Will Biochemical Catalysis Impact the Petroleum Refining Industry

Energy & Fuels 2002, 16, 1239-1250

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Will Biochemical Catalysis Impact the Petroleum Refining Industry? Rafael Vazquez-Duhalt,*,† Eduardo Torres,‡ Brenda Valderrama,† and Sylvie Le Borgne‡ Instituto de Biotecnologıá UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62250 Me´ xico, and Instituto Mexicano del Petro´ leo, Eje Central La´ zaro Ca´ rdenas 152, Me´ xico D.F., 07730 Me´ xico Received February 19, 2002

The potential application of biochemical catalysis in the petroleum refining industry is discussed by a prospective analysis of the available data on the microbial and enzymatic modification of oil products. The proposed biotechnological processes can be considered either alternative or complementary to conventional oil refining technologies. Desulfurization, denitrogenation, asphaltene upgrading, heavy metal removal, and effluent treatment by enzyme-mediated reactions are reviewed. The introduction of novel nonconventional techniques in the petroleum industry may improve its energetic efficiency and reduce its environmental impact.

Introduction Doubtless, history will describe our time as the oilbased society. Nature took 500 millions years to accumulate the world’s oil. A century ago the oil exploitation began, first as a source of energy and now as a source both of energy and raw material. Now, contemporary society is highly dependent on the oil supply for energy, transportation, food production, and in general, industrial production. Experts agree that the world’s petroleum will be consumed in two centuries.1 The inexorable production peak is estimated to occur sometime between 2010 and 2020, and then the oil resources will be drastically reduced at the end of this century.2 Shortly after the production peak, the more expensive fuel sources as hard-to-extract oil deposits, tarry sands, and synfuels from coal will be brought to the front of production. In addition to the expected development and implementation of new technologies for conventional processes, such as cracking, hydrogenation, isomerization, alkylation, polymerization, and hydrodesulfurization, the introduction of non-conventional new technologies could be expected, representing potential substitutes or processes complementary to traditional oil refining. These new technologies should improve the refining efficiency in energetic terms and reduce the environmental impact of the processes. Significant progress has been made in the past decades in technologies such as membrane separation,3 supercritical extractions,4-6 and many others. Biotechnology is among the new fields that * Author to whom correspondence should be addressed at Instituto de Biotecnologıá UNAM, Apartado Postal 510-3, Col. Chamilpa, Cuernavaca, Morelos, 62250 Mexico. Fax: (52) 555-622-7655. E-mail: [email protected]. † Instituto de Biotecnologıá UNAM. ‡ Instituto Mexicano del Petro ´ leo. (1) Kerr, R. A. Science 1998, 281, 1128-1131. (2) Campbell, C. J.; Laherre`re, J. H. Sci. Am. 1998, 278, 78-84. (3) Lai, W. C.; Smith, K. J. Fuel 2001, 80, 1121-1130.

might be introduced to the oil refining industry. The first contact between biotechnology and the oil industry was in environmental processes, such as wastewater treatment and soil bioremediation. However, there are other potential uses of biotechnological processes for the oil industry. It is important to point out that so far no enzymatic or biochemical processes exist in the oil refining industry, thus this work is not a conventional review of the published information on the field. This work is a prospective analysis of data on microbial and enzymatic transformations of petroleum products and their derivatives, to evaluate the possible impact of biotechnological processes on the petroleum refining. Bioremediation, reservoir engineering, and gas processing are not discussed in this work. This prospective analysis emphasizes biodesulfurization and asphaltene upgrading, but promising genetic and solvent engineering approaches are also discussed. Biodesulfurization Petroleum contains significant levels of sulfur, ranging from 0.2 to 5% in weight depending on the reservoir. The combustion of sulfur-containing fuels yields sulfur oxides (SOx) and environmental concerns govern the development of pre- and posttreatment technologies. Strict regulations on sulfur content in fuels have been recently implemented throughout the world to control these emissions.7 The Environmental Protection Agency of the United States (EPA) recently proposed a 97% reduction of the sulfur content in diesel to less than 15 ppm by 2006, down from the current specification of 500 ppm.8 (4) Scott, D. S.; Radlein, D.; Piskorz, J.; Majerski, P.; deBruijn, T. J. W. Fuel 2001, 80, 1087-1099. (5) Dadashev, M. N.; Stepanov, G. V. Chem. Technol. Fuels Oils 2000, 36, 8-13. (6) Park, S. J.; Kim, C. J.; Rhee, B. S. Ind. Eng. Chem. Res. 2000, 39, 4897-4900. (7) Monticello, D. J. Chemtech. 1998, 28, 38-45.

10.1021/ef020038s CCC: $22.00 © 2002 American Chemical Society Published on Web 07/16/2002

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Vazquez-Duhalt et al.

Figure 1. Some polyaromatic, sulfur, and nitrogen heteroaromatic compounds found in petroleum.

Up to 70% of the sulfur in diesel fuel is found as dibenzothiophene (DBT) and substituted DBTs (Figure 1).9 After crude oil distillation, these compounds are concentrated in the middle distillate fractions used to produce diesel. These compounds are the most resistant to the hydrodesulfurization (HDS) process currently used for the precombustion removal of sulfur in fuels.9,10 HDS involves the use of hydrogen gas to reduce sulfur, in the presence of metallic catalysts at high pressures (150-250 psi) and temperatures (200-425 °C) depending on the feedstock and the level of desulfurization required. To achieve the required sulfur content of less than 15 ppm using the HDS process, higher temperatures and pressures will be required, leading to an increase in the cost of this treatment, eventually higher CO2 emissions, and secondary reactions affecting diesel quality. Thus, new technologies are needed to remove sulfur from the most resistant molecules to meet the new specifications on sulfur content in diesel. An alternative approach is the use of microbial catalysts to perform the desulfurization reaction at low temperature and under atmospheric pressure. This process, known as biodesulfurization was first described in the 1950s by ZoBell, in which anaerobic sulfatereducing bacteria were used to perform reductive organic sulfur removal from petroleum.11 Since then, other anaerobic bacteria able to convert DBT to biphenyl and (8) Fletcher, S. US EPA proposes severe diesel sulfur limits. O. G. J. Online Story. May 17, 2000. (9) Schulz, H.; Bohringer, W.; Waller, P.; Ousmanov, F. Catal. Today 1999, 49, 87-97. (10) Shafi, R.; Hutchings, G. Catal. Today 2000, 59, 423-442. (11) ZoBell, C. E. Process for removing sulfur from petroleum hydrocarbons and apparatus. U.S. Patent 2,641,564, 1953.

hydrogen have been described.12-14 However, all these microorganisms presented very low desulfurizing activities not compatible with a practical process. Many aerobic bacteria are able to degrade DBT either by a ring destructive pathway without removal of the sulfur or by a completely destructive pathway in which DBT is mineralized.15 Such metabolic pathways are not applicable to petroleum desulfurization since they lead to a loss of the fuel energetic value. At the beginning of the 1990s, the research in biodesulfurization intensified due to the implementation of more stringent regulations on the sulfur content in fuels. Biodesulfurization became relevant only since 1992 when several aerobic bacteria were isolated, most of them belonging to the genus Rhodococcus, able to selectively extract the sulfur atom from DBT molecule without degrading its carbon skeleton.16 Therefore, the energetic value of the fuels is not affected since DBT is not degraded but only transformed into 2-hydroxybiphenyl (2-HBP). This product is recycled to the organic phase constituted by the fuel itself, while the sulfur is eliminated in the form of inorganic sulfate in the aqueous phase containing the biocatalyst (Figure 2). The first isolated and patented Rhodococcus strain, R. erythropolis IGTS8, is the basis of the biodesulfurization process proposed by ENCHIRA Biotechnology Corpora(12) Kim, T. S.; Kim, H. Y.; Kim, B. H. Biotechnol. Lett. 1990, 12, 757-760. (13) Lizama, H. M.; Wilkins, L. A.; Scott, T. C. Biotechnol. Lett. 1995, 17, 113-116. (14) Armstrong, S. M.; Sankey, B. M.; Voordouw, G. Biotechnol. Lett. 1995, 17, 1133-1136. (15) Oshiro, T.; Izumi, Y. Biosci., Biotechnol., Biochem. 1999, 63, 1-9. (16) Kilbane J. J. Mutant microorganisms useful for cleavage of organic C-S bonds. U.S. Patent 5,104,801, 1992.

