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Chloroperoxidase-mediated modifications of petroporphyrins and asphaltenes
Chloroperoxidase-mediated modifications of petroporphyrins and asphaltenes Phillip M. Fedorak, Kathleen M. Semple, Rafael Vazquez-Duhalt and Donald W. S. Westlake D e p a r t m e n t o f Microbiology, University o f Alberta, Edmonton, Alberta, Canada
Octaethylporphine, nickel octaethylporphine, vanadyl octaethylporphine, and a petroporphyrin-rich, low-molecular-weight fraction of asphaltenes from Cold Lake heavy oil were treated with chloroperoxidase from Caldariomyces fumago. Reactions in aqueous phosphate buffer (pH 3.0) or in a ternary solvent system of toluene, isopropanol, and water (3 mM phosphate buffer, p H 3.0) were absolutely dependent on the presence of hydrogen peroxide and chloride. Enzyme treatment resulted in reduction of the absorption of the Soret peak. The porphyrins and asphaltenes were insoluble in the aqueous buffer system; thus, mass transfer limited the reactions. These substrates were more soluble in the ternary system and the reactions were more complete, yielding decreases in metal recovery associated with the methylene chloride-soluble porphyrin-containing material. These decreases were: 93% of the Ni from nickel octaethylporphine, 53% of the V from vanadyl octaethylporphine, and20% of the total Ni and V from the asphaltene fraction. This work clearly demonstrated that an extracellular enzyme, chloroperoxidase, can alter components in the asphaltene fraction of petroleum. Because of the requirement for chloride, the enzyme-mediated reactions likely yield chlorinated products which would be undesirable in a refinery feedstock if this enzymatic process was used for the demetallation of petroleum.
Keywords:Asphaltenes; chloroperoxidase;demetallation;petroleum; petroporphyrins
Introduction
Address reprint requests to Dr. Fedorakat the Departmentof Microbiology,Universityof Alberta, Edmonton,AlbertaT6G 2E9, Canada Received l0 June 1992;revised 17 July 1992
bitumens that contain high amounts of asphaltenes, there is little clear evidence that microorganisms or biochemical processes can modify components of asphaltenes. For example, bacterial rods have been observed in freeze-fractured samples of Athabasca bitumen examined by transmission electron microscopy. J8 Wyndham and Costerton 19demonstrated bacterial colonization of bitumen particles suspended in the Athabasca River and reported that these microbes reduced the maltene content of bitumen but were unable to grow on the asphaltene fraction. Rontani et al. 2° reported the cometabolism of asphaltenes in a laboratory study with a mixed microbial population which grew on n-paraffins. They observed a 45.5% weight loss of asphaltenes during a 10-day incubation period. Infrared spectroscopic analysis of the residual asphaltenic fraction suggested the introduction of hydroxyl and carbonyl groups. However, their report did not describe analytical results of extractable materials recovered from appropriate sterile controls. The objective of this study was to determine whether enzyme reactions could bring about measurable changes in the asphaltene fraction of Cold Lake heavy
© 1993 Butterworth-Heinemann
Enzyme Microb. Technol., 1993, vol. 15, May
Petroleum is a complex mixture containing a vast number of organic compounds. Among these are the petroporphyrins ~-v which often complex with metals such as Ni and V. The role of porphyrins in petroleum has been reviewed I from the point of view of petroleum geochemistry, diagenesis, and maturation, 4'6'8 characterization of various fractions of oil, 1'9'1°and the inhibitory effect of metals and porphyrins on petroleum refining and upgrading. 11'12 Porphyrins are commonly associated with the pentane-insoluble fraction of petroleum known as asphaltenes, 6'N,13'14 which consists of very high-molecularweight compounds containing aromatic and aliphatic constituents, heteroatoms,15-1v and metals.3'9 Although microorganisms have been shown to associate with
429
Papers oil. Early investigations showed that chioroperoxidase (CPO, EC 1.11.1.10) in a phosphate buffer system could virtually eliminate the Soret peak in a low-molecular-weight fraction of these asphaltenes. This indicated that the petroporphyrins were being altered and prompted further studies with pure metalloporphyrins and various reaction conditions, including the use of a ternary solvent system, in which the porphyrins were soluble, to enhance the reaction. The effects of the enzyme-mediated reactions on the fate of the metals complexed with the porphyrins were also determined.
Materials and m e t h o d s Chemicals, CPO, a n d general reaction conditions Nickel octaethylporphine (NiOEP) and vanadyl tetraphenylporphine (VTPP) were obtained from Aldrich (Milwaukee, WI). Octaethylporphine (OEP) was obtained from Sigma (St. Louis, MO), and vanadyl octaethylporphine (VOEP) was synthesized according to the method of Bonnett et al. 2~ The isolation of asphaltenes from Cold Lake heavy oil (from Alberta, Canada) and their fractionation by gel permeation chromatography are described elsewhere. ~4 The low-molecularweight fraction, designated as fraction 5 asphaltenes, was rich in porphyrins and was used extensively in this study. A few experiments used the unfractionated asphaltenes. CPO (Rz = 1.4) from Caldariomycesfumago, a gift from Dr. M. A. Pickard (Department of Microbiology, University of Alberta), was stored as a frozen solution at 5 mg protein ml ~. Consistency in the enzyme activity of different preparations was measured using the monochlorodimedone assay, z2 Diluted solutions of CPO (1/20 dilution) in distilled H20 were made fresh daily, and the typical activity was 137 EU ml I (6.7 AA275 min ~/xg Lprotein). All aqueous solutions were made with water that had been purified through a MilliQ system (Millipore, Bedford, MA). Spectra were recorded using a Pye Unicam PU 8740 UV-visible spectrophotometer. Fraction 5 asphaltenes or individual porphyrins, dissolved in methylene chloride, were distributed into reaction vessels, the methylene chloride was evaporated, and then appropriate reaction mixtures were added. All reactions were done at room temperature (-22°C). When metals were to be recovered from the reaction mixtures, glassware was soaked in 10% HC1 to remove any metals that might interfere with the analyses. Methylene chloride used for extractions was first extracted with 9 M H2SO4 and distilled to remove any metals.
R e a c t i o n s in a q u e o u s buffer solutions The reaction methods for CPO in aqueous systems were developed using fraction 5 asphaltenes and NiOEP as chromogenic substrates, based on methods described by Pickard = and Carmichael et al. 23 Under optimum reaction conditions, the reaction mixture (2 ml) contained: 50 /~g fraction 5 asphaltenes or 2 /xg
43(1 Enzyme Microb. Technol., 1993, vol. 15, May
NiOEP, 9 mM KCI in a 3 mM KH2PO 4 (pH 3.0) buffer. Although this solution does not have a strong buffer capacity, the change in pH was less than 0. I unit during the course of the reaction. Higher concentrations of phosphate (0.1 M) were found to inhibit the reaction. For enzyme reactions, the H20 2 was added first and mixed in the buffer, then CPO was added to start the reaction. The optimum concentration of H20 2 for a given amount of enzyme was determined. When a reaction mixture received multiple additions of U202 and CPO, these were added at 5-min intervals. Three additions (designated as 3X) of 10/zg CPO ml i with 0.4 mM H202 or six additions (6X) 5 tzg CPO ml-I with 0.25 mM H202 were both effective at reducing the Soret peak of asphaltene fraction 5 and NiOEP. Sealed vials containing the reaction solution were mixed by attaching them to a tube roller (New Brunswick Scientific, Edison, N J) that had been modified so that the tubes were rotated about their vertical axis rather than their horizontal axis. Controls, including treatment with CPO alone and H202 alone, were run with each experiment. Methylene chloride (1 ml) was added to the small vials to stop the reaction and to extract the porphyrin. This layer was transferred into a cuvette to determine changes in the UV-visible spectrum. Several identical reaction mixtures were combined to ensure enough metals for analysis (30 to 50/xg V or Ni). Petroporphyrins were distributed into 30-ml screw cap test tubes or 125-ml Erlenmeyer flasks, and the petroporphyrins were deposited evenly over the sides and bottom of the container while the solvent evaporated. Reaction buffer (20 ml) was added to each tube and mixed to suspend particles of the insoluble petroporphyrins into the reaction mixture. An equivalent amount of porphyrin was demetallated with concentrated sulfuric acid at room temperature according to the method described by Buchler 24 to be used as a positive control for measuring metals removal. Samples were extracted with methylene chloride in a separatory funnel.
