Fate of Crude Oil by the Combination of Photooxidation and

Photooxidation controls were prepared as already described ... length and 10 mm in internal diameter). ... hopane as an internal control of the h-crud...
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Environ. Sci. Technol. 2000, 34, 1500-1505

Fate of Crude Oil by the Combination of Photooxidation and Biodegradation TAPAN K. DUTTA AND SHIGEAKI HARAYAMA* Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan

Photooxidation and biodegradation are the two most important factors involved in the transformation of crude oil or its products that are released into a marine environment. Natural microbial populations in seawater biodegraded 28% of crude oil within 8 weeks at 20 °C when sufficient nutrients were supplied to the seawater. Photooxidation mainly affected the aromatic compounds in crude oil and converted them to polar species. This treatment increased the amount of crude-oil components susceptible to biodegradation, and 36% of photooxidized crude oil could be degraded in 8 weeks at 20 °C. It is concluded that the susceptibility of crude oil to biodegradation is increased by its photooxidation.

Introduction The contamination of a marine environment with crude oil and/or its products through spillage or dumping causes sustained and serious problems to the natural environment. Although bioremediation is of worldwide interest as a potential cleanup option (1, 2), a fundamental understanding of natural processes, viz., evaporation, dispersion, photooxidation and microbial degradation of spilled oil, is essential for its design. Typical crude oil has a density of 0.85, and this factor combined with wind, wave action, and current leads to spreading of the spilled oil on the surface of seawater. Evaporation is one of the important abiotic processes and is enhanced by the formation of an oil slick due to its expanded surface area. As a result, those components of crude oil with a boiling point below 200 °C volatilize, leading to the removal of about 35% of the initial constituents (3). The composition of the floating oil is further affected by photooxidation, biodegradation, and dissolution into seawater. Photooxidation is one of the major events in the compositional change of crude oil. Nevertheless, this phenomenon is still poorly characterized. Most studies on the photooxidation of hydrocarbons have focused primarily on single compounds or on a simple mixture of hydrocarbons in the presence of sensitizer molecules (4-8). Studies on the photooxidation of crude oil and/or its fractions have been carried out (9-14), and the importance of photooxidation to the fate of spilled oil in a marine environment has been demonstrated. Several mechanisms for the photolytic decomposition of crude-oil components, e.g., direct photolysis, singlet oxygenation, and radical oxidation, have been proposed, but the relative importance of these processes is not yet clear (15). * Corresponding author phone: +81-193-26-6544; fax: +81-19326-6592; e-mail: [email protected]. 1500

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On the other hand, the microbial degradation of crude oil has been studied in great detail. It has been elucidated that the lighter fractions were degraded more rapidly than the heavier ones, that n-alkanes were degraded more rapidly than branched alkanes, and that aromatics with two to three rings were readily biodegraded through several pathways (1, 16-20). Studies on the synergism between photochemical and biological reactions for the in situ degradation of crude oil are of great ecological interest. However, such studies have not been systematically carried out although they can best describe the natural processes taking place in seawater after an oil spill. Changes in the fluorescence spectra led Literathy et al. (14) to suggest synergism between the biodegradation and photooxidation of crude oil. However, no significant effect of the solar radiation on the biodegradation of crude oil was observed (21). In contrast, the biodegradation of the aromatic compounds, anthracene, n-nonylbenzene, and 2-methylphenanthrene, was found to be enhanced by photooxidation (22-24). The major problem in studying the degradation of crude oil is its complexity because it comprises an enormous number of components. Among the analytical techniques available for structurally determining crude oil components or metabolites, gas chromatography in combination with mass spectrometry (GC-MS) has been the best choice so far and most widely used. The fractionation of crude oil and subsequent GC-MS analysis has characterized nearly 300 components comprising aliphatic, aromatic, and biomarker compounds (25, 26). Nevertheless, the major fractions of crude oil still remain uncharacterized since the majority of the components cannot be resolved and appear as a “hump” or “unresolved complex mixture (UCM)” in GC chromatograms (27). In this present study, we examine the combined effect of the photooxidation and biodegradation of crude oil to understand its fate when spilled in a marine environment. We use indigenous microbial populations for the biodegradation and artificial sunlight for the photooxidation.