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Figure 2. Metabolic pathway of DBT desulfurization by Rhodococcus erythropolis IGTS8.

tion (ENBC), formerly Energy Biosystems Corporation (EBC).16 The metabolic pathway for desulfurization has been elucidated and the enzymes and genes implicated have been isolated.17-19 The desulfurizing activity of R. erythopolis IGTS8 is repressed by sulfate and by sulfurcontaining amino acids.20 The first two enzymes of the pathway, DszA and DszC are FMNH2-dependent monooxygenases (Figure 2). FMNH2 is regenerated by a NADH-dependent flavin reductase, DszD.21 The need for cofactors and the complexity of the metabolic pathway implies working with whole cells. Other strains able to efficiently desulfurize substituted DBTs have been reported.22,23 As in the case of HDS, substituted DBTs in positions 4 and 6 are the most difficult to biodesulfurize due to steric hindrance.24 Surprisingly, a Sphingomonas strain was reported to desulfurize more efficiently the sterically hindered substituted DBTs than DBT.25 The application of a new gene shuffling method allowed the isolation of an evolved Dsz monooxigenase more active toward substituted DBTs.26 Recently, the Petroleum Energy Center (PEC) from Japan reported the isolation of new ther(17) Denome, S. A.; Olson, E. S.; Young, K. Appl. Environ. Microbiol. 1993, 59, 2837-2843. (18) Denome, S. A.; Oldfield, C.; Nash, L. J.; Young, K. D. J. Bacteriol. 1994, 176, 6707-6716. (19) Gray, K. A.; Pogrebinsky, O. S.; Mrachko, G. T.; Xi, L.; Monticello, D. J.; Squires, C. H. Nat. Biotechnol. 1996, 14, 1705-1709. (20) Li, M. Z.; Squires, C. H.; Monticello, D. J.; Childs, J. D. J. Bacteriol. 1996, 178, 6409-6418. (21) Xi, L.; Squires, C. H.; Monticello, D. J.; Childs, J. D. Biochem. Biophys. Res. Commun. 1997, 230, 73-75. (22) Lee, M. K.; Senius, J. D.; Grossman, M. J. Appl. Environ. Microbiol. 1995, 61, 4362-4366. (23) Darzins, A.; Mrachko, G. T. Sphingomonas biodesulfurization catalyst. U.S. Patent 6,133,016, 2000. (24) Oshiro, T.; Hirata, T.; Izumi, Y. FEMS Microbiol. Lett. 1996, 142, 65-70. (25) Monticello, D. J. Curr. Opin. Biotechnol. 2000, 11, 540-546.

mophilic bacterial strains, identified as Paenibacillus and able to selectively desulfurize DBT without degrading its hydrocarbon matrix.27 These strains follow the same metabolic pathway than R. erythropolis IGTS8.28 Paenibacillus enzymes are homologous to the Rhodococcus enzymes, however they are active at higher temperatures, from 50 to 60°C. These strains were proposed for the development of a biodesulfurization process for crude oil at high temperatures where crude oil viscosity is lower and mass transfer limitations are reduced. Biodesulfurization of middle distillates was reported using either wild-type or genetically engineered Rhodococcus strains, and the extent of sulfur removal showed to be dependent on the reaction conditions and the initial sulfur content of the fuel. For instance, the sulfur content of a middle-distillate not HDS treated was reduced from 20 000 to 14 000 ppm using wild type Rhodococcus sp. strain ECRD-1.29 When the same strain was used to desulfurize a middle-distillate already treated by an aggressive HDS, the sulfur content was reduced from 669 to 56 ppm.30 Furthermore, the sulfur content of a middle-distillate obtained after a partial HDS was reduced from 1850 to 615 ppm using R. erythropolis I-19 containing multiple copies of dszC, (26) Coco, W. M.; Levinson, W. E.; Crist, M. J.; Hektor, H. J.; Darzins, A.; Pienkos, P. T.; Squires, C. H.; Monticello, D. J. Nat. Biotechnol. 2001, 19, 314-315. (27) Konishi, J.; Ishii, Y.; Onaka, T.; Okumura, K.; Suzuki, M. Appl. Environ. Microbiol. 1997, 63, 3164-3169. (28) Ishii, Y.; Konishi, J.; Okada, H.; Hirasawa, K.; Onaka, T.; Suzuki, M. Biochem. Biophys. Res. Commun. 2000, 270, 81-88. (29) Grossman, M. J.; Lee, M. K.; Prince, R. C.; Garrett, K. K.; George, G. N.; Pickering, I. J. Appl. Environ. Microbiol. 1999, 65, 181188. (30) Grossman, M. J.; Lee, M. K.; Prince, R. C.; Minak-Bernero, V.; George, G. N.; Pickering, I. J. Appl. Environ. Microbiol. 2001, 67, 1949-1952.

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dszA, and dszD.31 Hydrogenation and hydrogenolysis (occurring during HDS) might facilitate the oxidative pathways. The sulfur compounds left after HDS are less reactive toward hydrogen, but probably they are more reactive toward oxygen, opening an opportunity niche or creating an advantageous position for enzymatic catalysis compared to conventional HDS. Three main factors have limited the implementation of the microbial desulfurization, including the technology proposed by Energy Biosystems Co.: (i) the large amounts of water necessary for the microbial metabolism, (ii) the long residence time in the process that makes the reactor volumes unthinkable, and (iii) the limited microbial metabolization on the large variety of chemical structures found in the organosulfur compounds existing in the petroleum. None of these problems have been solved yet. In microbial biodesulfurization, the biocatalyst, made by whole cells in an aqueous solution, is brought in contact with the fuel to be treated and the desulfurization reaction occurs at the interface between the aqueous and organic phases. As mentioned above, one of the limitations of microorganisms to be used in the oil industry is precisely the need of an aqueous phase to perform the desulfurization reaction. In the previous examples, the reaction systems presented very low oilto-water volumetric ratios (0.01 and 0.25). Important quantities of water have therefore to be handled, increasing the difficulty of mixing and further separation of oil/water emulsions, and one economic drawback of such a ratio is the throughput of the unit impacting the feasibility of the microbial process. Ideally, the desulfurization reactions should be performed in the absence of water or in a very low water medium. This limitation might be addressed by using enzymes instead of whole microorganisms. Enzymes require less water than microorganisms to be active and stable in organic solvents and, theoretically, only a film of water covering their surface should be sufficient for catalysis to occur. The fuel itself could be the organic solvent, avoiding or minimizing the addition of water. Moreover, the use of reaction media with low water content increases the solubility and the bioavailability of hydrophobic substrates. An enzymatic process for fuel desulfurization has been described.32 This method is based on the biocatalytic oxidation of organosulfides and thiophenes contained in the fuel with hemoproteins to form sulfoxides and sulfones, followed by a distillation step in which these oxidized compounds are removed from the fuel. Straightrun diesel fuel containing 1.6% of sulfur was biocatalytically oxidized with chloroperoxidase from Caldariomyces fumago in the presence of 0.25 mM hydrogen peroxide. The reaction was carried out at room temperature and the organosulfur compounds were effectively transformed to their respective sulfoxides and sulfones which were then removed by distillation. The resulting fraction after distillation contained only 0.27% of sulfur (Table 1). In addition to organosulfur compounds, chloroperoxidase is able to react with other fuel components, (31) Folsom, B.; Schieche, D. R.; DiGrazzia, P. M.; Werner, J.; Palmer, S. Appl. Environ. Microbiol. 1999, 65, 4967-4972. (32) Ayala, M.; Tinoco, R.; Hernandez, V.; Bremauntz, P.; VazquezDuhalt, R. Fuel Process. Technol. 1998, 57, 101-111.

