R.; Burggraf, S.; Hafenbradl, D.; Jannasch, H. W.; Stetter, K. O. Extremophiles 1997, 1, 14-21. (4) Kashefi, K.; Lovley, D. R. Sci. 2003, 301, 934. (5) Philippi, G. T. Geochim. Cosmochim. Acta 1977, 41, 33-52. (6) Wilhelms, A.; Larter, S. R.; Head, I.; Farrimond, P.; di Primio, R.; Zwach, C. Nature 2001, 411, 1034-1037.
(7) Magot, M.; Ollivier, B.; Patel, B. K. C. Antonie Van Leeuwenhoek 2000, 77, 103-116. (8) Ro¨ling, W. F. M.; Head, I. M.; Larter, S. R. Res. Microbiol. 2003, 154, 321-328. (9) Holba, A. G.; Dzou, L. I. P.; Hickey, J. J.; Franks, S. G.; May, S. J.; Lenney T.Org. Geochem. 1996, 24, 1179-1198. (10) Head, I. M.; Jones, D. M.; Larter, S. R. Nature 2003, 426, 344352. (11) Pallasser, R. J. Org. Geochem. 2000, 31, 1363-1373. (12) Sweeney, R. E.; Taylor, P. Presented at AAPG Hedberg Research Conference, Natural Gas Formation and Occurrence, Durango, CO, 1999. (13) Wilkes, H.; Vieth, A.; Scherf, A. K.; Rullko¨tter, J.; di Primio, R.; Horsfield, B. Presented at American Association of Petroleum Geologists Annual Meeting, Calgary, Canada, 2005.
10.1021/ef060148p CCC: $33.50 © 2006 American Chemical Society Published on Web 07/21/2006
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rather than physicochemical properties. Larter et al.14 showed that the net rates of degradation are not controlled by supply of hydrocarbons to the oil-water contact but are controlled by some other factors. The process is, thus, zero order with regard to hydrocarbon concentration. Head et al.10 suggested nutrient supply is the likely rate-controlling step. Limiting concentrations of nitrogen, phosphorus, and essential elements for biological systems frequently control the nature and extent of microbial activities in natural environments. The availability of nitrogen and phosphorus sources in oilfields has not been extensively studied from a nutrient perspective. If nitrogen gas is present, which it commonly is in oilfields at levels of a few mole % of produced gases, then it could be assimilated by nitrogen-fixing microorganisms if present. Nitrogen is also available in aromatic nitrogen-containing compounds, especially in the form of the nonbasic pyrrolic compounds such as carbazole and its alkyl and benzoderivatives15-17 and basic aromatic nitrogen compounds18 largely found in the high-molecular-weight fractions of the oil. Total nitrogen contents range from a few tenths of a percent up to several percents by weight of an oil for common oil types. The aim of this study was to investigate if the essential nutrient nitrogen is supplied by petroleum itself and if it controls the degree or rate of biological degradation. Materials and Methods Excellent natural biodegradation sequences, including petroleum from reservoir core extracts of three deep (1800 m) oil saturated turbidite sandstone reservoir systems (Es3 member) and one shallower (1500 m) oil column (Es1 member) were collected from the important oil-producing Liaohe Basin in NE China. The reservoir consists of the Eocene sandstones of the Es3 and Es1 members of the Shahajie Fm. The Es3 member oil columns cover a range of biodegradation from PM level 1 to 4/5 through the column from the top to the bottom,19 whereas the shallower (1500 m) Es1 member oil column is more highly degraded (PM level 5-8 through the oil column from top to bottom19). The three Es3 columns are located in the same oilfield block, with similar producing intervals at depths of ∼1800 m subsurface sharing the same oil-water system, whereas the burial depth of the Es1 section oil columns vary more widely and the oil-water relationships are more complicated.19 In addition to the reservoir sequences through these oil columns, a series of very viscous produced heavy oils from different wells in the Eocene Es3 reservoirs20 were also analyzed to study the variation of organic nitrogen content during biodegradation. The heavy oils were formed by biodegradation of originally normal gravity oils19,21 sourced by the Es3 mature deep-water lacustrine sediments down-dip. For more details, the reader is referred to earlier publications (e.g., refs 19 and 20). The organic carbon, nitrogen, and hydrogen analyses of these oils and reservoir core extracts were carried out using a Carlo (14) Larter, S.; Willhelms, A.; Head, I.; Koopmans, M.; Aplin, A.; di Primio, R.; Zwach, C.; Erdmann, M.; Telnæs, N. Org. Geochem. 