Experimental Section Organisms. Seawater was collected at a depth of 15 m from Kamaishi Bay in Japan. Crude Oil. Arabian light crude oil (38 L) was pretreated by distilling at 230 °C, and the unevaporated oil (22 L), called heated crude oil (h-crude oil), was used as the starting material. On the basis of the mass-reduction curve, this pretreatment is almost equivalent to the heat treatment at 100 °C for 48 h which has been used as an artificial weathering process (28). Medium and Cultivation. The natural seawater medium used for the biodegradation experiments consisted of 800 mL of nonsterilized natural seawater and 200 mL of an autoclaved solution (pH 7.6) containing 5 g/L of NH4NO3, 1 g/L of K2HPO4, and 0.1 g/L of ferric citrate. h-Crude oil was added at a concentration of 1 g/L as the carbon source. The natural seawater medium containing crude oil was cultivated at 20 °C for different periods of time under constant shaking (100 strokes/min) to promote the growth of indigenous oildegrading microorganisms. In the case of biodegradation of photooxidized oil, the chloroform extract of photooxidized h-crude oil at a concentration of 1 g/L was used as the carbon source. Each experimental set was cultivated in duplicate with a set of negative controls. For biodegradation controls, autoclaved seawater was used. 10.1021/es991063o CCC: $19.00

 2000 American Chemical Society Published on Web 03/09/2000

FIGURE 1. GC-MS traces of h-crude oil subjected to biodegradation and/or photooxidation. The sets of traces (A, B, C, D and a, b, c, d) represent total ion chromatograms and selected ion (m/z ) 191) chromatograms (distribution of hopanes), respectively. A and a, h-crude oil; B and b, biodegraded h-crude oil; C and c, photooxidized h-crude oil; D and d, photooxidized and biodegraded h-crude oil. UCM, unresolved complex mixture.

TABLE 1. Gravimetric Analysis of Different Fractions of Crude Oil Samples h-crude oil (%) fraction

n-hexane insoluble water soluble column fractions of maltene: n-hexane n-hexane:benzene (1:1) dichloromethane methanol chloroform subtotal reduction in control (reduction in sample) minus (reduction in control)

expt 1 4.2 0 52.4 31.0 3.7 5.9 2.8

biodegraded oil (%)

expt 2 mean expt 1 4.0 0

4.1 0

5.9 2.2

55.6 54.0 29.0 30.0 3.1 3.4 6.3 6.1 2.0 2.4 100

32.6 19.7 1.9 6.5 1.2

0

expt 2 6.7 3.2

photooxidized oil (%)

mean expt 1

expt 2

photooxidized and biodegraded oil (%)a

mean expt 1

expt 2

mean

6.3 2.7

36.5 5.1

40.5 4.3

38.5 4.7

31.6 5.7

29.2 4.3

30.4 5.0

28.2 30.4 21.1 20.4 2.5 2.2 6.1 6.3 2.2 1.7 70.0 2.0 ( 0.5 28.0 ( 1.8

32.8 2.5 4.8 13.6 1.2

29.6 31.2 3.3 2.9 5.0 4.9 12.8 13.2 1.0 1.1 96.5 5.0 ( 1.4 -1.5 ( 0.6

9.0 2.0 2.7 12.4 0

10.6 9.8 2.2 2.1 3.5 3.1 13.6 13.0 0 0 63.4 0.6 ( 0.2 36.0 ( 2.0

a The photooxidized sample (whose composition is shown in the “photooxidized oil” column) was extracted by chloroform and used for the biodegradation. The water-soluble fraction of the photooxidized sample was not included in this sample.

Photooxidation Treatment. The treatment by photooxidation was carried out on an oil slick of h-crude oil. Fifty milligrams of h-crude oil was dropped onto a 50 mL of distilled water in a glass Petri dish (90 mm in internal diameter) to allow the slick to form. Several samples were incubated without lid in a chamber whose temperature was controlled to 20 °C by ventilated air. Artificial sunlight was produced by a combination of four tin-halide lamps, four high-pressure mercury lamps, and four argon lamps and a heat-absorbing filter (Toshiba Corporation, Japan). Its emission spectrum and intensity (1200 µmol m-2 s-1) was very similar to that of natural sunlight. The oil slick was directly irradiated by the artificial sunlight for different times (typically 4 weeks). Photooxidation controls were prepared as already described and incubated in the dark.