Vazquez-Duhalt et al. Table 1. Sulfur Content of Straight-Run Diesel Fuel after Enzymatic Oxidation with Chloroperoxidase from Caldariomyces fumago Followed by a Distillation to 325 °C as Final Distillation Point (modified after Ayala et al.34) distillation

enzymatic + distillation

(%)

sulfur (%)

TPH (%)

sulfur (%)

1.27 3.21

71 29

0.27 5.51

TPHa distillate residue a

83 17

Total Petroleum Hydrocarbons.

such as polyaromatic hydrocarbons.33 However, in oil complex mixtures it is expected that organosulfur compounds will be preferentially oxidized.34 It is important to point out that this enzymatic process does not need cofactors or expensive chemicals, only the enzyme and very low concentrations of hydrogen peroxide. Sulfur removal from a very complex mixture, such as petroleum fractions, is far from being accomplished. Conventional hydrodesulfurization becomes more expensive and less efficient as lower sulfur levels are needed. The broad specificity and high activity of chloroperoxidase encourage further investigation in the use of this enzyme as an efficient catalyst in fuel desulfurization. This enzymatic process could be applied after a conventional hydrodesulfurization using a reactor with the immobilized enzyme. Thus, enzymatic oxidation of fuels appears as an interesting alternative for desulfurization. However, the challenge is still to have an enzymatic preparation able to perform the sulfur oxidation directly in the petroleum fraction without addition of water, and stable under the conditions found in the refinery. Biodenitrogenation As sulfur, nitrogen is a petroleum contaminant which contributes to acid rain since it is released to the atmosphere in the form of nitrogen oxides (NOx) during the combustion of petroleum-derived fuels. In addition, nitrogen compounds form a deposit on the refining catalysts, decreasing their activity and reducing the refining efficiency.35 The catalyst inactivation during the cracking and hydrodesulfurization processes is mainly due to the conversion of carbazole and other non basic nitrogen molecules into basic derivatives which are adsorbed on the catalyst active sites.35,36 The presence of nitrogen compounds in petroleum may also promote tank and pipe corrosion and the oil degradation during storage.37 The consequence of the removal of 90% of the nitrogen content from petroleum may be an increase of up to 20% of gasoline yields, and thus an economic gain for the refining process.38 (33) Vazquez-Duhalt, R.; Ayala, M.; Ma´rquez-Rocha, F. J. Phytochemistry 2001, 58, 929-933. (34) Ayala, M.; Robledo, N.; Lopez-Munguia, A.; Vazquez-Duhalt, R. Environ. Sci. Technol. 2000, 34, 2804-2809. (35) Furimsky, E.; Massoth, F. E. Catalysis Today 1999, 52, 381495. (36) Dong, D.; Jeong, S.; Massoth, F. E. Catalysis Today 1997, 37, 267-275. (37) Koboyashi, T.; Kurane, R.; Nakajima, K.; Nakamura, Y.; Kirimura, K.; Usami, S. Biosc. Biotechnol. Biochem. 1995, 59, 923933. (38) Benedik, M. J.; Gibbs, P. R.; Riddle, R. R.; Willson, R. C. Trends Biotechnol. 1998, 16, 390-395.



Figure 3. Suggested pathway for the degradation of qunoline by Pseudomonas ayucida IGTN9m according Kilbane et al.44

The nitrogen content in petroleum can vary from 0.1 to 2% according to the source, with high content in heavy oils.39 Nitrogen is found in heterocycles such as quinoline (structural equivalent to benzothiophene) and carbazole (structural equivalent to DBT) (Figure 1). Although, the nitrogen content of petroleum is lower than its sulfur content, the removal of nitrogen is also an important task. Nitrogen removal can be reached using high pressure and temperature physicochemical processes equivalent to HDS. Microbial or enzymatic approaches for petroleum denitrogenation could be beneficial for a deep denitrogenation in which the classical hydroprocessing methods are costly and non selective. Until recently most of the microbial degradation pathways for nitrogen heterocycles were ring destructive, leading to loss of the fuels’ energetic value.38,40,41 The economic importance of nitrogen-containing heterocycle loss remains to be evaluated. Bacterial isolates showed the capacity to degrade pyridine to ammonia,42 and similarly Pseudomonas aeruginosa showed selective transformation of quinolines and methylquinolines to products retaining the caloric value of the hydrocarbons within the fuel.43 Recently, a Pseudomonas ayucida strain, named IGTN9m, was reported to be able also to selectively remove nitrogen from quinoline without degrading the carbon skeleton of this molecule (Figure 3).44 Treatment of petroleum with this strain removed up to 5% of the nitrogen content without decreasing its energetic value. Despite the identification and cloning of the genes responsible for carbazole degradation,45 more microorganisms will have to be isolated with the ability to selectively remove nitrogen from other nitrogen compounds, such as carbazole. The limiting factors for microbial denitrogenation are the same as for microbial desulfurization, and the enzymatic denitrogenation is still to be explored. (39) Speight, J. G. The chemistry and Technology of Petrolum; Marcel Dekker. Inc.: Asphaltenes New York, 1998; Chapter 10. (40) Sugaya, K.; Nakayama, O.; Hinata, N.; Kamekura, K.; Ito, A.; Yamagiwa, K.; Ohkawa, A. J. Chem. Technol. Biotechnol. 2001, 76, 603-611. (41) Gieg, L. M.; Otter, A.; Fedorak, P. M. Environ. Sci. Technol. 1996, 30, 575-585. (42) Rhee, S. K.; Lee, K. S.; Chung, J. C.; Lee, S. T. Can. J. Microbiol. 1977, 43, 205-209. (43) Aislabie, J.; Bej, A. K.; Hurst, H.; Rothenburger, S.; Atlas, R. M. Appl. Environ. Microbiol. 1990, 56, 345-351. (44) Kilbane, J. J.; Ranganathan, R.; Cleveland, L.; Kayser, K. J.; Ribiero, C.; Linhares, M. M. Appl. Environ. Microbiol. 2000, 66, 68893. (45) Sato, S.; Nam, J.; Kasuga, K.; Nojiri, H.; Yamane, H.; Omori, T. J. Bacteriol. 1997, 179, 4850-4858.

