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Bioconversion Reactions in Asphaltenes and Heavy Crude Oils E. T. Premuzic,* M. S. Lin M. Bohenek, and W. M. Zhou Energy Science and Technology Division, Department of Applied Science, Brookhaven National Laboratory, Upton, New York 11973 Received November 2, 1998. Revised Manuscript Received December 30, 1998
Interactions between select microorganisms and heavy crude oils in which the microorganisms have been introduced under controlled conditions proceed via a complex set of multiple biochemical and chemical reactions. Extensive studies in this laboratory have shown that such reactions are not random and differ from those involved in biodegradation of oils and follow distinct trends which can be categorized by means of characteristic chemical markers. These markers include mass spectrometric fragmentation patterns of light and heavy hydrocarbons, heterocyclic, organometallic compounds, trace metals, and the contents of heteroatoms. Bioconversion of heavy crude oils depends on the distribution of polar compounds containing nitrogen (N), sulfur (S), oxygen (O), and trace metals. The overall effects of induced biochemical reactions are a significant lowering (24-40%) of the N, S, O, and trace metal contents. Concurrently, there is also a redistribution of hydrocarbons (HC). The reactions are both biocatalyst and crude oil dependent and, in terms of chemical mechanisms, appear to involve the asphaltene and the associated polar fractions. Recent studies dealing with biochemical reactions in heavy crudes will be discussed.
Introduction During the conversion of sedimentary organic matter from an initial input of natural products to oil, the original organic matter is subjected to multiple changes. Characteristic chemical changes continue to occur in the formed oil and are conventionally described in terms of the chemical composition of oils, for example, the content of major fractions such as saturates, aromatics, resins, and asphaltenes. Further classification of oils characterizes the oils as paraffinic, paraffinic-naphthenic, aromatic, and aromatic asphaltic. Although this organic matter is a complex reaction mixture and subject to multiple changes, its conversion during diagenesis and catagenesis follows observable trends which can be characterized and monitored by a distinct set of chemical markers.1,2 Although the chemical processes which lead to different types of oils are complex, the oils can be described in relatively simple terms such as light and heavy. In the transition of light to heavy oils, the ratio of hydrogen to carbon decreases and the ratio of the sum of heteroatoms, i.e., nitrogen, sulfur, and oxygen, to carbon increases.1,3 In addition, as the oil becomes richer in compounds containing nitrogen, sulfur, and oxygen, it also becomes richer in compounds containing metals, such as nickel and vanadium. Concurrently, the physical properties of an oil also change and may also be followed by monitoring * To whom correspondence should be addressed. E-mail: premuzic@ bnl.gov. (1) Hunt, J. M. Petroleum Geochemistry and Geology; Freeman: San Francisco, CA, 1979. (2) Tissot, B. P.; Welte, D. M. Petroleum and Occurrence; SpringerVerlag: New York, 1978. (3) Premuzic, E. T.; Lin, M. S. J. Pet. Sci. Eng. 1998, 22 (1-3), 171180.
distinct physical properties which include oAPI gravity, pour point, distillation, and others.1 Additional properties of oils of particular importance to the present topic of biochemical reactions and biocatalysis of crudes must also be considered. Thus, heavy crude oils can be distinguished by their reservoir history, i.e., they are heavy because they were biodegraded in the reservoir over geological periods of time or they are heavy because they are immature and their chemical composition has significant evidence of original input of organic matter. A convenient way to distinguish such properties is the use of biochemical markers. These markers are “chemical fossils” or molecules which have retained a resemblance to their natural product precursors during diagenesis, catagenesis, migration, and metamorphism.4 An extension of the “chemical marker” concept and its application to the characterization of bioconverted crude proved to be particularly useful in the studies of biocatalytic processing of oils.5 It is to be noted that biocatalytic conversion of heavy crude oil is due to the action of microbial biocatalysts introduced into the oil under controlled reaction conditions (e.g., time, temperature, pressure), and it is not due to indigenous microorganisms present in the oil and acting in a reservoir over geological periods of time and reservoir conditions. Furthermore, the action of biocatalysts is both biocatalyst and oil specific, resulting in considerable variations in the end-products. The significance of such variations in terms of heavy crudes and their chemical compositions will be discussed in the present paper. (4) Peters, U. E.; Moldowan, J. M. The Biomarker Guide; Prentice Hale: New York, 1993. (5) Premuzic, E. T.; Lin, M. S.; Lian, H.; Zhou, W. M.; Yablon, J. Fuel Process. Technol. 1997, 52, 207-223.