Analyses. At appropriate times, both the biodegraded and photooxidized samples were adjusted to pH 7.0 and extracted three times with half their volume of chloroform. The aqueous part was acidified with concentrated hydrochloric acid to pH 1.0 and extracted three times with half its volume of diethyl ether. Both the chloroform and diethyl ether extracts were evaporated in a rotary evaporator under reduced pressure. Each extracted sample was transferred to a preweighed glass vial and left under a fume hood to evaporate further to constant weight. Unless stated otherwise, the present results represent data from biodegradation and photooxidation experiments lasting for 8 and 4 weeks, respectively. Fractionation of the Crude Oil. Chloroform extracts of the biodegraded and/or photooxidized h-crude oil were precipitated by n-hexane to give asphaltenes (insoluble in VOL. 34, NO. 8, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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n-hexane) and maltenes (soluble in n-hexane). The maltene fraction was further fractionated by column chromatography. The silica gel (C-200, Wako Pure Chemicals, Japan) used for column chromatography was pretreated (25) by washing serially with 3 volumes of acetone, n-hexane, and dichloromethane in a column with a coarse-porosity fitted disk, left under a fume hood overnight, and then dried completely at 50 °C. The dried gel was activated at 180 °C for 20 h. Fractionation of the maltene fraction of crude oil used a chromatographic column with a glass stopcock (30 cm in length and 10 mm in internal diameter). The column was then dry-packed with 3 g of activated silica gel which was constantly tapped to settle it and topped with 1.5 g of anhydrous sodium sulfate. The column was conditioned with 20 mL of n-hexane, and, just before exposing the sodium sulfate layer to the air, a 300-µL aliquot of the maltene fraction (dissolved in n-hexane to a concentration of 100 mg/mL) from a biodegraded and/or photooxidized crude oil sample was applied to the column. All eluates up to this point were discarded. Next, the column was serially eluted with 3 bed volume of each solvent in the following order to provide the different classes of compounds indicated in parentheses: n-hexane (saturates plus long-chain alkylaromatics), 50% benzene in n-hexane (aromatics), dichloromethane (resins, nonacidic), methanol (resins, weakly acidic), and chloroform (remaining materials in the column). Each eluate was evaporated under reduced pressure to dryness and used for further analyses. Gravimetric Analysis. The amount of crude oil components in each sample or each chromatographic fraction was determined by measuring the weight of the sample on a sensitive digital balance. FT-IR Analysis. A Fourier transform infrared (FT-IR) spectroscopic analysis was carried out with an FT-IR-5000 spectrometer (JASCO, Japan). Each sample was prepared as a thin film on an NaCl plate by evaporating 25 µL of an applied carbon tetrachloride solution containing 50 mg/mL of crude oil components. Each spectrum was obtained by summing 128 scans at a resolution of 4 cm -1. GC-MS Analysis. An analysis of the crude oil by gas chromatography-mass spectrometry (GC-MS) was performed by using a QP-5000 instrument (Shimadzu, Japan) fitted with a fused silica capillary column (DB-5, 30 m × 0.25 mm, J&W Scientific, Japan). The temperature program gave a 2-min hold at 50 °C, an increase to 300 °C at 6 °C/min, and a 16-min hold at 300 °C. The injection volume was 1 µL, and the carrier gas was helium (1.7 mL/min). The mass-selective detector was operated in the scan mode to obtain spectral data for identifying the compounds and in the selected ion monitoring (SIM) mode for quantifying the target compounds. 1H- and 13C NMR Analyses. Spectra were recorded by a JNM FT-NMR system (JEOL, Japan) operated at 400 MHz for 1H and at 100 MHz for 13C data. For quantitative 13C NMR, a pulse delay of 1.0 s was used. The intensity of each chemical shift was determined relative to tetramethylsilane (TMS) as an internal standard, where CDCl3 was used to dissolve each sample. The aromaticity is defined as the ratio of aromatic carbons to total carbons, where the amounts of aromatic and aliphatic carbons were obtained from the integrated intensity of peaks in the ranges 100-170 ppm and 8-58 ppm, respectively (29).