Asphaltenes Upgrading Asphaltene, the highest-molecular-weight fraction of petroleum is a dark amorphous solid especially rich in heteroatoms (S, O, N), and metals (Fe, Ni, V).39,46,47 Many problems associated with recovery, separation, or processing of heavy oils and bitumens are related to the presence of high concentrations of asphaltenes. This fraction is thought to be largely responsible for other adverse oil properties such as high viscosity and the propensity to form emulsions, polymers, and coke. In crude oils the asphaltenic fraction may represent less than the detection limit, or as much as 50% of the total. Asphaltenes are particularly abundant in bitumens and oil sands. Cracking and metal removal by biotechnological processes on this fraction could be envisaged. The molecular structure of asphaltenes has been an enigma for seven decades.47 From numerous investigations there are indications that asphaltenes are condensed aromatic cores containing alkyl and alicyclic moieties. Heteroatoms, such as nitrogen, oxygen, and sulfur are present as non- and heterocyclic groups. A significant amount of porphyrins (petroporphyrins) can be found containing mainly nickel and vanadium. A hypothetical asphaltene molecule is shown in Figure 4. The complexity of the asphaltene chemical nature is evidenced by the difficulty of analysis of both their molecular weight and their molecular structure. Asphaltenes are a very complex mixture and are defined only by their solubility properties: the asphaltenic fraction is insoluble in short-chain n-alkanes, specially pentane. The complexity of asphaltene mixtures is evident considering that there has been a 20year controversy over the order of magnitude of asphaltene molecular weight.48,49 Estimations of the average molecular weight by different techniques differ by as much as a factor of 10. Size exclusion chromatography has yielded average molecular weights as high as 10 000 Da, vapor pressure osmometry showed values of 4 000 Da, with laser desorption mass spectroscopy values ranging from 200 to 1200 Da, field ionization mass spectroscopy yields 700 Da, while an average molecular weight of 500-1000 Da was obtained with the technique of fluorescence depolarization. The larger molecular (46) Speight, J. G.; Moschopedis, S. E. In Chemistry of Asphaltenes; Bunger, J. W., Li, N. C., Eds.; American Society for Advanced Chemistry Series 195: 1981. (47) Strausz, O. P.; Mojelsky, T. W.; Lown, E. M. Fuel 1992, 71, 1355-1363. (48) Groenzin, H.; Mullins, O. C. J. Phys. Chem. A 1999, 103, 11237-11245. (49) Groenzin, H.; Mullins, O. C. Energy Fuels 2000, 14, 677-684.

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Figure 4. Hypothetical asphaltene molecule; adapted from Strausz et al.47

weight obtained with some techniques could be explained by the evident aggregation of asphaltenes. This aggregation defines the solution-dispersion process of asphaltenes in oil. The asphaltene polymolecular structure is governed by a balance between the propensity of fused aromatic ring systems to stack via π-bonding, reducing solubility and the steric disruption of stacking due to alkane groups, increasing solubility.50 The asphaltenic fraction is recognized as the most resistant fraction of oil. So far, there is no clear evidence that asphaltenes are degraded by microbial activity. Some reports on oil biodegradation claim the degradation of asphaltenic fraction by mixed bacteria.51,52 However, these reports did not describe the analytical results of extractable materials recovered from appropriate sterile controls. On the other hand, although microorganisms have been found associated with bitumens that contain high amounts of asphaltenes,53 the asphaltenic fraction did not support bacterial growth (50) Buenrostro-Gonzalez, E.; Groenzin, H.; Lira-Galeana, C.; Mullins, O. C. Energy Fuels 2001, 15, 972-978. (51) Bertrand, J. C.; Rambeloarisoa, E.; Rontani, J. F.; Giusti, G.; Mattei, G. Biotechnol. Lett. 1983, 5, 567-572. (52) Rontani, J. F.; Bosser-Joulak, F.; Rambeloarisoa, E.; Bertrand, J. C.; Giusti, G.; Faure, R. Chemosphere 1985, 14, 1413-1422. (53) Wyndham, R. C.; Costerton, J. W. Appl. Environ. Microbiol. 1981, 41, 791-800.

and no changes in asphaltene content could be found after bioconversion of heavy oils and asphaltenes.54,55 Most of the asphaltene losses during microbial activity could be considered to be abiotic losses.56 Because the asphaltene content was usually determined gravimetrically after n-alkane precipitation, the reported changes could be attributed to the disruption of the asphaltenic matrix by the production of surfactants during bacterial growth, liberating trapped hydrocarbons. Nevertheless, some experimental evidence supports the idea of asphaltene biocracking. Fungal depolymerization of low rank coal has been reported.57,58 Evidence of coal depolymerization and desulfurization by thermophilic bacteria has been obtained with pyrolysis-gas chromatography-mass spectrometry (pyr-gc-ms) and X-ray absorption near edge structure (XANES).59 In (54) Premuzic, E. T.; Lin, M. S.; Bohenek, M.; Zhou, W. M. Energy Fuels 1999, 13, 297-304. (55) Thouand, G.; Bauda, P.; Oudot, J.; Kirsh, G.; Sutton, C.; Vidalie, J. F. Can. J. Microbiol. 1999, 45, 106-115. (56) Lacotte, D. J.; Mille, G.; Acquaviva, M.; Bertrand, J. C. Chemosphere 1996, 32, 1755-1761. (57) Cohen, M. S.; Gabriele, P. D. Appl. Environ. Microbiol. 1982, 44, 23-27. (58) Strandberg, G. W.; Lewis, S. N. J. Ind. Microbiol. 1987, 1, 371375. (59) Lin, M. S.; Premuzic, E. T.; Manowitz, B.; Jeon, Y.; Racaniello, L. Fuel 1993, 72, 1667-1672.


Energy & Fuels, Vol. 16, No. 5, 2002 1245 Table 3. Petroleum Refinery Wastewater before and after Enzymatic Treatment Wagener and Nicell98

total phenols (mM) COD (mg/L) BOD5 (mg/L) toxicity (Microtox TU50)

untreated

HPOtreateda

0.353 306 93.7 57

0.013 128 20 2.1

a HPO, horseradish peroxidase. peroxidase. NR. Not reported.

Figure 5. Absorption spectra of the petroporphyrin-rich fraction of asphaltenes before and after chloroperoxidase enzymatic treatment. The enzymatic reaction was performed according Fedorak et al.65 Table 2. Nickel and Vanadium Removal from Petroporphyrin-Rich Fractions of Asphaltenes by Chloroperoxidase-Mediated Reaction heavy metal

Fedorak et al.a 65

Mogollon et al.b 66

nickel vanadium

20% 19%

57% 52%

a The asphaltene fraction assayed contained 30 µg/g of Ni and 140 µg/g of V. b The asphaltene fraction assayed contained 306 µg/g of Ni and 2700 µg/g of V.

addition, enzymatic depolymerization of low-rank coal mainly by ligninolytic enzymes, such as fungal laccases and lignin- and manganese-peroxidases has been reported.60,64 However, the data with low-rank coal should be take considering the significant differences on chemical composition between low-rank coal and petroleum, especially carboxylic acids and esters contents. Clear experimental evidence that enzymes are able to modify asphaltene molecules has been reported.65 Chloroperoxidase from the fungus Caldariomyces fumago was able to degrade petroporphyrins and asphaltenes (Figure 5). This asphaltene modification is significantly higher in systems containing organic solvent than in aqueous systems.65,66 Because of the insolubility of asphaltenes and petroporphyrins, mass transfer limitations are expected in aqueous reactions. The destruction of petroporphyrins by chloroperoxidase in the presence of hydrogen peroxide leads to removal of Ni and V from asphaltene molecules (Table 2), as in the case of synthetic nickel and vanadium porphyrins.65 Enzymatic (60) Pyne, J. W.; Stewart, D. L.; Fredrickson, J.; Wilson, B. W. Appl. Environ. Microbiol. 1987, 53, 2844-2848. (61) Wondrack, L.; Szanto, M.; Wood, W. A. Appl. Biochem Biotechnol. 1989, 20/21, 765-779. (62) Kaufman, E. N.; Scott, C. D.; Woddward, C. A.; Scott, T. C. Appl. Biochem. Biotechnol. 1995, 54, 233-248. (63) Hofrichter, M.; Fritsche, W. Appl. Microbiol. Biotechnol. 1997, 47, 419-424. (64) Hofrichter, M.; Fritsche, W. Appl. Microbiol. Biotechnol. 1997, 47, 566-571. (65) Fedorak, P. M.; Semple, K. M.; Vazquez-Duhalt, R.; Westlake, D. W. S. Enzyme Microb. Technol. 1993, 15, 429-437. (66) Mogollon, L.; Rodriguez, R.; Larrota, W.; Ortiz, C.; Torres, R. Appl. Biochem. Biotechnol. 1998, 70-72, 765-777.