C P O reactions in ternary solvent s y s t e m s Optimization of reaction conditions in a ternary system. The initial experiments used a solvent system of a toluene : isopropanol : water, z5 Mixing appropriate proportions of these solvents results in clear microemulsions that can be used effectively for enzyme treatments of water-insoluble substrates. The optimum mixture for polyphenoloxidase activity 25 was chosen to test for activity of CPO on NiOEP. This mixture (No. 15, Table 1) was used and the level of enzyme activity was designated as 100%. Different ternary systems were also prepared using 5% (v/v) intervals (mixtures 19-45, Table 1) in order to find the highest enzyme activity in clear microemulsions. NiOEP was dissolved in toluene and added to the solvent mixture so that the final concentration was 2 txg NiOEP ml-J total volume. Additional toluene was added to make up the appropriate ratio. The aqueous portion was 3 mM KH2PO 4 buffer, pH 3.0, with 20 mM
Modifications of petroporphyrins and asphaltenes." P. M. Fedorak et al. Table I CPO-mediated reduction of the Soret peak of NiOEP in ternary solvent systems of various composition
Mixture No. a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Percent composition by volume Toluene Isopropanol Aqueous b 42.6 47.3 48.0 52.0 49.0 53.0 58.3 36.7 39.4 43.1 40.1 43.3 36.5 40.6 20.7 21.0 21.1 20.5 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 5 10 15 20 5 10 15 20 5 10
(0.25) d
(0.30) (0.33) (0.36) (0.36) (0.39) (0.44) (0.21) (0.25) (0.27) (0.28) (0.31) (0.27) (0.30) (0.11) (0.12) (0.13) (0.14) (0.13) (0.16) (0.20) (0.24) (0.03) (0.03) (0.08) (0.11) (0.14) (0.17) (0.21) (0.02) (0.05) (0.08) (0.10) (0.13) (0.14) (0.02) (0.04) (0.07) (0.09) (0.02) (0.04) (0.06) (0.08) (0.02) (0.04)
47.5 45.0 46.7 42.3 47.8 43.1 38.0 52.4 52.8 49.2 55.5 52.5 60.7 56.7 64.9 69.6 72.3 75.7 75 70 65 60 85 80 75 70 65 60 55 80 75 70 65 60 55 75 70 65 60 70 65 60 55 65 60
(0.40) (0.40) (0.45) (0.41) (0.49) (0.44) (0.40) (0.42) (0.46) (0.44) (0.54) (0.52) (0.62) (0.58) (0.46) (0.56) (0.63) (0.71) (0.68) (0.64) (0.60) (0.56) (0.65) (0.62) (0.58) (0.55) (0.52) (0.48) (0.45) (0.54) (0.51) (0.48) (0.45) (0.42) (0.39) (0.46) (0.43) (0.40) (0.38) (0.39) (0.36) (0.34) (0.31) (0.33) (0.31)
9.9 7.6 5.3 5.6 3.4 3.9 3.7 10.8 7.9 7.7 4.4 4.1 2.7 2.7 14.4 9.4 6.6 3.8 5 5 5 5 10 10 10 10 10 10 10 15 15 15 15 15 15 20 20 20 20 25 25 25 25 30 30
(0.35) (0.29) (0.22) (0.23) (0.15) (0.17) (0.16) (0.37) (0.29) (0.29) (0.18) (0.17) (0.12) (0.12) (0.43) (0.32) (0.24) (0.15) (0.19) (0.20) (0.20) (0.20) (0.32) (0.33) (0.33) (0.34) (0.34) (0.34) (0.35) (0.43) (0.44) (0.44) (0.45) (0.45) (0.46) (0.52) (0.52) (0.53) (0.53) (0.59) (0.60) (0.60) (0.60) (0.65) (0.66)
Percent activity c 2 2 14 0 1 1 1 20 16 13 0 0 0 0 100 15 0 0 0 0 0 0 2 2 4 17 21 18 6 18 28 40 95 79 124 97 86 96 137 89 96 125 77 0 0
a Mixtures 1-18 were used by Vulfson et aL 25 Mixtures 1-7 are transparent microemulsion, mixtures 8-14 are H-bonded aggregates of isopropanol and water dispersed in toluene, and mixtures 15-18 are normal ternary solutions. 25 b 3 mM phosphate buffer, pH 3.0 with 20 mM KCI c The reaction rate of mixture 15 (0.225 AA392 min -1/~g-1 protein) was assigned 100% d Mole fraction given in parentheses
KCI. Higher concentrations of phosphate resulted in the formation of a precipitate. The amount of chloride was adjusted by adding different amounts of 0.2 M KCI. The optimum chloride concentration (12 mM in ternary system) was determined for the ternary system using mixtures 15, 36, and 39. The amount of buffer added to initial mixtures was decreased by 10 ~l ml-~ to ac-
count for the addition of H202 and CPO and to ensure the final ratio was not altered. Isopropanol was added and the mixtures were shaken well to ensure that clear microemulsions were formed. The complete reaction mixture contained 0.5 mM H:O2 and 1.25/~g CPO ml- ~. This was the optimum ratio, equivalent to 14/zg H202 /.tg-I enzyme, determined using ternary mixtures 15, 36, and 39, and it gave a linear rate of loss of the Soret peak for about 3 rain reaction time. After the addition of H202 and CPO, the reaction mixtures were transferred to cuvettes to follow the rate of change in absorbance in the spectrophotometer. For reactions with NiOEP, the disappearance of the Soret peak at 392 nm was followed. The maximum rate of the reaction was determined from the linear portion of the curve. The appearance and disappearance of the visible peak at 445 nm was also monitored.
Scaleup of reactions in ternary systems. Porphyrins dissolved in methylene chloride were distributed into 500ml Erlenmeyer flasks and the solvent was allowed to evaporate. Reactions with NiOEP and VOEP used 5 /zg of the porphyrin ml- ~ of ternary solvent, whereas reactions with fraction 5 asphaltenes used 50/zg of the asphaltene fraction ml-1 of ternary solvent. A total of 0.5 mg of NiOEP or 0.6 mg of VOEP was reacted in order to have 50/zg metal for analysis. For fraction 5 asphaltenes, 20 mg were reacted in order to get approximately 28/zg V and 6/zg Ni.14 The petroporphyrin residue in the Erlenmeyer flask was then dissolved in toluene and appropriate volumes of isopropanol and buffer were added. Mixtures 36 and 39 (Table 1) gave very good enzyme activity, and these were chosen for scaleup reactions. H202 was added to give a final concentration of 0.5 mM, and the reaction was started by the addition of CPO to give a concentration of 1.25/zg of enzyme ml -~. The progress of the reaction was followed by placing a portion of the reaction mixture into a cuvette and following the disappearance of the Soret peak or the appearance and subsequent reduction of the product peaks (A = 445 nm for NiOEP). Further additions of CPO and H202 were made at 10-min intervals, as required in order to complete the reaction. An extraction blank, consisting of ternary system without porphyrin, and controls with CPO only or with H202 only were included with each experiment. At the end of the reaction, two volumes of water were added, thus yielding two phases and allowing for the extraction of the porphyrin material.
Analytical chemistry Porphyrins were extracted from the reaction mixtures in a separatory funnel with acid-extracted, redistilled methylene chloride. The methylene chloride layer was filtered through anhydrous sodium sulfate to remove water. A small portion of the sample in methylene chloride was taken for mass spectrometry analysis on an AEI MS-12 instrument operated at 70 eV with source temperature at 260°C and a DS-55 data acquisition system (University of Alberta, Chemistry DepartE n z y m e M i c r o b . T e c h n o l . , 1993, v o l . 15, M a y
431
Papers
4.0[,
. . . . . .
/'"'"~,,,~,
[
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o
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..D
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"~ FRACTION 5
"'",..
A S P H A k T ~ I ~ "-.
0.2
"
360
380
400
420
440
460
480
500
am Figure 1 UV-visible spectra of fraction 5 asphaltenes with and without treatment with CPO and H202. Absorbance was measured from a baseline drawn through the minimum on either side of the peak to eliminate the background absorbance 14'26
ment). The methylene chloride was evaporated and the remaining porphyrin was dissolved in 10 ml xylene with 10% base oil for metals analysis on an Inductively Coupled Plasma (ICP) simultaneous spectrophotometer (Model 34000 Applied Research Laboratory) at Alberta Research Council. The aqueous phase of the reaction was collected in an acid-washed beaker, concentrated more than 20-fold on a steam bath, and then the sample was dissolved in 2% HNO3. At this stage, insoluble material was found in CPO-containing reaction mixtures and CPO-containing controls. This precipitated protein was removed by filtering through glass wool and the filtrate was diluted to 10 ml in a volumetric flask. The presence of released metals (Ni or V) was analyzed by ICP atomic emission spectroscopy on a Lecoplasmaarray instrument (University of Alberta, Department of Chemistry).
Results and discussion
Reactions o f porphyrins in aqueous buffer Treatment of fraction 5 asphaltenes with CPO in the presence of H20 2 results in changes in the UV-visible spectrum (Figure 1). The prominent Soret peak at 410 nm, characteristic of vanadylpetroporphyrin molecules, 1A4'26 was reduced in size. The two peaks in the visible region (c~ = 572 nm, /3 = 530 nm)7'27-29were also eliminated. There was no effect seen when this fraction was treated with either CPO or H202 alone. Chloride ions were absolutely required for activity of CPO against petroporphyrins in the aqueous system. The enzyme reaction was very rapid under the experimental conditions, with the complete reduction of the
432
Enzyme Microb. Technol., 1993, vol. 15, May
Sorer peak often occurring in the first 30 s after addition of the complete reaction mixture. The reaction was inhibited if the peroxide concentration was too high, and the optimum ratio of H202 to CPO in the aqueous reaction mixture was determined to be between 1.1 and 1.6 p.g H2O2t~g -1 enzyme. In order to get the most complete reduction of the Soret peak, it was better to use multiple additions of small amounts of enzyme and peroxide rather than a single large addition. Successive additions of CPO and U202 produced a complete reduction of the Soret peak in both fraction 5 asphaltenes and NiOEP. The Soret peak of the unfractionated asphaltenes was also reduced by this treatment. The results obtained in the aqueous system were very inconsistent because of the insolubility of the porphyrin substrates. When using fraction 5 asphaltenes, the mixture contained a suspension of small asphaltene particles, providing a large surface area for the enzyme reaction. However, the mixture had mass-transfer limitations, and thus variability in the results was observed. For example, comparison of four experiments with fraction 5 asphaltenes conducted on different days showed that the amount of reduction of the Soret peak ranged from 33 to 93% for the same treatment. Variations were also found in tests with pure porphyrins. These compounds did not suspend in the buffer solution but adhered in a thin layer on the sides of the reaction vial. Spectrophotometric results from 10 replicate reactions with NiOEP showed that the reduction of the Soret peak ranged from 45 to 80%. The poor reproducibility of the reactions in aqueous buffer prompted us to look for a solvent system in which the petroporphyrins would be soluble and CPO would remain active.
Optimization o f CPO reaction conditions for porphyrins in the ternary system In order to eliminate the mass-transfer limitations in the system and improve the action of CPO on petroporphyrins, the reaction was carried out in ternary systems. Treatment of NiOEP with CPO in the presence of H202 resulted in the reduction and disappearance of the Soret peak at 392 nm and two peaks in the visible region (c~ = 552 nm,/3 = 517 rim) (Figure 2). There was a corresponding appearance of a peak at 445 nm and a solution color change from pink to yellow. After the Soret peak was completely removed, the reaction continued further with the reduction of this product peak. In some reaction mixtures, the product peak subsequently disappeared, leaving a colorless solution with no UV-visible absorbance peaks (Figure 2). Monitoring the maximum rate of decrease in the absorbance at 392 nm was used as a measure of the enzyme activity in order to determine the optimum composition of the ternary solvent system. A number of different mixtures of toluene : isopropanol : buffer were tested to determine the optimum ratio of solvents to use for CPO-mediated reactions. Mixture 15 of Vulfson et al. z5 was used as a reference and the activity was designated 100% (Table 1). In this system,
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. various monophase organic solvents. 33-36 In this work, the ternary solvent system of toluene : isopropanol : buffer enhanced the CPO-catalyzed modifications of water-insoluble porphyrins. In addition, the reaction in the ternary system required less enzyme than the aqueous system for complete removal of the Soret peak. For example, 1.25 tzg of CPO ml-~ was adequate for treatment of porphyrins in the ternary system, whereas multiple additions of 5 to 10/xg of CPO ml -j were required in the aqueous system.