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Experimental Section Preparations of Biocatalyst and Biotreatment of Oil. Bacterial catalysts were prepared by culturing selected strains in nutrient broth (B3 DIFCO Manual).6 One volume of biocatalyst stock was stirred into 19 volumes of an oil and water (1:10 w/v) mixture and incubated at 40-65 °C for 48 h under aerobic conditions. The choice of temperature depends on the density of the oil. The reaction mixture was then extracted with methylene chloride. A portion of the extracted sample was analyzed directly by GC-MS, the solvent was removed from the remaining sample, and the residue was kept for further studies. Oil Samples. OSC (Off Shore California), oAPI 15.9, is an immature heavy crude from Santa Barbara, CA; MWS (Midway Sunset), oAPI 16.5, is a biodegraded heavy crude which was steam-recovered from Kern county, CA; CN (Cerro Negro) o API 8.2, is a biodegraded heavy crude from Venezuela; A851, o API 11.9, is a biodegraded on-shore Monterey, CA, heavy crude. Solvent Extraction. In the development of a new process and/or chemical reaction pathways, a good mass balance is of critical importance. However, in very viscous systems, such as heavy crude oils, “recovery” becomes experimentally difficult to accomplish, because such materials stick to surfaces. In bioprocessing, this becomes further complicated by formation of emulsions. Because of these experimental difficulties, solvent extraction, at the present time, appears to be the best solution. The choice of methylene chloride was based on the fact that it does not introduce sulfur into the system, which would compromise analysis, and the oil mixture has good solubility properties in this solvent, ensuring that no interaction between peptizing agents, such as resins and asphaltenes, can occur.7 In addition, methylene chloride volatilizes at 40 °C and fully evaporates and makes possible a minimum loss of volatiles and a water-free (C25) begin to elute. A comparison of untreated with treated OSC in Figure 1 allows the detection of hydrocarbons in the lighter and heavier fractions. In this figure, an increase of the kerosene fraction (C12-16) and gas-oil fraction (>C26) in the biotreated OCS oil is observed. It is to be noted that conventional gas chromatography is not the best tool for analyzing the heavy crude oils because of retention of large molecular weight components. Therefore, complimentary pyrolysis GC-MS of the corresponding pentane-precipitated asphaltene is needed. As shown in Figure 2, Py-GC-MS analysis of OSC reveals further biochemical conversion of the heavy ends of the crude. A substantial increase in the yield of hydrocarbons (C8C32) is also an indication of cleavage of the macromo-
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Energy & Fuels, Vol. 13, No. 2, 1999 301 Table 9. List of Organosulfur Compounds Identified 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
H2S and other sulfur gases thiophene C1-substituted thiophenes C4-C8 sulfides C3-C6 mercaptans unidentified sulfur compounds unidentified sulfur compounds C2-substituted thiophenes C3-substituted thiophenes C4-substituted thiophenes C5-substituted thiophenes benzothiophene C1-substituted benzothiophenes C2-substituted benzothiophenes bithiophene dibenzothiophene dibenzothiophene sulfone 1,2-benzodiphenylene
Table 10. Effect of Different Biocatalysts on the SARA Distribution in a Single, Biodegraded (A851) Crudea A851 BNL-Z-3 BNL-TH-31 BNL-TH-29 BNL-4-21 BNL-4-22 BNL-4-23 BNL-4-24 a
Figure 3. FPD (sulfur-specific detector) chromatogram trace of untreated and treated OSC crude.
Figure 4. Pyrolysis-gas chromatograph analysis (Py-GC) FPD (sulfur-specific detector) trace of asphaltenes from OSC crude treated with BNL-4-23.
lecular matrix. Direct comparison of data is possible because the same experimental conditions (e.g., detector response, sample size, etc.) were used in all analyses. Extension of this analytical approach to sulfur analysis using a sulfur-specific detector (FPD) indicated that, in
saturate %
aromatic %
resin %
asphaltene %
19.19 23.62 22.01 24.72 32.29 28.72 34.42 29.21
45.15 31.64 35.27 28.62 32.04 33.87 29.72 19.59
31.23 38.94 37.88 43.44 32.00 33.31 32.71 38.20
4.44 5.79 4.84 3.41 3.67 4.09 3.57 2.99
A851 ) Monterey 851 untreated.