b

Ibrahim et al.99 APOuntreated treatedb 2.02 482 NR NR

0.606 180 NR NR

APO, Arthromyces ramosus

treatment of asphaltenes is an interesting alternative for removing heavy metals in order to reduce catalyst poisoning in hydrotreatment and cracking processes. On the other hand, enzymatic cracking of asphaltene molecules should not be excluded. The reaction mechanism of chloroperoxidase and ligninolytic enzymes involves free radical production. Depolymerization of both natural aromatic polymers such as lignin and lowrank coal by peroxidases is mediated by free radicals.67 Free organic radicals are present in asphaltenes.68,69 From these data and from the activity of chemically generated radicals,70 it seems reasonable to propose an enzymatic free radical reaction potentially able to cleave carbon-carbon or carbon-sulfur bonds in a biocracking process. The huge energetic resource found as asphaltene-rich deposits is the driving force to investigate and to innovate upgrading technologies. Enzymatic Treatment of Oil Industry Effluents Biological wastewater treatment is a well-established technology in the petroleum refineries. Much experience has been accumulated in the petroleum industry regarding activated sludge processes. Untreated petroleum industry wastewaters contain, in addition to pollutants similar to those found in municipal wastewaters, oil, grease, various hydrocarbons, phenolics, sulfides, and metals (Table 3). Organic chemicals from waste streams that include petroleum products are potentially problematic for the wastewater biological treatment plants for two reasons: First, if organic compounds, such as phenols, are present at high concentrations, they may be potentially toxic to microorganisms. Second, the various organic compounds found in the waste streams, such as polycyclic aromatic hydrocarbons (PAHs), have differing susceptibilities to aerobic and anaerobic degradation processes, increasing the difficulty of establishing the conditions for biological treatment. Nowadays there is a growing recognition that enzymes can be used in many remediation processes for treatment of pollutants such as PAHs, phenols, dyes, and similar compounds. The low energy requirements for operation and easy process control are two of the advantages that make the enzymatic treatment an interesting and attractive technology over conventional treatments.71 Nevertheless, the enzymatic processes should be considered only for the treatment (67) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6255-6257. (68) Niizuma, S.; Iwaizumi, M.; Strausz, O. P. AOSTRA J. Res. 1991, 7, 217-223. (69) Montanari, L.; Clericuzio, M.; Del Piero, G.; Scotti, R. Appl. Magn. Reson. 1998, 14, 81-100. (70) Ignasiak, T.; Kemp-Jones, A. V.; Strausz, O. P. J. Org. Chem. 1977, 42, 312-320.

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of special effluents that cannot be treated by conventional activated sludge technologies, due to the presence of highly toxic materials or resistant compounds. Polycyclic Aromatic Hydrocarbon Oxidation. Several oxidative enzymes have been reported to be able to catalyze the modification of pollutants produced from the oil industry. Peroxidases and polyphenol oxidases are enzymes that can act on specific pollutants by transforming them to less toxic or easier degradable products.72,73 PAHs are oxidized by peroxidases such as lignin peroxidase,74,75 manganese peroxidase,76 and by non-peroxidase hemoproteins such as cytochrome P450,77 cytochrome c,78 and hemoglobin79 in the presence of hydrogen peroxide. Polyphenol oxidases, like laccases, are also able to oxidize PAHs.80,81 The oxidation products from both hemoprotein and laccase reactions on PAHs are mainly quinones and hydroxylated derivatives. Several quinones from PAHs are not mutagenic or significantly less mutagenic than the parental hydrocarbons.72 In addition, these oxidized products are more biodegradable.73 Thus, the enzymatic oxidation of PAHs can be considered as a detoxification process. Unfortunately, not all PAHs are substrates for peroxidases. A correlation has been found between the ionization potential (IP) of PAHs and the specific activity of manganese peroxidase, lignin peroxidase, hemoglobin, and chloroperoxidase. A threshold value of ionization potential was found for each enzyme. Lignin peroxidase oxidizes PAHs with IP e 7.55 eV,75 while manganese peroxidase oxidizes PAHs with IP up to 8.2 eV,76,82 the highest IP for hemoglobin-mediated oxidation of PAHs was 8.0 eV 83 and chloroperoxidase was able to modify PAHs with IP lower than 8.2 eV.34 On the other hand, no correlation was found between oxidation of PAHs and their IP with laccase from Trametes versicolor.80 Another limiting factor for enzymatic oxidation of PAHs is the substrate partition between the enzyme active site and the bulk solvent.84 Different chemical modifications have been applied to hemoproteins in order to produce a more hydrophobic enzyme with improved activity against aromatic substrates in organic solvents.85,86 Tinoco and Vazquez(71) Karam, J.; Nicell, J. A. J. Chem. Technol. Biotechnol. 1997, 69, 141-153. (72) Durant, J. L.; Busby, W. F.; Lafleur, A. L.; Penman, B. W.; Crespi, C. L. Mutation Res. 1996, 371, 123-157. (73) Meulenberg, R.; Rijnaarts, H. H. M.; Doddema, H. J.; Field, J. A. FEMS-Microbiol. Lett. 1997, 152, 145-149. (74) Hammel, K. E.; Kalyanaraman, B.; Kirk, K. J. Biol. Chem. 1986, 261, 16948-16952. (75) Vazquez-Duhalt, R.; Westlake, D. W. S.; Fedorak, P. M. Appl. Environ. Microbiol. 1994, 60, 459-466. (76) Bogan, B.W.; Lamar, R.T.; Hammel, K. E. Appl. Environ. Microbiol. 1996, 62, 1788-1792. (77) Masaphy, S.; Levanon, D.; Henis, Y.; Venkateswarlu, K.; Kelly, S. L. Biotechnol. Lett. 1995, 17, 969-974. (78) Torres, E.; Sandoval, J. V.; Rosell, F. I.; Mauk, A. G.; VazquezDuhalt, R. Enzyme Microb. Technol. 1995, 17, 1014-1020. (79) Ortiz-Leon, M.; Velasco, L.; Vazquez-Duhalt, R. Biochem. Biophys. Res. Commun. 1995, 215, 968-973. (80) Majcherczyk, A.; Johannes, C.; Hu¨ttermann, A. Enzyme Microb. Technol. 1998, 22, 335-341. (81) Pickard, M. A.; Roman, R.; Tinoco, R.; Vazquez-Duhalt, R. Appl. Environ. Microbiol. 1999, 65, 3805-3809. (82) Bogan, B. W.; Lamar, R. T. Appl. Environ. Microbiol. 1995, 61, 2631-2633. (83) Torres, E.; Vazquez-Duhalt, R. Biochem. Biophys. Res. Commun. 2000, 273, 820-823. (84) Torres, E.; Tinoco, R.; Vazquez-Duhalt, R. J. Biotechnol. 1996, 49, 59-67. (85) Tinoco, R.; Vazquez-Duhalt, R. Enzyme Microb. Technol. 1998, 22, 8-12.