0.4 Soret peak 392 nm
0.3
¢, o t-, r5
- Control NiOEP 0.2
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CPO-treated
Demetallation o f model metalloporphyrins
0.1
0 350
400
450
500
550
600
nm Figure 2 Changes in the UV-visible spectrum of NiOEP by CPO treatment in ternary mixture 36. Labels (1X and 4X) indicate the number of additions of CPO and H202
the CPO showed an activity of 0.225 AA392 min ~/xg-l protein over a 3- to 4-min interval. The rate of disappearance of the Sorer peak of NiOEP in this ternary system was proportional to the amount of CPO used. Different ratios were plotted on a ternary graph (Figure 3), based on molar fraction, which showed the region of different phases of the ternary system as determined by Lund and Holt. 3° The highest enzyme activities, which were 75 to 137% of that observed with mixture 15, were found in a range of combinations of solvents corresponding to the formation of a normal ternary system where the amount of toluene ranged from 5 to 20% by volume and the water content was 15 to 25% (Table 1). Mixture 15, which gave the maximum activity for polyphenoloxidase,25 was included in this region. Among the mixtures that gave high CPO activity (i.e., a rapid reduction in the Soret peak of NiOEP) the overall extent of the reactions differed. Reaction mixtures with 20% water (mixtures 36 to 39) were chosen for further study because they showed a more complete reaction, with the reduction of both the Soret peak and the peak at 445 nm. Some reactions stopped with the formation of a product with a maximum absorption at 445 nm. This could be removed with multiple additions of CPO and H2Oz. Chloride was absolutely required for activity of CPO on the NiOEP and the other porphyrins (data not shown) in the ternary reaction systems, and the optimum concentration was 12 mM CI-. There was no loss of Soret peak when the reaction mixtures lacked CPO or H202. Detergentless microemulsions based on toluene or hexane have been used successfully for enzyme reactions on water-insoluble substrates. 25'31'32 Free radical production by peroxidases has been demonstrated in
The optimum mixture of solvents chosen for demetallation experiments was 5% toluene:75% isopropanol: 20% buffer (mixture 36). Figure 2 shows the spectral changes of NiOEP solutions after treatment with CPO in the ternary system. The reaction labeled IX was treated with a single addition of CPO and H202. When the Soret peak was completely removed and the yellow product was at the maximum absorbance, the reaction was stopped by addition of excess water. The sample labeled 4X (Figure 2) was reacted further with a total of four additions of CPO and H202 until the yellow color and the absorbance at 445 nm were removed. The results of Ni analyses of the extracted NiOEP and the reaction products (organic layer) and the remaining aqueous phase are shown in Table 2. The amount of Ni that remained in the organic phase when the yellow product was present was 25% less than in the control
O
0.2
0.4
0.6
0.8
1.O
TOLUENE
Figure 3 Mole fraction compositions of ternary systems used to test the CPO on NiOEP. According to Lund and Holt, 3° zone A contains normal ternary systems, zone B contains H-bonded aggregates of water and isopropanol distributed in toluene, zone C contains mixtures with transparent microemulsions, and zone D contains unstable macroemulsions. ( . ) Enzyme activities that were 77 to 137% of that in mixture 15; (0) 10 to 40% of that in mixture 15; (©) <10% of that in mixture 15. There were no mixtures that gave enzyme activities between 40 and 77%
Enzyme Microb. Technol., 1993, vol. 15, M a y
433
Papers Table 2
Nickel analysis of CPO-treated NiOEP in ternary mixture 36 Amounts of Ni Organic phase
Aqueous phase
Total recovered b
Sample
mg
% decreasea
mg
% found a
mg
Control CPO-treated, 1Xc CPO-treated, 4X d Reagent blank
5.86 4.41 0.39 0.01
0 25 93
0.012 0.44 3.12 <0.01
0.2 8 53
5.87 4.85 3.51
a Based on Ni content in organic phase from control b Expected total Ni was 5 mg c One addition of CPO and hydrogen peroxide showed complete reduction of Soret peak, producing a yellow solution with peak absorbance at 445 nm d Four additions of CPO and hydrogen peroxide removing the absorbance at 445 nm, producing a colorless solution
in which there was no enzyme reaction. Further CPO treatment that gave a clear reaction mixture resulted in the loss of almost all (93%) the Ni from the porphyrin extract. Other workers who have studied chemical demetallation of petroporphyrins measured only the metal content of the organic residues. 37-4° In contrast, we have also determined the metal content of the aqueous phase. The data in Table 2 clearly show that the CPO treatments increased the amount of water-soluble Ni. Unfortunately, the analyses could only account for 3.51 mg of the expected 5.89 mg of Ni. Losses of metal may have occurred by complexation with protein precipitate and in the filtration, evaporation, and transfer steps of the aqueous phase. In spite of the low overall metal recoveries, the results clearly showed that Ni partitioned into the aqueous phase after CPO treatment. Treatment of NiOEP in an aqueous system achieved only 50 to 80% reduction of the Soret peak and a resultant 25 to 50% loss of Ni from the organic-phase extracts. VOEP was also treated with CPO in the ternary system resulting in a decrease of the Sorer band at 407 nm. The reaction product had a strong absorbance at 447 nm. Multiple additions of CPO and H20 2 were also able to remove this peak. Table 3 shows that the V content of the organic extract was reduced after one addition of CPO and H20 2. More metal was released after multiple treatments (4X). Reaction of CPO with VOEP was slower and not as complete as with NiOEP. However, the reaction was much better in the ternary system than in the aqueous system, where only 35% reduction in the Soret peak of VOEP and, at most, a 25% reduction in V content of the organic phase were observed. VTPP was not susceptible to modification in the ternary system under the reaction conditions used for the other porphyrins, even with increased concentrations of CPO. The bulky phenyl groups block the meso position of the molecule, which was the site of chlorination by chlorinating agents 38'4~'42or oxidation by peroxidative activity. 43'44 However, in the aqueous system, there was an average reduction of 40 to 60% of the
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Enzyme Microb. Technol., 1993, vol. 15, May
Soret peak at 424 nm with a 45 to 50% decrease in the amount of V in the methylene chloride-extractable material. CPO has a peroxidase activity in the absence of chloride at pH 3 and 5. 23'45 However, no reduction in the Soret peak was observed without chloride with the model metalloporphyrins used in this study. Because the enzyme reaction requires chloride, the products were likely chlorinated. Based on steady-state kinetic studies, Dunford et al. 46 concluded that the liberation of free hypochlorous acid was not important in the CPO-catalyzed chlorination of monochlorodimedone. Rather the reaction proceeds from compound I to formation of iron (III)-OC1 by chloride ion addition to the ferryl oxygen atom. Thus the enzyme controls the chlorination of monochlorodimedone. The same may be true for the CPO-catalyzed modifications of metalloporphyrins. Sugihara et a/. 37'38described the oxidative demetallation of vanadyl porphyrins in asphaltenes with the chlorinating agents chlorine and sulfuryl chloride. They described a green product with a maximum absorbance at 450 nm, consistent with a chlorinated VOEP. The porphyrin was attacked at the meso position, and therefore VTPP was much less reactive than the VOEP, as we also found with CPO reactions. Metal release by CPO is likely due to the disruption of the integrity of the chelating system, rather than a simple release of metal, as is the case with strong-acid demetallation. The molecular weight of NiOEP is 590. After treatment of NiOEP with concentrated sulfuric acid, low resolution mass spectrometry showed that the product had a molecular weight of 534, which is consistent with the loss of Ni and the addition of two hydrogen atoms. Indeed, the mass spectrum of the product was virtually identical to that of octaethylporphyrin reported by Smith. 47 The mass spectrum of the solvent-extracted products after demetallation of NiOEP with CPO in the ternary system showed no ion at m/z 534. The highest mass ion observed was m/z 446, and there were many other fragments. McDonagh 48 reported that electron impact mass spectrometry of porphyrins produces weak molecular ions and that
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. Table 3
Vanadium analysis of CPO-treated VOEP in ternary mixture 36 Amounts of V Organic phase
Aqueous phase
Sample
mg
% decreasea
Control CPO-treated, 1Xc CPO-treated, 4X d
4.20 2.76 1.96
0 34 53
mg 0.006 0.035 0.825
Total recoveredb
% found a
mg
0.1 0.8 20
4.21 2.80 2.79
a Based on V content in organic phase from control b Expected total V was 5 mg c One addition of CPO and hydrogen peroxide showed complete reduction of Soret peak, producing a green solution with peak absorbance at 448 nm d Four additions of CPO and hydrogen peroxide removing the absorbance at 448 nm, producing a colorless solution
thermal decomposition of the sample in the source gives misleading spectra. It is most likely that the CPO reaction yields a variety of products, which we did not attempt to identify. Schaefer et al. 43 described the enzymatic peroxidative degradation of porphyrins and determined the effect of the chelated metal on the reaction. They determined that a redox active metal was required for porphyrin degradation, and thus no reaction occurred between H202 and metal-free OEP. However, in our experiments, OEP was susceptible to attack by CPO. During the treatment of OEP with CPO in the ternary system, the Soret peak shifted from 396 to 408 nm, then was slowly degraded to a colorless product. The visible spectrum was also shifted from peaks at 497, 530, and 567 nm to peaks at 508, 543, and 584 nm. Based on these results, the degradation products of OEP were likely chlorinated products. For example, monochlorooctaethylporphine has a Soret peak at 406 nm and absorptions at 507,540, and 578 nm. Dichlorooctaethylporphine has a Sorer peak at 411 nm and absorptions at 514, 548, and 586 r i m . 41'42 However, it was difficult to isolate and identify these intermediates because they were further degraded to colorless products by CPO.