Results and Discussion Hopanes are crude-oil components that are known to be resistant to both biodegradation and photooxidation and are used as internal standards for estimating the extent of biodegradation and photooxidation (12, 30). We confirmed this conclusion by the following two observations. (a) Quantitative GC-MS analyses in the SIM (m/z ) 191) mode 1502

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TABLE 2. Abundance of Alkanes and Aromatics in h-Crude Oil before and after Biodegradation abundancea compoundb C11-n-alkane C12-n-alkane C13-n-alkane C14-n-alkane C15-n-alkane C16-n-alkane C17-n-alkane pristane C18-n-alkane phytane C19-n-alkane C20-n-alkane C21-n-alkane C22-n-alkane C23-n-alkane C24-n-alkane C25-n-alkane C26-n-alkane C27-n-alkane C28-n-alkane C29-n-alkane C30-n-alkane C31-n-alkane C32-n-alkane C33-n-alkane C34-n-alkane C35-n-alkane C0-Naph C1-Naph C2-Naph C3-Naph C4-Naph C0-DBT C1-DBT C2-DBT C3-DBT C4-DBT C0-Phn C1-Phn C2-Phn C3-Phn C4-Phn C5-Phn C6-Phn C7-Phn C0-Fluorene C1-Fluorene C2-Fluorene

h-crude biodegradation biodegraded biodegradation oil controlc h-crude oil (%)d 10 320 3470 12900 23500 29400 30600 4600 28500 8480 26100 22400 20500 17600 14800 12300 10100 8760 6560 5700 4800 4020 2990 2080 1400 803 581 0 203 2860 5860 4020 1170 4160 8540 6640 3950 540 1430 2370 1790 1190 1060 1440 1640 86 351 730

0 0 58 1050 6120 14200 18800 2960 20000 5890 19500 18000 16600 15100 13400 11600 9490 8370 6230 5290 4620 3890 2890 1970 1360 790 592 0 0 0 1000 2060 706 3010 6690 5640 3140 324 1030 1840 1640 1070 962 1370 1620 31 180 563

0 0 0 0 0 74 82 26 90 132 35 35 9 27 27 29 34 22 50 22 5 41 10 0 0 15 34 0 0 0 0 0 0 0 341 622 1080 0 0 0 68 15 157 761 1570 0 0 0

NAe NA 100 100 100 99.5 99.6 99.1 99.6 97.8 99.8 99.8 99.9 99.8 99.8 99.8 99.6 99.7 99.2 99.6 99.9 98.9 99.7 100 100 98.1 94.3 NA NA NA 100 100 100 100 94.9 89.0 65.6 100 100 100 95.9 98.6 83.7 44.5 3.1 100 100 100

a Abundance of each compound was determined by GC-MS in SIM mode and normalized with that of 17R(H),21β(H)-hopane as internal standard. b Naph, DBT, and Phn represent naphthalene, dibenzothiophene, and phenanthrene, respectively. C0 to C7 represent carbon numbers of the alkyl groups in alkylated polycyclic aromatic hydrocarbons. c h-Crude oil was incubated in sterilized seawater. d The percentage of biodegradation was calculated by the eq 100-100 × (A/B), where A is the abundance of a compound in biodegraded sample while B is that in its control sample. e Not applicable.

demonstrated that the ratio of 17R(H),21β(H)-hopane (the largest peak with a retention time of 40.62 min in Figure 1) to the other major hopane components remained unaltered by biodegradation and photooxidation, indicating the resistance of hopanes to such processes. (b) The concentration of 17R(H),21β(H)-hopane in each h-crude oil sample was inversely related to the weight of the sample after biodegradation and/or photooxidation. These observations can best be explained if hopane is essentially resistant to biodegradation and photooxidation. We therefore used 17R(H),21β(H)hopane as an internal control of the h-crude oil.