addition to the lowering of the total sulfur content, consistent with elemental analysis (e.g., Table 4), there are fine structural changes, i.e., lowering of the sulfur peaks in substituted thiophenes (peaks 9, 10, 11), benzothiophenes (peaks 13, 14), and dibenzothiophenes (peak 16). For peak identification, see Table 9. FPD GC chromatograms as shown in Figure 3. The corresponding pyrolysis gas chromatograph (Py-GC) analysis of the OSC pentane-precipitated asphaltenes given in Figure 4 shows a reduction of sulfur peaks, in general, particularly noticeable in unidentified sulfur peaks (peaks 6 and 7), C2-C5-substituted thiophenes (peaks 8-11), C1-C2-substituted benzothiophenes (peaks 13 and 14), and dibenzothiophenes (peak 16) and is consistent with X-ray analysis.9 Comparable results have been obtained with a Cerro Negro crude and are shown in Figures 5-8. Cerro Negro is a biodegraded and much heavier crude. It is to be noted that this set of experimental data also indicates that the effect of the biocatalysts on the asphaltene fraction of Cerro Negro is significantly more than that in the whole oil (vide infra). Conventional GC analyses shown in Figures 5 and 7 for alkanes and sulfur compounds are not well resolved because of the low volatility of the starting material. However, Py-GC analyses of the biotreated Cerro Negro shows a substantial increase in the hydrocarbon yield (Figure 6) and an overall reduction in the contents of sulfur compounds (Figure 8). The latter is also consistent with the decrease of the total sulfur content (see Table 4). Preliminary results of comparable analyses of nitrogencontaining fractions follow a similar pattern and have been discussed elsewhere.3,5 The efficiency of biocatalytic activity in heavy ends of crudes is of key importance in the development of new technology based on biocatalysis. In terms of biochemical and chemical mechanisms of biocatalysis, an understanding of de-
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Figure 5. GC-MS analysis: m/e 57 gas chromatogram trace of untreated and treated Cerro Negro crude.
Figure 7. FPD (sulfur-specific detector) chromatogram trace of untreated and treated Cerro Negro crude.
Figure 6. Py GC-MS: m/e 57 gas chromatogram trace of asphaltenes derived from Cerro Negro crude.
tailed compositional and structural changes in chemical markers due to biocatalysis is of paramount importance. Toward this goal, asphaltenes isolated from Cerro Negro crude have been subjected to liquid chromatographymass spectrometric analysis (LC-MS). Preliminary results of these analyses are presented in Figures 9-11. The solvent mixtures used in liquid chromatography (LC) were programmed to elute nonpolar compounds first, then compounds with increasing polarity. In Figure 9, liquid chromatography trace of the BNL 4-23treated sample shows five new peaks which appear before 9 min in the less-polar region of the chromatogram and are not present in the untreated samples. The biochemical reactions are expected to convert the polar macromolecular resin and asphaltene fractions into
Figure 8. Pyrolysis-gas chromatograph analysis (Py-GC) FPD (sulfur-specific detector) trace of asphaltenes from Cerro Negro crude treated with BNL-4-23.
smaller, less polar, or nonpolar fragments. At the same time, if heteroatoms were also removed from these fragments, the products should become even less polar. Analysis of the higher polarity region of treated samples, e.g., retention time of 30.23 min and above (Figure 11),
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Figure 9. Liquid chromatograph (LC-MS) of asphaltene isolated from Cerro Negro heavy crude.
Figure 12. Formation of molecular solutions. Colored circles represent active sites.
Discussion
Figure 11. Mass spectrum of polar fractions at retention times of 30.91, 32.27, and 36.57 min.