CPO treatment and demetallation o f fraction 5 asphaltenes Initial experiments showed that CPO had good activity against fraction 5 asphaltenes in an aqueous reaction system. Studies with the ternary solvent system showed that the asphaltene fraction was not completely soluble in this system, and it gave a slightly cloudy mixture. However, the Soret peak was still discernible to monitor the progress of the reaction. Multiple additions of CPO and H 2 0 2 reduced the Soret peak and the shoulder which was formed at about 436 nm (Figure 1). There was a 20% decrease in the amount of both Ni and V content of the organic phase containing the asphaltene fraction (Table 4). The metals contents of the aqueous phase was significantly higher than that of the control which had not been reacted with CPO, but the amounts of metal released from the asphaltenes
were not recovered in the aqueous phase. Fraction 5 asphaltenes treated in the aqueous system also showed a complete loss of the Sorer peak and about 20% decrease in the amount of V in the methylene chlorideextractable material. Sugihara et al. 38 studied the use of chlorine in the demetallation of two fractions of asphaltenes from Boscan crude oil. In one fraction, all of the metal content was accountable as metalloporphyrins, and this fraction was more readily demetallated than the other fraction that had a much higher nonporphyrin metal content. Similarly, G o u l d 39 compared the relative susceptibilities of porphyrin and nonporphyrin metals to demetallation. He treated Cold Lake asphaltenes with various proportions of peroxyacetic acid and measured the loss of the Soret peak absorbance and the amount of V and Ni remaining in the asphaltenes. The greatest amounts of demetallation were associated with the decrease in the Sorer peak absorbance. At the peroxyacetic acid dose that exceeded that required to completely remove the Soret peak, there was little additional release of V but a more significant release of Ni. G o u l d 39 suggested that the peroxyacetic acid acts rapidly to destroy the metalloporphyrin and liberate their associated metals, while the metals held by nonporphyrin ligands are somewhat more slowly attacked and removed. Semple et al.J4 showed that only about 40% of the total V in the Cold Lake fraction 5 asphaltenes was complexed with porphyrins. Based on the 20% demetallation of V from this fraction (Table 4), and assuming that the metalloporphyrins are more susceptible to demetallation by CPO than the nonporphyrin-complexed metals, 38'39 about one-half of the porphyrin-bound metal was released by CPO treatment. CPO treatment of the fraction 5 asphaltene does not appear to alter the solubility of this material. In contrast, sulfuric acid treatment used for demetallation produced methylene chloride-insoluble precipitates. CPO treatment of porphyrins in petroleum may offer an alternative method to remove metals from oil to avoid problems with poisoning catalysts. Organometallic compounds determine more or less the detrimental effects of asphaltenes and resins on catalysts.17 Com-
Enzyme Microb. Technol., 1993, vol. 15, May
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Papers Table 4 Metals analysis of CPO-treated fraction 5 asphaltenes in ternary mixture 36. A sample of 20 mg of fraction 5 asphaltenes was treated, and this was expected to contain approximately 0.6 mg Ni and 2.8 mg V Amounts of Ni Organic phase
Amount of V
Aqueous phase
Organic phase
Aqueous phase
Sample
mg
% decreasea
mg
% found a
mg
% decreasea
mg
% found a
Control CPO-treated b Reagent blank
0.80 0.64 0.02
0 20
0.036 0.098 0.003
5 12
3.72 3.02 0.01
0 19
<0.01 0.036 <0.01
<0.2 1
a Based on Ni or V content in organic phase from control b Treatment resulted in complete reduction of Soret peak at 410 nm and reduction of resulting shoulder at 435 nm
plexes of Ni and V are concentrated in the asphaltenes, 3'~ and Ni and V seriously affect catalytic cracking catalysts. Because metalloporphyrins are the most volatile metal-containing molecules in petroleum and they cause the greatest problems, 11 the destruction of the porphyrins and removal of the metals is beneficial. The objective is to destroy the porphyrins with little effect on the petroleum character. Oxidative demetallation of vanadyl porphyrins has been investigated. 38'39 Sugihara eta/. 37"38used mild oxidating agents such as chlorine and sulfuryl chloride, and Gould 39 used hypochlorite bleach and peroxyacetic acid. Indeed, Gould 39 used Cold Lake asphaltenes and observed that hypochloride bleach released 78% of the V and 37% of the Ni, whereas peroxyacetic acid released 73% of the V and 49% of the Ni from the asphaltenes. A number of methods described by Kukes and Aldag4° cause changes in the character of petroleum, and some methods, using phosphorus compounds, remove only V but not Ni. Chlorination methods 3s demetallate the porphyrins but cause incorporation of chlorine into the feed material. 39 Because of the absolute requirement for chloride in the CPO reactions, this enzymatic reaction likely yields chlorinated products.
other nonchlorinating enzymatic reactions in nonaqueous solvents in attempts to upgrade heavy oils.
Acknowledgements This work was funded by a contract from the Alberta Oil Sands Technology and Research Authority. We thank V. Foubister and J. Gerard for technical assistance and M. A. Pickard for the CPO and valuable discussions during the course of this study.
References 1 2 3 4 5 6 7
Conclusions This work has demonstrated that the extracellular enzyme CPO can alter petroporphyrins associated with the asphaltene fraction of a heavy oil. We are not aware of any other clear demonstration of modification of components of asphaltenes by a biological product or reaction. As a consequence of the reaction that results in removal of the Soret peak, CPO can release metals from pure metalloporphyrins and the porphyrin-rich fraction of Cold Lake asphaltenes. The ternary system of toluene, isopropanol, and buffer was superior to the aqueous buffer system in that the reaction is more complete, with further reduction of the reaction products, and the extent of the reaction can be seen directly in the color change before extraction is done. CPO treatment was effective for metal removal from the porphyrin-rich fraction of the asphaltenes. It is postulated that the enzyme-mediated reactions would yield chlorinated products which would be undesirable as a refinery feedstock. We are currently investigating
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8 9 10 11 12 13 14 15 16
17 18
Baker, E. W. in Organic Geochemistry (Eglinton, G. and Murphy, M. T. J., eds.) Springer-Verlag, Berlin, 1969, pp. 464-497 Yen, T. F., Boucher, L. J., Dickie, J. P., Tynan, E. C. and Vaughan, G. B. J. Inst. Petrol., London 1969, 55, 87-99 Filby, R. H. in The Role o f Trace Metals in Petroleum (Yen, T. F., ed.) Ann Arbor Science Publishers, Ann Arbor, MI, 1975, pp. 31-58 Baker, E. W. and Palmer, S. in The Porphyrins. Vol H (Dolphin, D. A., ed.) Academic Press, New York, 1978, pp. 485-550 Fish, R. H., Kolenic, J. J. and Wines, B. K. Anal. Chem. 1984, 56, 2452-2460 Filby, R. H. and VanBerkel, G. J. in Metal Complexes in Fossil Fuels (Filby, R. H. and Branthaver, J. F., eds.) American Chemical Society, Washington, DC, 1987, pp. 2-39 Frakman, Z., Ignasiak, T. M., Montgomery, D. S. and Strausz, O. P. AOSTRA J. Res. 1988, 4, 171-179 Hodgson, G. W. Ann. N Y A c a d . Sci. 1973, 206, 670-684 Yen, T. F. in The Role o f Trace Metals in Petroleum (Yen, T. F., ed.) Ann Arbor Science Publishers, Ann Arbor, MI, 1975, pp. 1-30 Gallegos, E. J. and P. Sundararaman. Mass Spect. Rev. 1985, 4, 55-85 Branthaver, J. F. in Metal Complexes in Fossil Fuels (Filby, R. H. and Branthaver, J. F., eds.) American Chemical Society, Washington, DC, 1987, pp. 188-204 Reynolds, J. G. and Biggs, W. R. Accounts Chem. Res. 1988, 21, 319-326 Baker, E. W., Yen, T. F., Dickie, J. P., Rhodes, R. E. and Clark, L. F. J. Am. Chem. Soc. 1967, 89, 3631-3639 Semple, K. M., Cyr, N., Fedorak, P. M. and Westlake, D. W. S. Can. J. Chem. 1990, 68, 1092-1099 Yen, T. F. Energy Sources 1974, 1, 447-463 Speight, J. G. and Moschopedis, S. E. in Chemistry o f Asphaltenes. Adv. Chem. Series 195 (Bunger, J. W. and Li, N. C., eds.) American Chemical Society, Washignton, DC, 1981, p. 1 Pelet, R., Behar, F. and Monin, J. C. Org. Geochem. 1986, 10, 481-498 Zajic, J. E., Cooper, D. G., Marshall, J. A. and Gerson, D. F. Fuel 1981, 60, 619-623
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Wyndham, R. C. and Costerton, J. W., Appl. Environ. Microbiol. 1981, 41, 791-800 Rontani, J. F., Bosser-Joulak, F., Ramberloarisoa, E., Bertrand, J. C. and Giusti, G. Chemosphere 1985, 14, 1413-1422 Bonnett, R., Brewer, P., Noro, K. and Noro, T. Tetrahedron 1978, 34, 379-385 Pickard, M. A. Can. J. Microbiol. 1981, 27, 1298-1305 Carmichael, R., Fedorak, P. M. and Pickard, M. A. Biotechnol. Lett. 1985, 7, 289-294 Buchler, J. W. in Porphyrins and Metalloporphyrins (Smith, K. M., ed.) Elsevier, New York, 1975, pp. 195-198 Vulfson, E. N., Ahmed, G., Gill, l., Kozlov, I. A., Goodenough, P. W. and Law, B. A. Biotechnol. Lett. 1991, 13, 91-96 Sugihara, J. M. and Bean, R. M. J. Chem. Eng. Data 1962, 7, 269-271 Millson, M. F., Montgomery, D. S. and Brown, R. S. Geochim. Cosmochim. Acta 1966, 30, 207-221 Spencer, W. A., Galobardes, J. F., Curtis, M. A. and Rogers, L. B. Separation Sci. Technol. 1982, 17, 797-819 Champlin, J. B. F. and Dunning, H. N. Economic Geol. 1960, 55, 797-804 Lund, G. and Holt, S. L. J. Am. Oil Chemists Soc. 1980, 57, 264-267 Khmelnitsky, Y. L., Hilhorst, R. and Veeger, C. Eur. J. Biochem. 1988, 170, 265-271 Smith, G. D. and Barden, R. E. in Solution Behavior o f Surfactants (Mittal, K. L. and Fendler, E. J., eds.) Plenum Press, New York, 1982, pp. 1225-1235 Khmelnitsky, Y. L., Levanshov, A. V., Klyachko, N. L. and Martinek, K. Enzyme Microb. Technol. 1988, 10, 710-724