2003, 34, 601-613. (15) Helm, R. V.; Latham, D. R.; Ferrin, C. R.; Ball, J. S. Anal. Chem. 1960, 32, 1765-1767. (16) Seifert, W. K. Anal. Chem. 1969, 41, 62-68. (17) Larter, S. R.; Bowler, B. F. J.; Li, M.; Chen, M.; Brincat, D.; Bennett, B.; Noke, K.; Donohoe, P.; Simmons, D.; Kohnen, M.; Allan, J.; Telnæs, N.; Horstad, I. Nature 1996, 383, 593-597. (18) Oldenburg, T. B. P.; Huang, H.; Donohoe, P.; Willsch, H.; Larter, S. R. Org. Geochem. 2004, 35, 665-678. (19) Huang, H.; Bowler, B. F. J.; Zhang, Z.; Oldenburg, T. B. P.; Larter, S. R. Org. Geochem. 2003, 34, 951-969. (20) Koopmans, M. P.; Larter, S. R.; Zhang, C.; Mei, B.; Wu, T.; Chen, Y. Am. Assoc. Pet. Geol. Bull. 2002, 86, 1833-1843.
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Figure 1. Variation in the saturated hydrocarbon content with depth within a heavily biodegraded Es1 oil column and three less degraded Es3 oil columns (after Huang et al.19).
Erba 1106 elemental analyzer. The samples were analyzed as duplicates. Levels of biodegradation of sampled oils and petroleums extracted from reservoir cores were assessed using standard organic geochemical techniques. Briefly, a finely crushed reservoir core sample was added to dichloromethane (DCM)/ methanol (93:7) and extracted in an ultrasonic bath as described by Huang et al.19 After asphaltene precipitation and the addition of standard compounds to the maltenes (for details, see ref 19), the maltene fractions were separated using solid-phase extraction (SPE) into hydrocarbon and non-hydrocarbon fractions.19 The hydrocarbon fractions were separated into aliphatic and aromatic hydrocarbons using a silver nitrate silica gel SPE method we have published previously.22 Detailed analysis of the aliphatic and aromatic fractions and non-hydrocarbon fractions were carried out using GC-MS techniques.19 Results Hydrocarbon Biodegradation. The degree of biodegradation within the oil columns varied from the top to the bottom of the reservoir from incipient degradation (loss of short-chain nalkanes near the top of the deep Es3 reservoir columns) to very heavy degradation (loss of all n-alkanes, acyclic isoprenoids (pristine, phytane), steranes, and hopanes at the bottom of the shallower Es1 reservoir column), covering a range of 1-8 on the Peters and Moldowan1 scalesPM level 1-8. Whereas the bulk C15+ petroleum composition data from the three deeper Es3 wells (A, B, and C) show a consistent decrease in saturated hydrocarbon content from 45% w/w to about 25% w/w with increasing depth (Figure 1, data from Huang et al.19), the hydrocarbon content in the shallower Es1 column decreases only slightly through well D with complete removal of all n-alkanes, even at the top of the column. Huang et al.19 showed, using several conventional biodegradation-related molecular ratios, that the degree of biodegradation ranges from PM level 1 to level 4-5 in the three deep Es3 reservoir columns and from level 5 to level 8 through the shallower Es1 column. Influence of Biodegradation on Nitrogen-Compound Distributions in Reservoired Oils. Free carbazole and benzocarbazole components are common constituents in petroleum systems (e.g., refs 15 and 23-26), with the concentrations and distributions being affected by source maturity (e.g., refs 27 and 28) and migration processes (e.g., refs 17, 29, and 30). Alteration of carbazoles during biodegradation as a source of nitrogen for biodegradation is an important process to (21) Lu, S.; He, W.; Huang, H. In AdVances in Organic Geochemistry 1989; Baker, E. W., Douglas, A. G., Eds.; Pergamon Press: Oxford, U.K., 1990; pp 437-449. (22) Bennett, B.; Larter, S. R. Anal. Chem. 2000, 72, 1039-1044.