Duhalt85 modified the free amino and carboxylic groups of horse heart cytochrome c by chemical reaction with methyl, trimethylsilyl (TMS), and poly(ethylene)glycol (PEG) moieties. As a consequence of the chemical modification, the heme environment (active site) was altered. Cytochrome c molecules with a double modification, PEG on free amino groups, and methyl esters on free carboxylic groups were able to oxidize 17 aromatic compounds from 20 tested, while the unmodified protein could oxidize only 8 compounds. These results show that it is possible to overcome the problem of substrate partition by a chemical modification of the enzyme. Thus, enzymatic oxidation of PAHs may be envisaged as an alternative for effluent detoxification. However, experimental work is still necessary on the enzymatic oxidation of substituted PAHs which are the real constituents of oil. Phenol Oxidation. Phenolic compounds are present in many industrial wastewaters, specially in petroleum industry effluents (Table 3). Due to their persistence, toxicity, and health risk, phenolic compounds have become a very important target in environmental biotechnology. The oxidation of phenolic compounds, including phenols, chlorophenols, and dimethoxyphenols is readily catalyzed by a number of extracellular fungal and plant oxidative enzymes. Enzyme-catalyzed oxidation has been proposed as an alternative method for removing phenol from wastewater.87 The oxidation of phenols has been reported to be catalyzed by lignin peroxidase,88 chloroperoxidase,89 horseradish peroxidase,90 Coprinus cinereus peroxidase,91 laccase,92 and tyrosinase.93 The treatment of aqueous phenols using peroxidases results in the formation of insoluble polymeric products, which arise from the interaction of phenoxyl radicals.94 However, it has been reported that the trace amounts of soluble products that remain after the removal of the polymeric precipitates can still retain a significant toxicity.90 The treatment of phenols using tyrosinase and laccase results in the formation of o-quinones, which are toxic compounds, and other colored compounds that do not easily precipitate from solution.95 To solve this problem, an additional strategy has been applied to enzymatic processes. Color and toxicity have been decreased with tyrosinase-catalyzed oxidation in the presence of chitosan.96 It seems that the enzyme promotes the chemical interaction between the products and chitosan, followed by a coagulation process. A similar procedure was (86) Modi, S.; Primrose, W. V.; Lian, L. Y.; Roberts, G. C. K. Biochem. J. 1995, 310, 939-943. (87) Durań, N.; Esposito, E. Appl. Catal. B, Environ. 2000, 28, 8399. (88) Aitken, M. D.; Venkatadri, R.; Irvine, R. Water Res. 1989 23, 443-450. (89) Casella, L.; Poli, S.; Gullotti, M.; Selvaggini, C.; Beringhelli, T.; Marhcesini, A. Biochemistry 1994, 33, 6377-6386. (90) Aitken, M. D.; Massey, I. J.; Chen, T. P.; Heck, P. E. Water Res. 1994, 28, 1879-1889. (91) Flock, C.; Bassi, A.; Gijzen, M. J. Chem. Technol. Biotechnol. 1999, 74, 303-309. (92) Bollag, J. M.; Shuttleworth, K. L.; Anderson, D. H. Appl. Environ. Microbiol. 1988, 53, 3086-3091. (93) Wada, S.; Ichikawa, H.; Tatsumi, K. Biotechnol. Bioeng. 1993, 42, 854-858. (94) Klibanov, A. M.; Alberti, B. N.; Morris, E. D.; Felshin, L. M. J. Appl. Biochem. 1980, 2, 414-421. (95) Walker, J. D. J. Water Pollut. Control Fed. 1988, 60, 11061121. (96) Ikehata, K.; Nicell, J. A. Biotechnol. Prog. 2000, 16, 533-540.


assayed with peroxidases and additives such as poly(ethylene glycol).97 It seems that additives combine with polymerization products, leading to both a toxicity and color reduction. In addition, additives may protect the enzyme against the inactivation caused by the interaction of the phenoxyl radicals with the enzyme. Enzymatic phenol removal from petroleum refinery wastewater has been assayed.98,99 Phenol content in a petroleum refinery wastewater has been reduced below the discharge limit following an enzymatic treatment with horseradish peroxidase and hydrogen peroxide (Table 3). After the enzymatic treatment, 58% of the chemical oxygen demand, 78% of the biological oxygen demand, and 95% of the toxicity were removed along with the phenols.98 Phenol removal did not appear to be adversely affected by the presence of hydrocarbons, and the use of polymeric additives improve the enzymatic efficiency. In another study on phenol removal from petroleum refinery wastewater in batch and continuous flow system, from 95 to 99% of phenols was removed after treatment with Arthromyces ramosus peroxidase.99 Thus, as in the case for PAHs oxidation, phenolics oxidation, and polymerization could be envisaged as an attractive technique to reduce the environmental impact of wastewaters from the petroleum industry. New Source of Enzymes So far more than 3000 enzymatic proteins are known, and it is estimated that the number of enzymatic proteins on earth could be close to 15 000. This estimation is supported by the fact that only from 2 to 5% of all microorganisms can be cultivated,100 and that insects carry specific microflora absent in other habitats.101 In addition, new microorganisms are currently discovered from extreme environments such as thermal vents in the ocean deep and fossilized salt rocks. All these unknown organisms are a potential source of new enzyme forms with different physicochemical properties: the potential source of biocatalytic activity for the oil industry is there. “Harsh” environments are colonized by especially adapted microorganisms, called extremophilic microorganisms or extremophiles.102 These environments include ecosystems with very high or low temperatures, high pressures, extreme pH (pH < 3 or pH > 10) and high salt concentrations (from 5% to saturated sodium chloride). As many of these environments are similar to those found in chemical processes, extremophiles and their enzymes are attractive catalysts to be used in petroleum refining. The enzymes isolated from extremophilic microorganisms are extremely thermostable and generally resistant to organic solvents and extreme pH. In the last two to three decades, microorganisms able to grow at temperatures up to 110 °C have been (97) Wu, Y.; Taylor, K. E.; Biswas, N.; Bewtra, J. K. Enzyme Microb. Technol. 1998, 22, 315-322. (98) Wagner, M.; Nicell, J. A. Water Sci. Technol. 2001, 43, 253260. (99) Ibrahim, M. S.; Ali, H. I.; Biswas, N.; Bewtra, J. K. Water Environ. Res. 2001, 73, 165-172. (100) Cowan, D. A. Trends Biotechnol. 2000, 18, 14-16. (101) Baumann, P.; Moran, N. A. Antoine Leeuwenhoek 1997, 72, 38-48. (102) Aguilar, A. FEMS Microbiol. Rev. 1996, 18, 89-92.