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Dordick, J. S. Enzyme Microb. Technol. 1989, 11, 194-211 Kazadjian, R. Z., Dordick, J. S. and Klibanov, A. M. Biotechnol. Bioeng. 1986, 28, 417-421 Vazquez-Duhalt, R., Fedorak, P. M. and Westlake, D. W. S. Enzyme Microb. Technol. 1992, 14, 837-841 Sugihara, J. M., Branthaver, J. F. and WiUcox, K. W. Prepr. Am. Chem. Soc. Div. Petrol. Chem. 1973, 18, 645-647 Sugihara, J. M., Branthaver, J. F. and Willcox, K. W. in The Role o f Trace Metals in Petroleum (Yen, T. F., ed.) Ann Arbor Science Publishers, Ann Arbor, MI, 1975, pp. 183-193 Gould, K. A. Fuel 1980, 59, 733-736 Kukes, S. G. and Aldag, A. W. Prepr. Am. Chem. Soc. Div. Petrol. Chem. 1985, 30, 119-129 Furhop, J. H. in The Porphyrins. Vol. H (Dolphin, D. A., ed.) Academic Press, New York, 1978, pp. 152-153 Bonnett, R., Gale, I. A. D. and Stephanson, G. F. J. Chem. Soc. (C) 1966, 1600-1604 Schaefer, W. H., Harris, T. M. and Guengerich, F. P. Biochemistry 1985, 24, 3254-3263 Schmid, R. and McDonagh, A. F. in The Porphyrins. Vol. VI (Dolphin, D. A., ed.) Academic Press, New York, 1979, pp. 257-292 Pickard, M. A., Kadima, T. A. and Carmichael, R. D. J. Ind. Microbiol. 1991, 7, 235-242 Dunford, H. B., Lambeir, A.-M., Kashem, M. A. and Pickard, M. Arch. Biochem. Biophys. 1987, 252, 292-302 Smith, K. M. in Porphyrins and Metalloporphyrins (Smith, K. M., ed.) Elsevier Science Publishing Co., Amsterdam, 1975, pp. 381-398 McDonagh, A. F. in The Porphyrins. Vol. V1 (Dolphin, D. A., ed.) Academic Press, New York, 1979, pp. 293-472
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Octaethylporphine, nickel octaethylporphine, vanadyl octaethylporphine, and a petroporphyrin-rich, low-molecular-weight fraction of asphaltenes from Cold Lake heavy oil were treated with chloroperoxidase from Caldariomyces fumago. Reactions in aqueous phosphate buffer (pH 3.0) or in a ternary solvent system of toluene, isopropanol, and water (3 mM phosphate buffer, p H 3.0) were absolutely dependent on the presence of hydrogen peroxide and chloride. Enzyme treatment resulted in reduction of the absorption of the Soret peak. The porphyrins and asphaltenes were insoluble in the aqueous buffer system; thus, mass transfer limited the reactions. These substrates were more soluble in the ternary system and the reactions were more complete, yielding decreases in metal recovery associated with the methylene chloride-soluble porphyrin-containing material. These decreases were: 93% of the Ni from nickel octaethylporphine, 53% of the V from vanadyl octaethylporphine, and20% of the total Ni and V from the asphaltene fraction. This work clearly demonstrated that an extracellular enzyme, chloroperoxidase, can alter components in the asphaltene fraction of petroleum. Because of the requirement for chloride, the enzyme-mediated reactions likely yield chlorinated products which would be undesirable in a refinery feedstock if this enzymatic process was used for the demetallation of petroleum.
Keywords:Asphaltenes; chloroperoxidase;demetallation;petroleum; petroporphyrins
Introduction
Address reprint requests to Dr. Fedorakat the Departmentof Microbiology,Universityof Alberta, Edmonton,AlbertaT6G 2E9, Canada Received l0 June 1992;revised 17 July 1992
bitumens that contain high amounts of asphaltenes, there is little clear evidence that microorganisms or biochemical processes can modify components of asphaltenes. For example, bacterial rods have been observed in freeze-fractured samples of Athabasca bitumen examined by transmission electron microscopy. J8 Wyndham and Costerton 19demonstrated bacterial colonization of bitumen particles suspended in the Athabasca River and reported that these microbes reduced the maltene content of bitumen but were unable to grow on the asphaltene fraction. Rontani et al. 2° reported the cometabolism of asphaltenes in a laboratory study with a mixed microbial population which grew on n-paraffins. They observed a 45.5% weight loss of asphaltenes during a 10-day incubation period. Infrared spectroscopic analysis of the residual asphaltenic fraction suggested the introduction of hydroxyl and carbonyl groups. However, their report did not describe analytical results of extractable materials recovered from appropriate sterile controls. The objective of this study was to determine whether enzyme reactions could bring about measurable changes in the asphaltene fraction of Cold Lake heavy
© 1993 Butterworth-Heinemann
Enzyme Microb. Technol., 1993, vol. 15, May
Petroleum is a complex mixture containing a vast number of organic compounds. Among these are the petroporphyrins ~-v which often complex with metals such as Ni and V. The role of porphyrins in petroleum has been reviewed I from the point of view of petroleum geochemistry, diagenesis, and maturation, 4'6'8 characterization of various fractions of oil, 1'9'1°and the inhibitory effect of metals and porphyrins on petroleum refining and upgrading. 11'12 Porphyrins are commonly associated with the pentane-insoluble fraction of petroleum known as asphaltenes, 6'N,13'14 which consists of very high-molecularweight compounds containing aromatic and aliphatic constituents, heteroatoms,15-1v and metals.3'9 Although microorganisms have been shown to associate with
429
Papers oil. Early investigations showed that chioroperoxidase (CPO, EC 1.11.1.10) in a phosphate buffer system could virtually eliminate the Soret peak in a low-molecular-weight fraction of these asphaltenes. This indicated that the petroporphyrins were being altered and prompted further studies with pure metalloporphyrins and various reaction conditions, including the use of a ternary solvent system, in which the porphyrins were soluble, to enhance the reaction. The effects of the enzyme-mediated reactions on the fate of the metals complexed with the porphyrins were also determined.
Materials and m e t h o d s Chemicals, CPO, a n d general reaction conditions Nickel octaethylporphine (NiOEP) and vanadyl tetraphenylporphine (VTPP) were obtained from Aldrich (Milwaukee, WI). Octaethylporphine (OEP) was obtained from Sigma (St. Louis, MO), and vanadyl octaethylporphine (VOEP) was synthesized according to the method of Bonnett et al. 2~ The isolation of asphaltenes from Cold Lake heavy oil (from Alberta, Canada) and their fractionation by gel permeation chromatography are described elsewhere. ~4 The low-molecularweight fraction, designated as fraction 5 asphaltenes, was rich in porphyrins and was used extensively in this study. A few experiments used the unfractionated asphaltenes. CPO (Rz = 1.4) from Caldariomycesfumago, a gift from Dr. M. A. Pickard (Department of Microbiology, University of Alberta), was stored as a frozen solution at 5 mg protein ml ~. Consistency in the enzyme activity of different preparations was measured using the monochlorodimedone assay, z2 Diluted solutions of CPO (1/20 dilution) in distilled H20 were made fresh daily, and the typical activity was 137 EU ml I (6.7 AA275 min ~/xg Lprotein). All aqueous solutions were made with water that had been purified through a MilliQ system (Millipore, Bedford, MA). Spectra were recorded using a Pye Unicam PU 8740 UV-visible spectrophotometer. Fraction 5 asphaltenes or individual porphyrins, dissolved in methylene chloride, were distributed into reaction vessels, the methylene chloride was evaporated, and then appropriate reaction mixtures were added. All reactions were done at room temperature (-22°C). When metals were to be recovered from the reaction mixtures, glassware was soaked in 10% HC1 to remove any metals that might interfere with the analyses. Methylene chloride used for extractions was first extracted with 9 M H2SO4 and distilled to remove any metals.
R e a c t i o n s in a q u e o u s buffer solutions The reaction methods for CPO in aqueous systems were developed using fraction 5 asphaltenes and NiOEP as chromogenic substrates, based on methods described by Pickard = and Carmichael et al. 23 Under optimum reaction conditions, the reaction mixture (2 ml) contained: 50 /~g fraction 5 asphaltenes or 2 /xg
43(1 Enzyme Microb. Technol., 1993, vol. 15, May
NiOEP, 9 mM KCI in a 3 mM KH2PO 4 (pH 3.0) buffer. Although this solution does not have a strong buffer capacity, the change in pH was less than 0. I unit during the course of the reaction. Higher concentrations of phosphate (0.1 M) were found to inhibit the reaction. For enzyme reactions, the H20 2 was added first and mixed in the buffer, then CPO was added to start the reaction. The optimum concentration of H20 2 for a given amount of enzyme was determined. When a reaction mixture received multiple additions of U202 and CPO, these were added at 5-min intervals. Three additions (designated as 3X) of 10/zg CPO ml i with 0.4 mM H202 or six additions (6X) 5 tzg CPO ml-I with 0.25 mM H202 were both effective at reducing the Soret peak of asphaltene fraction 5 and NiOEP. Sealed vials containing the reaction solution were mixed by attaching them to a tube roller (New Brunswick Scientific, Edison, N J) that had been modified so that the tubes were rotated about their vertical axis rather than their horizontal axis. Controls, including treatment with CPO alone and H202 alone, were run with each experiment. Methylene chloride (1 ml) was added to the small vials to stop the reaction and to extract the porphyrin. This layer was transferred into a cuvette to determine changes in the UV-visible spectrum. Several identical reaction mixtures were combined to ensure enough metals for analysis (30 to 50/xg V or Ni). Petroporphyrins were distributed into 30-ml screw cap test tubes or 125-ml Erlenmeyer flasks, and the petroporphyrins were deposited evenly over the sides and bottom of the container while the solvent evaporated. Reaction buffer (20 ml) was added to each tube and mixed to suspend particles of the insoluble petroporphyrins into the reaction mixture. An equivalent amount of porphyrin was demetallated with concentrated sulfuric acid at room temperature according to the method described by Buchler 24 to be used as a positive control for measuring metals removal. Samples were extracted with methylene chloride in a separatory funnel.