Relative concentrations of compounds containing sulfur, nitrogen, oxygen, and trace metals vary with different oils, and while present in all fractions of oil, they are predominantly concentrated in the heavy ends, resins, and asphaltenes. For the purposes of this discussion, it is reasonable to assume12,13 that an average elemental formula of C420H496N6S14O4V and an H/C ratio of 1.18 and (N + S + O)/C of 0.057 is representative of an asphaltene “molecule”. A three-dimensional structure of the model molecule is shown in Figure 12 (“initial molecule”). Such structures, together with resins, aromatic, and saturated hydrocarbons,3,5 are capable of forming inclusion complexes, clathrates, charge-transfer complexes, coordination compounds, as well as multiplebridged “polymeric” structures, stacked and connected via weak (complexes) and strong (heteroatom bridges) bonds. Resulting mixed structures form clusters, micelles, and ultimately molecular solutions, as shown in Figure 12. The relative distribution of such structures varies with low abundance in light oils and progressively increasing abundance as oils become heavier. However, where present, these structures may serve as anchors for biocatalysts. More specifically, the biocatalysts will attach themselves to available heteroatom sites.14 It is a well-known phenomenon that in complex mixtures of natural products15,16 containing multifunctional sites
also showed more peaks. While detailed analyses are in progress, preliminary data reported in this paper are consistent with biochemical pathways which cause a breakdown of large polar fractions into smaller fragments of varying polarity, indicating chemical differences in the treated and untreated high molecular weight fractions of the crude. However, until an adequate database has been established, these results must be considered diagnostic.
(11) Premuzic, E. T.; Lin, M. S. Microbial Enhancement of Oil RecoverysRecent Advances; Elsevier: Amsterdam, 1991; pp 277-296. (12) Strausz, O. P.; Mojelsky, T. W.; Lown, E. M. Fuel 1992, 72, 2355-1363. (13) Yen, T. F. Advances in the Applications of Membrane-Mimetic Chemistry; Plenum Press: New York, 1994; pp 255-279. (14) Tuovinen, O. H. Biological fundamentals of Mineral Leaching Processes Microbial Mineral Recovery; McGraw-Hill: New York, 1990. (15) Karrer, P. Organic Chemistry: Natural Products Section; Elsevier Publishing Co., Inc.: New York, 1950. (16) Lehninger, A. L. Biochemistry, 2nd ed.; Electron Transport Section; Worth Publishers: New York, 1975.
Figure 10. Mass spectrum of less polar fractions at retention times of 3.51, 5.03, and 8.45 min.
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(e.g., those containing functional groups such as ketones, esters, aldehydes, sulfur, and nitrogen functions as well as molecular entities such as those bound by van der Waals forces, coordination, change-transfer, inclusion, clathrates etc.) can be affected by reactions with low energies of activation. Such reactions may be caused by changes in acidity, alkalinity, and enzymatic reactions, even when intervening molecules involved in the electron transport are not clearly definable. Such reactions may lead to multiple and simultaneous inter- and intramolecular structural rearrangements within the organic phase and the aqueous phase containing electrolytes and biocatalysts, ultimately leading to unfolding and destacking of the initial, three-dimensional structures, in a manner which schematically may be a reversal of the process represented in Figure 12. The net result is a formation of smaller fragments, release of entrapped molecules with simultaneous redistribution of hydrocarbons, and fragmentation at the heteroatom sites. In this process the heteroatom moieties appear to be converted to water-soluble species and are extracted into the aqueous phase. Extensive and continuing studies of mechanistic pathways using multiparameter analyses will allow for a better understanding of the complex processes associated with catalysis and biocatalysis.17 A better understanding of the basic mechanisms will also make possible better optimization strategies, essential to the development of new biocatalysis technology.
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Conclusions The use of chemical markers makes it possible to monitor the effects of biocatalysts on various crude oils. The biochemical mechanisms by which biocatalysts interact with crude oils appear to involve reactions at heteroatoms and organometallic sites. The present information also indicates that biocatalysts act in a manner which leads to a redistribution of hydrocarbons and fragmentation of heavy polar fractions. The overall effects of the biocatalytic conversion of the crudes are a reduction in sulfur and nitrogen concentrations, a reduction in the concentration of trace metals, and redistribution of hydrocarbons. Acknowledgment. This work is supported by the U.S. Department of Energy, Division of Fossil Fuels, under Contract Nos. AS-405-ESTD and DEAC02-98CH10886. We express our gratitude to Christine Liu for technical assistance, a participant in the Women and Minorities in Science program. We also acknowledge B. Manowitz of BNL for valuable comments and advice. EF9802375 (17) Dolbear, G. E.; Tang, A.; Moorehead, E. L. Metal Complexes in Fossil Fuels; ACS Symposium Series 344; American Chemical Society: Washington, DC, 1987; pp 220-232.