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Figure 2. Variation in the absolute abundance of carbazoles, benzocarbazoles, and dibenzocarbazoles or naphthocarbazoles with depth within an Es1 oil column and three Es3 oil columns (after Huang et al.19).
consider, as carbazoles typically represent the most abundant single N-containing compound group and are, thus, a major component of the organic nitrogen, in petroleum.23,26 Some early general observations about the ability of microbes to metabolize petroleum compounds, indicating that heteroaromatic NSO compounds with a small number of rings might be biodegradable, were provided by Atlas31 and Fedorak and Westlake.32 Recently, Zhang et al.33 and Oldenburg et al.30,34 also demonstrated the effect of biodegradation on the concentration and distribution of alkylcarbazoles in petroleum. Huang and co(23) Snyder, L. R.; Buell, B. E.; Howard, H. E. Anal. Chem. 1968, 40, 1303-1317. (24) Dorbon, M.; Schmitter, J. M.; Garrigues, P.; Ignatiadis, I.; Ewald, M.; Arpino, P.; Guiochon, G. Org. Geochem. 1984, 7, 111-120. (25) Frolov, Y. B.; Smirnov, M. B.; Vanyukova, N. A.; Sanin, P. I. Pet. Chem. U.S.S.R. 1989, 29, 87-102. (26) Bakel, A. J.; Philp, R. P. Org. Geochem. 1990, 16, 353-367. (27) Horsfield, B.; Clegg, H.; Wilkes, H.; Santamarı´a-Orozco, D. Naturwissenschaften 1998, 85, 233-237. (28) Li, M.; Fowler, M. G.; Stasiuk, L. D. Org. Geochem. 1998, 29, 163-182. (29) Terken, J. M. J.; Frewin, N. L. Am. Assoc. Pet. Geol. Bull. 2000, 84, 523-544. (30) Oldenburg, T. B. P.; Horsfield, B.; Wilkes, H.; van Duin, A. C. T.; Stoddart, D.; Wilhelms, A. Presented at 20th International Meeting on Organic Geochemistry, Nancy, France, 2001. (31) Atlas, R. M. Microbiol. ReV. 1981, 45, 180-209. (32) Fedorak, P. M.; Westlake, D. W. S. Appl. EnViron. Microbiol. 1984, 47, 858-862. (33) Zhang, C.; Zhao, H.; Mei, B.; Chen, M.; Xiao, Q.; Wu, T. Shiyou Yu Tianranqi Dizhi (Oil and Gas Geology) 1999, 20, 341-343. (34) Oldenburg, T. B. P.; Horsfield, B.; Wilkes, H.; Stoddart, D.; Wilhelms, A. Earth Syst. Process. 2001, 126; The Geological Society of America and The Geological Society of London, Edinburgh.
Figure 3. Change of the nitrogen content with increasing degree of biodegradation [BP2 ) C30 R,β-hopane/(pristane + phytane); see also Koopmans et al.20] in a series of produced oils from the Liaohe Basin (solid cycles); the triangles illustrate the comparative concentrations of C0-2 carbazoles in a larger set of oils versus the same parameter (black highlighted triangles show the oil samples selected for the whole oil nitrogen determination).
workers35,19 found that benzocarbazole and methylbenzocarbazole concentrations and isomer distributions are also clearly affected by biodegradation. (35) Huang, H.; Ren, F.; Larter, S. R. Chin. Sci. Bull. 2002, 47, 17341739.
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Figure 4. Total nitrogen content changes in the whole oils and separated asphaltenes from the three Es3 oil columns and the Es1 oil column petroleum.