C P O reactions in ternary solvent s y s t e m s Optimization of reaction conditions in a ternary system. The initial experiments used a solvent system of a toluene : isopropanol : water, z5 Mixing appropriate proportions of these solvents results in clear microemulsions that can be used effectively for enzyme treatments of water-insoluble substrates. The optimum mixture for polyphenoloxidase activity 25 was chosen to test for activity of CPO on NiOEP. This mixture (No. 15, Table 1) was used and the level of enzyme activity was designated as 100%. Different ternary systems were also prepared using 5% (v/v) intervals (mixtures 19-45, Table 1) in order to find the highest enzyme activity in clear microemulsions. NiOEP was dissolved in toluene and added to the solvent mixture so that the final concentration was 2 txg NiOEP ml-J total volume. Additional toluene was added to make up the appropriate ratio. The aqueous portion was 3 mM KH2PO 4 buffer, pH 3.0, with 20 mM
Modifications of petroporphyrins and asphaltenes." P. M. Fedorak et al. Table I CPO-mediated reduction of the Soret peak of NiOEP in ternary solvent systems of various composition
Mixture No. a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Percent composition by volume Toluene Isopropanol Aqueous b 42.6 47.3 48.0 52.0 49.0 53.0 58.3 36.7 39.4 43.1 40.1 43.3 36.5 40.6 20.7 21.0 21.1 20.5 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 5 10 15 20 5 10 15 20 5 10
(0.25) d
(0.30) (0.33) (0.36) (0.36) (0.39) (0.44) (0.21) (0.25) (0.27) (0.28) (0.31) (0.27) (0.30) (0.11) (0.12) (0.13) (0.14) (0.13) (0.16) (0.20) (0.24) (0.03) (0.03) (0.08) (0.11) (0.14) (0.17) (0.21) (0.02) (0.05) (0.08) (0.10) (0.13) (0.14) (0.02) (0.04) (0.07) (0.09) (0.02) (0.04) (0.06) (0.08) (0.02) (0.04)
47.5 45.0 46.7 42.3 47.8 43.1 38.0 52.4 52.8 49.2 55.5 52.5 60.7 56.7 64.9 69.6 72.3 75.7 75 70 65 60 85 80 75 70 65 60 55 80 75 70 65 60 55 75 70 65 60 70 65 60 55 65 60
(0.40) (0.40) (0.45) (0.41) (0.49) (0.44) (0.40) (0.42) (0.46) (0.44) (0.54) (0.52) (0.62) (0.58) (0.46) (0.56) (0.63) (0.71) (0.68) (0.64) (0.60) (0.56) (0.65) (0.62) (0.58) (0.55) (0.52) (0.48) (0.45) (0.54) (0.51) (0.48) (0.45) (0.42) (0.39) (0.46) (0.43) (0.40) (0.38) (0.39) (0.36) (0.34) (0.31) (0.33) (0.31)
9.9 7.6 5.3 5.6 3.4 3.9 3.7 10.8 7.9 7.7 4.4 4.1 2.7 2.7 14.4 9.4 6.6 3.8 5 5 5 5 10 10 10 10 10 10 10 15 15 15 15 15 15 20 20 20 20 25 25 25 25 30 30
(0.35) (0.29) (0.22) (0.23) (0.15) (0.17) (0.16) (0.37) (0.29) (0.29) (0.18) (0.17) (0.12) (0.12) (0.43) (0.32) (0.24) (0.15) (0.19) (0.20) (0.20) (0.20) (0.32) (0.33) (0.33) (0.34) (0.34) (0.34) (0.35) (0.43) (0.44) (0.44) (0.45) (0.45) (0.46) (0.52) (0.52) (0.53) (0.53) (0.59) (0.60) (0.60) (0.60) (0.65) (0.66)
Percent activity c 2 2 14 0 1 1 1 20 16 13 0 0 0 0 100 15 0 0 0 0 0 0 2 2 4 17 21 18 6 18 28 40 95 79 124 97 86 96 137 89 96 125 77 0 0
a Mixtures 1-18 were used by Vulfson et aL 25 Mixtures 1-7 are transparent microemulsion, mixtures 8-14 are H-bonded aggregates of isopropanol and water dispersed in toluene, and mixtures 15-18 are normal ternary solutions. 25 b 3 mM phosphate buffer, pH 3.0 with 20 mM KCI c The reaction rate of mixture 15 (0.225 AA392 min -1/~g-1 protein) was assigned 100% d Mole fraction given in parentheses
KCI. Higher concentrations of phosphate resulted in the formation of a precipitate. The amount of chloride was adjusted by adding different amounts of 0.2 M KCI. The optimum chloride concentration (12 mM in ternary system) was determined for the ternary system using mixtures 15, 36, and 39. The amount of buffer added to initial mixtures was decreased by 10 ~l ml-~ to ac-
count for the addition of H202 and CPO and to ensure the final ratio was not altered. Isopropanol was added and the mixtures were shaken well to ensure that clear microemulsions were formed. The complete reaction mixture contained 0.5 mM H:O2 and 1.25/~g CPO ml- ~. This was the optimum ratio, equivalent to 14/zg H202 /.tg-I enzyme, determined using ternary mixtures 15, 36, and 39, and it gave a linear rate of loss of the Soret peak for about 3 rain reaction time. After the addition of H202 and CPO, the reaction mixtures were transferred to cuvettes to follow the rate of change in absorbance in the spectrophotometer. For reactions with NiOEP, the disappearance of the Soret peak at 392 nm was followed. The maximum rate of the reaction was determined from the linear portion of the curve. The appearance and disappearance of the visible peak at 445 nm was also monitored.
Scaleup of reactions in ternary systems. Porphyrins dissolved in methylene chloride were distributed into 500ml Erlenmeyer flasks and the solvent was allowed to evaporate. Reactions with NiOEP and VOEP used 5 /zg of the porphyrin ml- ~ of ternary solvent, whereas reactions with fraction 5 asphaltenes used 50/zg of the asphaltene fraction ml-1 of ternary solvent. A total of 0.5 mg of NiOEP or 0.6 mg of VOEP was reacted in order to have 50/zg metal for analysis. For fraction 5 asphaltenes, 20 mg were reacted in order to get approximately 28/zg V and 6/zg Ni.14 The petroporphyrin residue in the Erlenmeyer flask was then dissolved in toluene and appropriate volumes of isopropanol and buffer were added. Mixtures 36 and 39 (Table 1) gave very good enzyme activity, and these were chosen for scaleup reactions. H202 was added to give a final concentration of 0.5 mM, and the reaction was started by the addition of CPO to give a concentration of 1.25/zg of enzyme ml -~. The progress of the reaction was followed by placing a portion of the reaction mixture into a cuvette and following the disappearance of the Soret peak or the appearance and subsequent reduction of the product peaks (A = 445 nm for NiOEP). Further additions of CPO and H202 were made at 10-min intervals, as required in order to complete the reaction. An extraction blank, consisting of ternary system without porphyrin, and controls with CPO only or with H202 only were included with each experiment. At the end of the reaction, two volumes of water were added, thus yielding two phases and allowing for the extraction of the porphyrin material.
Analytical chemistry Porphyrins were extracted from the reaction mixtures in a separatory funnel with acid-extracted, redistilled methylene chloride. The methylene chloride layer was filtered through anhydrous sodium sulfate to remove water. A small portion of the sample in methylene chloride was taken for mass spectrometry analysis on an AEI MS-12 instrument operated at 70 eV with source temperature at 260°C and a DS-55 data acquisition system (University of Alberta, Chemistry DepartE n z y m e M i c r o b . T e c h n o l . , 1993, v o l . 15, M a y
431
Papers
4.0[,
. . . . . .
/'"'"~,,,~,
[
Soret peak
I\..,-,,( \
Control
-,x%.\
o
CPO-treated
..D
<
0,4
"~ FRACTION 5
"'",..
A S P H A k T ~ I ~ "-.
0.2
"
360
380
400
420
440
460
480
500
am Figure 1 UV-visible spectra of fraction 5 asphaltenes with and without treatment with CPO and H202. Absorbance was measured from a baseline drawn through the minimum on either side of the peak to eliminate the background absorbance 14'26
ment). The methylene chloride was evaporated and the remaining porphyrin was dissolved in 10 ml xylene with 10% base oil for metals analysis on an Inductively Coupled Plasma (ICP) simultaneous spectrophotometer (Model 34000 Applied Research Laboratory) at Alberta Research Council. The aqueous phase of the reaction was collected in an acid-washed beaker, concentrated more than 20-fold on a steam bath, and then the sample was dissolved in 2% HNO3. At this stage, insoluble material was found in CPO-containing reaction mixtures and CPO-containing controls. This precipitated protein was removed by filtering through glass wool and the filtrate was diluted to 10 ml in a volumetric flask. The presence of released metals (Ni or V) was analyzed by ICP atomic emission spectroscopy on a Lecoplasmaarray instrument (University of Alberta, Department of Chemistry).
Results and discussion
Reactions o f porphyrins in aqueous buffer Treatment of fraction 5 asphaltenes with CPO in the presence of H20 2 results in changes in the UV-visible spectrum (Figure 1). The prominent Soret peak at 410 nm, characteristic of vanadylpetroporphyrin molecules, 1A4'26 was reduced in size. The two peaks in the visible region (c~ = 572 nm, /3 = 530 nm)7'27-29were also eliminated. There was no effect seen when this fraction was treated with either CPO or H202 alone. Chloride ions were absolutely required for activity of CPO against petroporphyrins in the aqueous system. The enzyme reaction was very rapid under the experimental conditions, with the complete reduction of the
432
Enzyme Microb. Technol., 1993, vol. 15, May
Sorer peak often occurring in the first 30 s after addition of the complete reaction mixture. The reaction was inhibited if the peroxide concentration was too high, and the optimum ratio of H202 to CPO in the aqueous reaction mixture was determined to be between 1.1 and 1.6 p.g H2O2t~g -1 enzyme. In order to get the most complete reduction of the Soret peak, it was better to use multiple additions of small amounts of enzyme and peroxide rather than a single large addition. Successive additions of CPO and U202 produced a complete reduction of the Soret peak in both fraction 5 asphaltenes and NiOEP. The Soret peak of the unfractionated asphaltenes was also reduced by this treatment. The results obtained in the aqueous system were very inconsistent because of the insolubility of the porphyrin substrates. When using fraction 5 asphaltenes, the mixture contained a suspension of small asphaltene particles, providing a large surface area for the enzyme reaction. However, the mixture had mass-transfer limitations, and thus variability in the results was observed. For example, comparison of four experiments with fraction 5 asphaltenes conducted on different days showed that the amount of reduction of the Soret peak ranged from 33 to 93% for the same treatment. Variations were also found in tests with pure porphyrins. These compounds did not suspend in the buffer solution but adhered in a thin layer on the sides of the reaction vial. Spectrophotometric results from 10 replicate reactions with NiOEP showed that the reduction of the Soret peak ranged from 45 to 80%. The poor reproducibility of the reactions in aqueous buffer prompted us to look for a solvent system in which the petroporphyrins would be soluble and CPO would remain active.