discovered. Enzymes from these microorganisms working in nonaqueous systems at temperatures higher as 200 °C (operating conditions found in refineries) could be expected. Moreover at high temperatures, the hydrocarbons bioavailability and solubility is increased. Enzymes from halophiles should be interesting too. Since halophilic environments present low water activities, enzymes from halophiles might work particularly well in low water activity reaction media such as organic solvents.103 Biocatalysis in Organic Media Enzymatic activity in nonaqueous media seems to be the most important challenge for the introduction of biotechnology in the oil industry. As demonstrated in the case of microbial desulfurization process, abovediscussed, large amounts of water are incompatible with the refining processes. Thus, the biocatalysis success in the petroleum industry depends on the development of biocatalysts able to perform the transformations of oil products in nonaqueous systems, and they should be stable under the conditions usually found in the refineries. Microbial activity occurs in aqueous media. Most of the enzymes were designed by nature to work in aqueous media, but fortunately it is possible to have enzymatic activity in nonaqueous systems with very low water content, almost anhydrous. Biocatalysis in nonaqueous media has increased significantly the range of practical applications of enzymes.104 The abundant information on enzymatic activity in hydrophobic solvents, such as hexane, toluene, and many other organic solvents has been extensively reviewed,104,105 and enzymatic activity in petroleum can be expected. In addition, very hydrophobic substrates, such as asphaltenes, can be modified by enzymatic reactions performed in organic solvents.65 Since petroleum is a hydrophobic material, it is suitable to speculate that new enzymatic processes for the oil industry should be carried out in nonaqueous systems. The use of reaction mixtures containing organic solvents reduces mass transfer limitations, promoting the establishment of productive interactions between the enzyme and the hydrophobic substrates (oil-derived compounds). In addition, a biocatalyst placed in a nonaqueous medium shows interesting properties, such as improved thermostability, higher substrate accessibility, adjustable selectivity, and high storage stability.106,107 Enzymatic reactions can be performed at more than 120 °C in organic solvents, even if the enzyme is not thermostable in aqueous media.108 Enzymes are far more efficient than chemical catalysts: high specificity, low substrate concentration, and mild reaction conditions are the most interesting properties of enzymes in this regard. However, the industrial use of enzymatic catalysts is limited by their instability under harsh conditions, which are usually found in large-scale (103) Kim, J.; Dordick, J. S. Biotechnol. Bioeng. 1997, 55, 471-479. (104) Klibanov, A. M. Nature 2001, 409, 241-246. (105) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubblolts, M.; Witholt, B. Nature 2001, 409, 258-268. (106) Dordick, J. S. Enzyme Microb. Technol. 1989, 11, 192-211. (107) Dordick, J. S.; Khmelnitsky, Y. L.; Sergeeva, M. Curr. Opin. Microbiol. 1998, 1, 311-318. (108) Zaks, A.; Klibanov, A. M. Science 1984, 224, 1249-1253.

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processes. Nevertheless, chemical and genetic modifications of enzymes to improve both activity and stability, together with the new research field of “Solvent Engineering” will provide better biocatalysts for the specific needs of the petroleum industry. Solvent engineering is focused on the study of the relationships between enzyme performance and the physicochemical properties of the reaction solvent, such as the dielectric constant109 and the solvent hydrophobicity.110,111 However, the validity of these correlations remains case specific.112,113 Water thermodynamic activity and solvent hydrophobicity seem to be important parameters for biocatalysis in nonaqueous media84,114,115 and especially for further kinetic calculations, since these activity-based kinetic parameters can be applied to all solvents.116 Enzymatic catalysis in nonaqueous media is mainly governed at two physicochemical levels: first, reaction phase and equilibrium, and second biocatalyst activity and stability. The thermodynamics of enzyme activity in nonaqueous systems has not been fully elucidated,115 although it is evident that a better understanding of the biocatalysts behavior under these conditions will lead to an improvement of their performance in organic media. The protonation state of ionizable side-chain groups in the enzyme is another factor affecting biocatalysis in non conventional media. This protonation state can be also controlled by the addition of an appropriate solid buffer directly into the organic solvent system,117,118 or by manipulating the pH memory of the enzyme before lyophilization, which eliminates the need for pH adjustment prior to the addition of organic solvents.119 The interaction of counterions with proteins has been reported as an important parameter to enhance the functionality of some enzymes in low dielectric-constant media. For example, the addition of salts such as sodium acetate or sodium chloride during the lyophilization process can positively influence the activity and the enantioselectivity of some enzymes such as proteases and lipases.117,120,121 The binding energy between the enzyme and the substrate is the most important driving force for enzymatic catalysis. In aqueous reaction mixtures, two forces are mainly involved in hydrophobic substrate binding: one inducing the compound drawn into the active site (109) Affleck, R.; Haynes, C. A.; Clark, D. S. PNAS 1992, 89, 51675170. (110) Laane, C.; Boeren, S.; Veeger, C. Biotechnol. Bioeng. 1987, 30, 81-87. (111) Torres, E.; Siminovich, B.; Barzana, E.; Vazquez-Duhalt, R. J. Mol. Catal. B, Enzymatic 1998, 4, 155-159. (112) Laroute, V.; Willemot, R. Enzyme Microb. Technol. 1992, 4, 528-534. (113) van Tol, J. B. A.; Jongejan, J. A.; Duine, J. A.; Kierkels, H. G. T.; Gelade, E. F. T.; Mosterd, F.; van der Tweel, W. J. J.; Kamphuis, J. Biotechnol. Bioeng. 1995, 48, 179-189. (114) Ryu, K.; Dordick, J. S. J. Am. Chem. Soc. 1989, 111, 80268027. (115) Halling, P. J. Enzyme Microb. Technol. 1994, 16, 178-204. (116) Sandoval, G. C.; Marty, A.; Condoret, J. S. AIChE J. 2001, 47, 718-726. (117) Khmelnitsky, Y. L.; Welch, S. H.; Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1994, 116, 2647-2648. (118) Zacharis, E.; Moore, B. D.; Halling, P. J. J. Am. Chem. Soc. 1997, 119, 12396-12397. (119) Zaks, A.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3192-3196. (120) Sasaki, T.; Kise, H. Bull. Chem. Soc. Jpn. 1999, 72, 13211325. (121) Okamoto, T.; Ueji, S. Chem. Commun. 1999, 939-940.


and another expelling it from the aqueous medium.122 In most cases the predominant factor is the ability of the active site to draw the hydrophobic substrate from the aqueous solvent. Replacing water with an organic solvent considerably affects this binding energy because the hydrophobic substrate is desolvated with more difficulty from an organic solvent than from an aqueous medium, and because the hydrophobic interactions are eliminated in a nonpolar environment. Thus, the more hydrophobic the solvent, the weaker the binding energy; in consequence, the enzymatic reaction slows down. Proper selection of solvent is therefore expected to promote unfavorable solvent-substrate interactions, and to improve both the substrate partition between the enzyme active-site and the bulk organic solvent84 and the biocatalytic rate.123 In addition, the solvent may affect the enzyme structure and the substrate ground state.124 Several approaches have been successfully applied to enhance the performance of enzymes in organic solvents. Chemical modification of the enzyme surface has been performed to improve the catalytic activity in organic solvents.125-127 Amphiphilic and surfactant molecules, such as poly(ethylene)glycol and Brij, respectively, have been covalently bound to reactive groups on the protein surface. The modified enzymes are soluble and showed high enzymatic activity in organic solvents, such as benzene, toluene, and chlorinated hydrocarbons, which suggests that these preparations may be soluble and active in oil and oil derivatives. Catalase and peroxidases efficiently catalyze their respective reactions in organic solvents, while hydrolytic enzymes, such as lipases and proteases, are able to catalyze the reverse hydrolysis reaction in low-watercontent media. The complex formed between enzymes and surfactants also forms a soluble catalyst which is highly active in nonpolar organic solvents.128 In addition, incorporation of enzymes into a polymeric matrix by chemical modification, a preparation known as biocatalytic plastics, has been successfully assayed for reactions in nonaqueous systems.129,130 Biocatalytic plastics have been shown to be more reactive, more thermostable, and more resistant to nonpolar organic solvent than the native enzyme. The enhancement of the enzyme activity in nonaqueous systems containing oil products and an oil-soluble biocatalyst, which can be obtained by chemical modification, support the great potential for enzyme application in the oil industry. Molecular imprinting techniques have been used to improve the enzyme affinity for a given substrate. This technique involves a complex formation between the (122) Backers, W. L.; Cawley, G.; Eyer, C. S.; Means, M.; Causey, K. M.; Canady, W. J. Arch. Biochem. Biophys. 1993, 304, 27-37. (123) Klibanov, A. M. Trends Biotechnol. 1997, 15, 97-100. (124) Ryu, K.; Dordick, J. S. Biochemistry 1992, 31, 2588-2598. (125) Inada, Y.; Takahashi, K.; Yoshimoto, T.; Ajima, A.; Matsushima, A.; Saito, Y. Trends Biotechnol. 1986, July, 190-194. (126) Gaertner, H. F.; Puigserver, A. J. Eur. J. Biochem. 1989, 181, 207-213. (127) Vazquez-Duhalt, R.; Fedorak, P. M.; Westlake, D. W. S. Enzyme Microb. Technol. 1992, 14, 837-841. (128) Sergeeva, M. V.; Paradkar, V. M.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 70-76. (129) Yang, Z.; Mesiano, A. J.; Venkatasubramanian, S.; Gross, S. H.; Harris, J. M.; Russel, A. J. J. Am. Chem. Soc. 1995, 117, 48434850. (130) Wang, P.; Sergeeva, M. V.; Lim, L.; Dordick, J. Nat. Biotechnol. 1997, 15, 789-793.