Although alkylcarbazoles are generally regarded as resistant to biodegradation at low to moderate levels of biodegradation, at levels of biodegradation greater than PM level 4 they are altered in a similar way to that observed for aromatic hydrocarbons (Figures 2 and 3, triangles). During the early stages of biodegradation, the concentrations of carbazole compounds in the oils increase as hydrocarbons are destroyed, followed by a decrease in concentration after PM level 4. In addition, the alkylcarbazoles are preferentially depleted compared to benzoand dibenzocarbazoles or naphthocarbazoles, as shown in Figure 2. This indicates that the order of susceptibility to biodegradation for carbazoles decreases with increasing ring number. Change of Nitrogen Content during Biodegradation. This is the first time that petroleum nitrogen contents have been measured throughout oil columns with wide ranges of degree of biodegradation, to the best of our knowledge. The goal was to investigate if the organic nitrogen content changes with varying degrees of biodegradation and if organic nitrogen is potentially available as a nutrient for the petroleum-degrading biosphere. Although C0-C3 carbazoles are an important nitrogen-bearing compound group in oils, the nitrogen from the C0-C3 carbazoles contributes less than ∼1‰ of the whole amount of the nitrogen in a crude oil. An assessment of the total nitrogen content changes in the oils occurring within each oil column was, thus, based on analysis of the nitrogen content of whole-oil extracts and the asphaltenes. At low degrees of biodegradation when normal hydrocarbons are being destroyed, an increase in nitrogen is observed up to PM level 3 in both the core extracts (Figure 4) and in the whole oils (Figure 3). This reflects degradation of hydrocarbons, while nitrogen-containing compounds are still largely unaffected. At higher degrees of biodegradation, the nitrogen content of the whole oils (Figures 3 and 4) declines slowly, which reflects degradation of N-containing compounds in addition to hydrocarbons. The nitrogen content of the separated asphaltenes shows no consistent trend through the whole
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biodegradation sequence. The only minor increase in nitrogen content in the core extract petroleums in the Es1 section at degradation levels above PM 5 suggests little nitrogen is either added to the oil or removed during biodegradation. The change of the nitrogen content with increasing degree of biodegradation is qualitatively similar to the change in carbazole concentrations (Figures 2 and 3, triangles), suggesting that C0-C3 carbazole degradation is a proxy for overall carbazole structures pool alteration in the crude oils. At high levels of biodegradation, nitrogen derived from oil may, thus, be accessible in mineral form and a nutrient by the petroleum-degrading biosphere, but at low degrees of biodegradation, a source of nitrogen other than organic petroleum nitrogen has to be used for microbial growth. In summary, the oils from the Liaohe Basin, NE China, are strongly affected by biodegradation (Figure 1). At a low degree of biodegradation, first an increase in the nitrogen content was observed followed by a sharp decrease in concentration in the lower part of the Es3 columns (Figure 4) and higher C30 R,βhopane/(pristane + phytane) values (BP2) of the oils (Figure 3) where degradation is more severe. This may be the result of degradation of the nitrogen-containing compounds to other components, including low-molecular-weight inorganic compounds. Thus, small amounts of petroleum nitrogen could potentially be available for use during petroleum biodegradation at levels above PM level 3, but at lower levels of biodegradation nitrogen, as an essential nutrient, it would have to be supplied from other sources such as nitrogen gas or ammonium ion in formation waters. A recent study from Manning and Hutcheon36 showed that ammonium ion is abundant in oilfield waters and that the distribution of ammonium ion (NH4+) is controlled by cation exchange with illite or micas. This system could supply the essential nutrient nitrogen at lower degrees of biodegradation and possibly even throughout the whole degradation process. Dinitrogen gas is also a common component of oilfields, and nitrogen-fixing microorganisms may convert molecular N2 to ammonia, which hydrocarbon-degrading organisms could perhaps use as a nitrogen source below PM level 3. While nitrogenfixing organisms have not yet been reported in petroleum reservoirs, to the best of our knowledge, the anaerobic conditions and abundance of N2 gas in oilfields at the mole % level, plus the nonavailability of significant oil-derived organic nitrogen below PM level 4, suggest to us it is likely that N-fixing organisms do exist in oil reservoirs. Thus, while carbazole-derived nitrogen may be a component of nutrient supply for hydrocarbondegrading organisms at intermediate and high levels of biodegradation, this cannot be proven with the current study. Conclusion Because hydrocarbon supply is not limiting in subsurface biodegradation,14 a limiting nutrient must limit degradation rate and extent. Of the common nutrients, nitrogen and phosphorus are the most likely to be limiting. Because nitrogen for biodegradation could be provided from nitrogen gas, ammonium ion in formation waters, or, above biodegradation levels PM 3, from the crude oil itself through carbazole structure degradation, we conclude that nitrogen sources are sufficiently available and that other essential nutrients such as phosphorus are more likely biodegradation-limiting elements. Acknowledgment. The work was encouraged and supported by the Bacchus Consortia, who are thanked: Agip, BP, Chevron, ConocoPhillips, Norsk Hydro, Petrobras, Saudi Aramco, Shell, Statoil, Total, and Woodside. EF060148P (36) Manning, D. A. C.; Hutcheon, I. E. Appl. Geochem. 2004, 19, 14951503.