Optimization o f CPO reaction conditions for porphyrins in the ternary system In order to eliminate the mass-transfer limitations in the system and improve the action of CPO on petroporphyrins, the reaction was carried out in ternary systems. Treatment of NiOEP with CPO in the presence of H202 resulted in the reduction and disappearance of the Soret peak at 392 nm and two peaks in the visible region (c~ = 552 nm,/3 = 517 rim) (Figure 2). There was a corresponding appearance of a peak at 445 nm and a solution color change from pink to yellow. After the Soret peak was completely removed, the reaction continued further with the reduction of this product peak. In some reaction mixtures, the product peak subsequently disappeared, leaving a colorless solution with no UV-visible absorbance peaks (Figure 2). Monitoring the maximum rate of decrease in the absorbance at 392 nm was used as a measure of the enzyme activity in order to determine the optimum composition of the ternary solvent system. A number of different mixtures of toluene : isopropanol : buffer were tested to determine the optimum ratio of solvents to use for CPO-mediated reactions. Mixture 15 of Vulfson et al. z5 was used as a reference and the activity was designated 100% (Table 1). In this system,
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. various monophase organic solvents. 33-36 In this work, the ternary solvent system of toluene : isopropanol : buffer enhanced the CPO-catalyzed modifications of water-insoluble porphyrins. In addition, the reaction in the ternary system required less enzyme than the aqueous system for complete removal of the Soret peak. For example, 1.25 tzg of CPO ml-~ was adequate for treatment of porphyrins in the ternary system, whereas multiple additions of 5 to 10/xg of CPO ml -j were required in the aqueous system.
0.4 Soret peak 392 nm
0.3
¢, o t-, r5
- Control NiOEP 0.2
..Q <
CPO-treated
Demetallation o f model metalloporphyrins
0.1
0 350
400
450
500
550
600
nm Figure 2 Changes in the UV-visible spectrum of NiOEP by CPO treatment in ternary mixture 36. Labels (1X and 4X) indicate the number of additions of CPO and H202
the CPO showed an activity of 0.225 AA392 min ~/xg-l protein over a 3- to 4-min interval. The rate of disappearance of the Sorer peak of NiOEP in this ternary system was proportional to the amount of CPO used. Different ratios were plotted on a ternary graph (Figure 3), based on molar fraction, which showed the region of different phases of the ternary system as determined by Lund and Holt. 3° The highest enzyme activities, which were 75 to 137% of that observed with mixture 15, were found in a range of combinations of solvents corresponding to the formation of a normal ternary system where the amount of toluene ranged from 5 to 20% by volume and the water content was 15 to 25% (Table 1). Mixture 15, which gave the maximum activity for polyphenoloxidase,25 was included in this region. Among the mixtures that gave high CPO activity (i.e., a rapid reduction in the Soret peak of NiOEP) the overall extent of the reactions differed. Reaction mixtures with 20% water (mixtures 36 to 39) were chosen for further study because they showed a more complete reaction, with the reduction of both the Soret peak and the peak at 445 nm. Some reactions stopped with the formation of a product with a maximum absorption at 445 nm. This could be removed with multiple additions of CPO and H2Oz. Chloride was absolutely required for activity of CPO on the NiOEP and the other porphyrins (data not shown) in the ternary reaction systems, and the optimum concentration was 12 mM CI-. There was no loss of Soret peak when the reaction mixtures lacked CPO or H202. Detergentless microemulsions based on toluene or hexane have been used successfully for enzyme reactions on water-insoluble substrates. 25'31'32 Free radical production by peroxidases has been demonstrated in
The optimum mixture of solvents chosen for demetallation experiments was 5% toluene:75% isopropanol: 20% buffer (mixture 36). Figure 2 shows the spectral changes of NiOEP solutions after treatment with CPO in the ternary system. The reaction labeled IX was treated with a single addition of CPO and H202. When the Soret peak was completely removed and the yellow product was at the maximum absorbance, the reaction was stopped by addition of excess water. The sample labeled 4X (Figure 2) was reacted further with a total of four additions of CPO and H202 until the yellow color and the absorbance at 445 nm were removed. The results of Ni analyses of the extracted NiOEP and the reaction products (organic layer) and the remaining aqueous phase are shown in Table 2. The amount of Ni that remained in the organic phase when the yellow product was present was 25% less than in the control
O
0.2
0.4
0.6
0.8
1.O
TOLUENE
Figure 3 Mole fraction compositions of ternary systems used to test the CPO on NiOEP. According to Lund and Holt, 3° zone A contains normal ternary systems, zone B contains H-bonded aggregates of water and isopropanol distributed in toluene, zone C contains mixtures with transparent microemulsions, and zone D contains unstable macroemulsions. ( . ) Enzyme activities that were 77 to 137% of that in mixture 15; (0) 10 to 40% of that in mixture 15; (©) <10% of that in mixture 15. There were no mixtures that gave enzyme activities between 40 and 77%
Enzyme Microb. Technol., 1993, vol. 15, M a y
433
Papers Table 2
Nickel analysis of CPO-treated NiOEP in ternary mixture 36 Amounts of Ni Organic phase
Aqueous phase
Total recovered b
Sample
mg
% decreasea
mg
% found a
mg
Control CPO-treated, 1Xc CPO-treated, 4X d Reagent blank
5.86 4.41 0.39 0.01
0 25 93
0.012 0.44 3.12 <0.01
0.2 8 53
5.87 4.85 3.51
a Based on Ni content in organic phase from control b Expected total Ni was 5 mg c One addition of CPO and hydrogen peroxide showed complete reduction of Soret peak, producing a yellow solution with peak absorbance at 445 nm d Four additions of CPO and hydrogen peroxide removing the absorbance at 445 nm, producing a colorless solution
in which there was no enzyme reaction. Further CPO treatment that gave a clear reaction mixture resulted in the loss of almost all (93%) the Ni from the porphyrin extract. Other workers who have studied chemical demetallation of petroporphyrins measured only the metal content of the organic residues. 37-4° In contrast, we have also determined the metal content of the aqueous phase. The data in Table 2 clearly show that the CPO treatments increased the amount of water-soluble Ni. Unfortunately, the analyses could only account for 3.51 mg of the expected 5.89 mg of Ni. Losses of metal may have occurred by complexation with protein precipitate and in the filtration, evaporation, and transfer steps of the aqueous phase. In spite of the low overall metal recoveries, the results clearly showed that Ni partitioned into the aqueous phase after CPO treatment. Treatment of NiOEP in an aqueous system achieved only 50 to 80% reduction of the Soret peak and a resultant 25 to 50% loss of Ni from the organic-phase extracts. VOEP was also treated with CPO in the ternary system resulting in a decrease of the Sorer band at 407 nm. The reaction product had a strong absorbance at 447 nm. Multiple additions of CPO and H20 2 were also able to remove this peak. Table 3 shows that the V content of the organic extract was reduced after one addition of CPO and H20 2. More metal was released after multiple treatments (4X). Reaction of CPO with VOEP was slower and not as complete as with NiOEP. However, the reaction was much better in the ternary system than in the aqueous system, where only 35% reduction in the Soret peak of VOEP and, at most, a 25% reduction in V content of the organic phase were observed. VTPP was not susceptible to modification in the ternary system under the reaction conditions used for the other porphyrins, even with increased concentrations of CPO. The bulky phenyl groups block the meso position of the molecule, which was the site of chlorination by chlorinating agents 38'4~'42or oxidation by peroxidative activity. 43'44 However, in the aqueous system, there was an average reduction of 40 to 60% of the
434
Enzyme Microb. Technol., 1993, vol. 15, May
Soret peak at 424 nm with a 45 to 50% decrease in the amount of V in the methylene chloride-extractable material. CPO has a peroxidase activity in the absence of chloride at pH 3 and 5. 23'45 However, no reduction in the Soret peak was observed without chloride with the model metalloporphyrins used in this study. Because the enzyme reaction requires chloride, the products were likely chlorinated. Based on steady-state kinetic studies, Dunford et al. 46 concluded that the liberation of free hypochlorous acid was not important in the CPO-catalyzed chlorination of monochlorodimedone. Rather the reaction proceeds from compound I to formation of iron (III)-OC1 by chloride ion addition to the ferryl oxygen atom. Thus the enzyme controls the chlorination of monochlorodimedone. The same may be true for the CPO-catalyzed modifications of metalloporphyrins. Sugihara et a/. 37'38described the oxidative demetallation of vanadyl porphyrins in asphaltenes with the chlorinating agents chlorine and sulfuryl chloride. They described a green product with a maximum absorbance at 450 nm, consistent with a chlorinated VOEP. The porphyrin was attacked at the meso position, and therefore VTPP was much less reactive than the VOEP, as we also found with CPO reactions. Metal release by CPO is likely due to the disruption of the integrity of the chelating system, rather than a simple release of metal, as is the case with strong-acid demetallation. The molecular weight of NiOEP is 590. After treatment of NiOEP with concentrated sulfuric acid, low resolution mass spectrometry showed that the product had a molecular weight of 534, which is consistent with the loss of Ni and the addition of two hydrogen atoms. Indeed, the mass spectrum of the product was virtually identical to that of octaethylporphyrin reported by Smith. 47 The mass spectrum of the solvent-extracted products after demetallation of NiOEP with CPO in the ternary system showed no ion at m/z 534. The highest mass ion observed was m/z 446, and there were many other fragments. McDonagh 48 reported that electron impact mass spectrometry of porphyrins produces weak molecular ions and that
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. Table 3
Vanadium analysis of CPO-treated VOEP in ternary mixture 36 Amounts of V Organic phase
Aqueous phase
Sample
mg
% decreasea
Control CPO-treated, 1Xc CPO-treated, 4X d
4.20 2.76 1.96
0 34 53
mg 0.006 0.035 0.825
Total recoveredb
% found a
mg
0.1 0.8 20
4.21 2.80 2.79
a Based on V content in organic phase from control b Expected total V was 5 mg c One addition of CPO and hydrogen peroxide showed complete reduction of Soret peak, producing a green solution with peak absorbance at 448 nm d Four additions of CPO and hydrogen peroxide removing the absorbance at 448 nm, producing a colorless solution
thermal decomposition of the sample in the source gives misleading spectra. It is most likely that the CPO reaction yields a variety of products, which we did not attempt to identify. Schaefer et al. 43 described the enzymatic peroxidative degradation of porphyrins and determined the effect of the chelated metal on the reaction. They determined that a redox active metal was required for porphyrin degradation, and thus no reaction occurred between H202 and metal-free OEP. However, in our experiments, OEP was susceptible to attack by CPO. During the treatment of OEP with CPO in the ternary system, the Soret peak shifted from 396 to 408 nm, then was slowly degraded to a colorless product. The visible spectrum was also shifted from peaks at 497, 530, and 567 nm to peaks at 508, 543, and 584 nm. Based on these results, the degradation products of OEP were likely chlorinated products. For example, monochlorooctaethylporphine has a Soret peak at 406 nm and absorptions at 507,540, and 578 nm. Dichlorooctaethylporphine has a Sorer peak at 411 nm and absorptions at 514, 548, and 586 r i m . 41'42 However, it was difficult to isolate and identify these intermediates because they were further degraded to colorless products by CPO.