enzyme and the substrate, then the complex is dried and washed with an organic solvent to remove the substrate. In this manner, the protein acquires the ligand-induced conformation, which is retained in anhydrous solvents, enhancing the specificity toward the substrate and the overall enzyme activity.131,132 In addition, the potential of genetic engineering for the design of enzyme catalysts for nonaqueous reactions is discussed below. Genetic Engineering In nature, enzymes catalyze chemical reactions under physiological conditions, which can be reduced to mean in aqueous solution at mild temperatures. The properties of water have shaped the course of bio-molecule evolution, even water-insoluble components of the cell, such as membrane lipids, interact with each other in ways dictated by the polar properties of water. In particular, the forces that provide stability and specificity to the three-dimensional structure of proteins are mostly hydrophobic interactions, which are significantly strong in aqueous solutions. The transient nature of these noncovalent interactions within a protein provides the critical flexibility for catalysis. When in a highly polar solvent, a concentrated salt solution for example, the stability of any protein fold is driven by the favorable entropic effect of clustering hydrophobic side chains into a stable core. Organic solvents unfold proteins by disrupting the hydrophobic interactions that make up the core. Even a partial loss of three-dimensional structure is sufficient to cause loss of function in most enzymes. The poor performance of enzymes in organic solvents reduces their technological utility in the oil industry. Current knowledge regarding the structural and catalytic behavior of enzymes in nonconventional media comes from the study of aqueous-organic mixtures. Although it is tempting to assume that if enzymes denature in the former medium, they will certainly suffer the same fate in pure organic solvents, this assumption has proven to be wrong. Not only it is possible to catalyze reactions in anhydrous organic solvents but also the synthesis of specific products becomes thermodynamically favorable in the absence of water.104 This ability has been demonstrated for oxidative enzymes, whose activities are potentially useful for the oil industry.133 In addition, the thermal stability of lyophilized enzymes in anhydrous solvents is improved, probably due to the absence of the prevalent covalent reactions responsible for irreversible thermal inactivation in aqueous solution. Finally, the possibility of catalyzing reactions in pure organic solvent might alleviate mass transfer limitations between the solvent and the active sites. Alternatively, it is feasible, in principle, to select enzyme variants with higher stability toward organic solvents. This stabilizing effect must arise from the establishment of a new set of interactions, probably involving the replacement of hydrophobic interactions (131) Ohya, Y.; Miyaoka, J.; Ouchi, T. Macromol. Rapid Commun. 1996, 17, 871-874. (132) Slade, C. J.; Vulfson, E. N. Biotechnol. Bioeng. 1998, 57, 211215. (133) Dai, L.; Klibanov, A. M. PNAS USA 1999, 96, 9475-9478.


for ionic pairs, which are less sensitive to the media polarity. Multiple changes are expected to occur before the protein presents significant stability in organic solvents. The complexity of the task is beyond routine selection methods. In response, directed evolution methods have been used to obtain improved biocatalysts.134-136 In an early application of directed evolution, the protease subtilisin E was subjected to sequential generations of random mutagenesis and screening to yield a variant that is active in 60% dimethylformamide (DMF) almost as efficiently as the wild type is in water, an increase of nearly 500 times in the kcat/KM ratio.137 More recently, both sequential random mutagenesis and gene recombination were used to evolve p-nitrobenzyl esterase for deprotection of an antibiotic synthetic intermediate in aqueous DMF. The total activity in 30% DMF was increased more than 100 times.138,139 In an excellent contribution, researchers at Maxygen (USA) and Novo Nordisk (Denmark) simultaneously screened for four properties (activity at 23 °C, thermostability, organic-solvent tolerance, and pH-profile) in a library of family shuffled subtilisins, and reported variants with considerably improved characteristics for all parameters.140 In the only example of molecular evolution of a desulfurizing enzyme, dibenzothiophene monooxygenase, a novel gene-shuffling method was used to develop a Rhodococcus/Nocardia dszC chimeric gene which combines the high affinity of one parent with the high turnover rate of the other parent.26 The use of genetic tools for catalysis in nonaqueous systems is in its infancy, and the possibilities of genetic techniques on the design of enzymes for oil refining are still unexplored. Future Directions A main challenge for the petroleum industry, as for all industries, in this century is to apply more energetically efficient production processes and with reduced environmental impact. In addition to the improvement of conventional processes, the use of new and non conventional techniques for petroleum refining should be evaluated. Biotechnology is among the non conventional techniques to be explored. Enzymatic catalysis with high transformation efficiency, high specificity, and mild reaction conditions offers a wide range of possibilities. The available data on microbial and enzymatic transformations of oil products shows several opportunities for some sectors of the petroleum industry, such as deep desulfurization and denitrogenation, and asphaltene upgrading. However, many enzymes and enzymatic proteins are still to be discovered. In addition, over the past two decades people have seen many examples of the improvement of biocatalysts by chemical (134) Tobin, M. B.; Gustafsson, C.; Huisman, G. W. Curr. Opinion Struct. Biol. 2000, 10, 421-427. (135) Arnold, F. H. Nature 2001, 409, 253-257. (136) Bornscheuer, U. T.; Pohl, M. Curr. Opinion Chem. Biol. 2001, 5, 137-143. (137) Chen, K.; Arnold, F. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5618-5622. (138) Moore, J. C.; Arnold, F. H. Nat. Biotechnol. 1996, 14, 458467. (139) Moore, J. C.; Jin, H. M.; Kuchner, O.; Arnold, F. H. J. Mol. Biol. 1997, 272, 336-347. (140) Ness, J. E.; Welch, M.; Giver, L.; Bueno, M.; Cherry, J. R.; Borchert, T. V.; Stemer, W. P.; Minshull, J. Nat. Biotechnol. 1999, 17, 893-896.

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and genetic techniques. These techniques may be applied to the particular needs of the petroleum industry. We see three main research fields in which to propose the use of enzyme catalysts in the petroleum industry: (i) the search of new enzymatic activities upon petroleum products, specially from extreme environments; (ii) the improvement of the enzymatic activities in very low water systems, to increase the transformation rates using petroleum fractions without further addition of water. The study of the relationship between the solvent properties and the enzyme activity seems to be essential to understand and to improve the biocatalytic processes. (iii) The enzyme design by genetic and chemical meth-


ods. In all fields, two main goals should be addressed: activity enhancement and protein stabilization under the actual conditions found in the petroleum refining industry. Acknowledgment. We thank Prof. Michael A. Pickard for the critical reading and discussion on petroleum biotechnolgy. This work was supported by the grants from the Mexican Institute of Petroleum (Research Project D.00020 and FIES 98-110-VI). EF020038S

Will Biochemical Catalysis Impact the Petroleum Refining Industry

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