CPO treatment and demetallation o f fraction 5 asphaltenes Initial experiments showed that CPO had good activity against fraction 5 asphaltenes in an aqueous reaction system. Studies with the ternary solvent system showed that the asphaltene fraction was not completely soluble in this system, and it gave a slightly cloudy mixture. However, the Soret peak was still discernible to monitor the progress of the reaction. Multiple additions of CPO and H 2 0 2 reduced the Soret peak and the shoulder which was formed at about 436 nm (Figure 1). There was a 20% decrease in the amount of both Ni and V content of the organic phase containing the asphaltene fraction (Table 4). The metals contents of the aqueous phase was significantly higher than that of the control which had not been reacted with CPO, but the amounts of metal released from the asphaltenes
were not recovered in the aqueous phase. Fraction 5 asphaltenes treated in the aqueous system also showed a complete loss of the Sorer peak and about 20% decrease in the amount of V in the methylene chlorideextractable material. Sugihara et al. 38 studied the use of chlorine in the demetallation of two fractions of asphaltenes from Boscan crude oil. In one fraction, all of the metal content was accountable as metalloporphyrins, and this fraction was more readily demetallated than the other fraction that had a much higher nonporphyrin metal content. Similarly, G o u l d 39 compared the relative susceptibilities of porphyrin and nonporphyrin metals to demetallation. He treated Cold Lake asphaltenes with various proportions of peroxyacetic acid and measured the loss of the Soret peak absorbance and the amount of V and Ni remaining in the asphaltenes. The greatest amounts of demetallation were associated with the decrease in the Sorer peak absorbance. At the peroxyacetic acid dose that exceeded that required to completely remove the Soret peak, there was little additional release of V but a more significant release of Ni. G o u l d 39 suggested that the peroxyacetic acid acts rapidly to destroy the metalloporphyrin and liberate their associated metals, while the metals held by nonporphyrin ligands are somewhat more slowly attacked and removed. Semple et al.J4 showed that only about 40% of the total V in the Cold Lake fraction 5 asphaltenes was complexed with porphyrins. Based on the 20% demetallation of V from this fraction (Table 4), and assuming that the metalloporphyrins are more susceptible to demetallation by CPO than the nonporphyrin-complexed metals, 38'39 about one-half of the porphyrin-bound metal was released by CPO treatment. CPO treatment of the fraction 5 asphaltene does not appear to alter the solubility of this material. In contrast, sulfuric acid treatment used for demetallation produced methylene chloride-insoluble precipitates. CPO treatment of porphyrins in petroleum may offer an alternative method to remove metals from oil to avoid problems with poisoning catalysts. Organometallic compounds determine more or less the detrimental effects of asphaltenes and resins on catalysts.17 Com-
Enzyme Microb. Technol., 1993, vol. 15, May
435
Papers Table 4 Metals analysis of CPO-treated fraction 5 asphaltenes in ternary mixture 36. A sample of 20 mg of fraction 5 asphaltenes was treated, and this was expected to contain approximately 0.6 mg Ni and 2.8 mg V Amounts of Ni Organic phase
Amount of V
Aqueous phase
Organic phase
Aqueous phase
Sample
mg
% decreasea
mg
% found a
mg
% decreasea
mg
% found a
Control CPO-treated b Reagent blank
0.80 0.64 0.02
0 20
0.036 0.098 0.003
5 12
3.72 3.02 0.01
0 19
<0.01 0.036 <0.01
<0.2 1
a Based on Ni or V content in organic phase from control b Treatment resulted in complete reduction of Soret peak at 410 nm and reduction of resulting shoulder at 435 nm
plexes of Ni and V are concentrated in the asphaltenes, 3'~ and Ni and V seriously affect catalytic cracking catalysts. Because metalloporphyrins are the most volatile metal-containing molecules in petroleum and they cause the greatest problems, 11 the destruction of the porphyrins and removal of the metals is beneficial. The objective is to destroy the porphyrins with little effect on the petroleum character. Oxidative demetallation of vanadyl porphyrins has been investigated. 38'39 Sugihara eta/. 37"38used mild oxidating agents such as chlorine and sulfuryl chloride, and Gould 39 used hypochlorite bleach and peroxyacetic acid. Indeed, Gould 39 used Cold Lake asphaltenes and observed that hypochloride bleach released 78% of the V and 37% of the Ni, whereas peroxyacetic acid released 73% of the V and 49% of the Ni from the asphaltenes. A number of methods described by Kukes and Aldag4° cause changes in the character of petroleum, and some methods, using phosphorus compounds, remove only V but not Ni. Chlorination methods 3s demetallate the porphyrins but cause incorporation of chlorine into the feed material. 39 Because of the absolute requirement for chloride in the CPO reactions, this enzymatic reaction likely yields chlorinated products.
other nonchlorinating enzymatic reactions in nonaqueous solvents in attempts to upgrade heavy oils.
Acknowledgements This work was funded by a contract from the Alberta Oil Sands Technology and Research Authority. We thank V. Foubister and J. Gerard for technical assistance and M. A. Pickard for the CPO and valuable discussions during the course of this study.
References 1 2 3 4 5 6 7
Conclusions This work has demonstrated that the extracellular enzyme CPO can alter petroporphyrins associated with the asphaltene fraction of a heavy oil. We are not aware of any other clear demonstration of modification of components of asphaltenes by a biological product or reaction. As a consequence of the reaction that results in removal of the Soret peak, CPO can release metals from pure metalloporphyrins and the porphyrin-rich fraction of Cold Lake asphaltenes. The ternary system of toluene, isopropanol, and buffer was superior to the aqueous buffer system in that the reaction is more complete, with further reduction of the reaction products, and the extent of the reaction can be seen directly in the color change before extraction is done. CPO treatment was effective for metal removal from the porphyrin-rich fraction of the asphaltenes. It is postulated that the enzyme-mediated reactions would yield chlorinated products which would be undesirable as a refinery feedstock. We are currently investigating
436
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8 9 10 11 12 13 14 15 16
17 18
Baker, E. W. in Organic Geochemistry (Eglinton, G. and Murphy, M. T. J., eds.) Springer-Verlag, Berlin, 1969, pp. 464-497 Yen, T. F., Boucher, L. J., Dickie, J. P., Tynan, E. C. and Vaughan, G. B. J. Inst. Petrol., London 1969, 55, 87-99 Filby, R. H. in The Role o f Trace Metals in Petroleum (Yen, T. F., ed.) Ann Arbor Science Publishers, Ann Arbor, MI, 1975, pp. 31-58 Baker, E. W. and Palmer, S. in The Porphyrins. Vol H (Dolphin, D. A., ed.) Academic Press, New York, 1978, pp. 485-550 Fish, R. H., Kolenic, J. J. and Wines, B. K. Anal. Chem. 1984, 56, 2452-2460 Filby, R. H. and VanBerkel, G. J. in Metal Complexes in Fossil Fuels (Filby, R. H. and Branthaver, J. F., eds.) American Chemical Society, Washington, DC, 1987, pp. 2-39 Frakman, Z., Ignasiak, T. M., Montgomery, D. S. and Strausz, O. P. AOSTRA J. Res. 1988, 4, 171-179 Hodgson, G. W. Ann. N Y A c a d . Sci. 1973, 206, 670-684 Yen, T. F. in The Role o f Trace Metals in Petroleum (Yen, T. F., ed.) Ann Arbor Science Publishers, Ann Arbor, MI, 1975, pp. 1-30 Gallegos, E. J. and P. Sundararaman. Mass Spect. Rev. 1985, 4, 55-85 Branthaver, J. F. in Metal Complexes in Fossil Fuels (Filby, R. H. and Branthaver, J. F., eds.) American Chemical Society, Washington, DC, 1987, pp. 188-204 Reynolds, J. G. and Biggs, W. R. Accounts Chem. Res. 1988, 21, 319-326 Baker, E. W., Yen, T. F., Dickie, J. P., Rhodes, R. E. and Clark, L. F. J. Am. Chem. Soc. 1967, 89, 3631-3639 Semple, K. M., Cyr, N., Fedorak, P. M. and Westlake, D. W. S. Can. J. Chem. 1990, 68, 1092-1099 Yen, T. F. Energy Sources 1974, 1, 447-463 Speight, J. G. and Moschopedis, S. E. in Chemistry o f Asphaltenes. Adv. Chem. Series 195 (Bunger, J. W. and Li, N. C., eds.) American Chemical Society, Washignton, DC, 1981, p. 1 Pelet, R., Behar, F. and Monin, J. C. Org. Geochem. 1986, 10, 481-498 Zajic, J. E., Cooper, D. G., Marshall, J. A. and Gerson, D. F. Fuel 1981, 60, 619-623
Modifications of petroporphyrins and asphaltenes: P. M. Fedorak et al. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
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