TABLE 3. Abundance of Alkanes and Aromatics in Photooxidized h-Crude Oil before and after Biodegradation abundancea

abundance

compoundsb

photooxidation control

photooxidized h-crude oil

photooxidation (%)c

biodegradation control of ph-crude oild

biodegraded ph-crude oil

biodegradation (%)c

C11-n-alkane C12-n-alkane C13-n-alkane C14-n-alkane C15-n-alkane C16-n-alkane C17-n-alkane pristane C18-n-alkane phytane C19-n-alkane C20-n-alkane C21-n-alkane C22-n-alkane C23-n-alkane C24-n-alkane C25-n-alkane C26-n-alkane C27-n-alkane C28-n-alkane C29-n-alkane C30-n-alkane C31-n-alkane C32-n-alkane C33-n-alkane C34-n-alkane C35-n-alkane C0-Naph C1-Naph C2-Naph C3-Naph C4-Naph C0-DBT C1-DBT C2-DBT C3-DBT C4-DBT C0-Phn C1-Phn C2-Phn C3-Phn C4-Phn C5-Phn C6-Phn C7-Phn C0-Fluorene C1-Fluorene C2-Fluorene

0 0 0 0 50 600 2080 494 7030 3440 13100 15000 14600 13700 12200 11000 8960 8100 5900 5090 4520 3840 2800 1920 1350 759 577 0 0 0 0 0 49 435 2420 3410 2480 0 165 855 1300 969 952 1310 1590 0 0 195

0 0 0 111 230 1830 6880 1210 13600 4930 18300 16200 15200 13800 12400 11030 8950 8040 5780 4890 4480 3700 2680 1890 1330 738 568 0 0 0 0 0 113 1220 2020 627 224 121 110 121 0 58 68 38 0 0 0 84

NAe NA NA NA -360 -205 -231 -145 -93.5 -43.3 -39.7 -8.0 -4.1 -0.7 -1.6 -0.3 0.1 0.7 2.0 3.9 0.9 3.6 4.3 1.6 1.5 2.8 1.6 NA NA NA NA NA -131 -180 16.5 81.6 91.0 NA 33.3 85.8 100 94.0 92.9 97.1 100 NA NA 56.9

0 0 0 0 43 942 4390 815 9940 3500 13100 13700 13100 12600 11500 10500 8790 7880 5710 4830 4500 3770 2640 1860 1310 736 570 0 0 0 0 0 60 798 1720 582 180 0 78 107 0 53 64 37 0 0 0 65

0 0 0 0 0 46 45 45 55 21 84 95 93 84 71 106 40 44 37 24 23 34 23 9 6 20 28 0 0 0 0 0 0 0 199 267 73 0 0 0 0 24 39 21 0 0 0 0

NA NA NA NA 100 95.1 99.0 94.5 99.4 99.4 99.4 99.3 99.3 99.3 99.4 99.0 99.5 99.4 99.4 99.5 99.5 99.1 99.1 99.5 99.5 97.3 95.1 NA NA NA NA NA 100 100 88.4 54.1 59.4 NA 100 100 NA 54.7 39.1 43.2 NA NA NA 100

a Abundance of each compound was determined by GC-MS in SIM mode and normalized with that of 17R(H),21β(H)-hopane as internal standard. The abundance in the initial sample is indicated in the “h-crude oil” column of Table 2. b Naph, DBT, and Phn represent naphthalene, dibenzothiophene, and phenanthrene, respectively. C0 to C7 represent carbon numbers of the alkyl groups in alkylated polycyclic aromatic hydrocarbons. c The percentage of biodegradation/photooxidation was calculated by the eq 100-100 × (A/B), where A is the abundance of a compound in a photooxidized or biodegraded sample while B is that in its control sample. d Chloroform extract of photooxidized h-crude oil (ph-crude oil) was used for biodegradation. e Not applicable.

Silica gel column chromatography separated the h-crude oil into five fractions (Table 1) according to their differential solubility in organic solvents. The total recovery of h-crude oil after the column chromatography was found to be 99 ( 1%. Losses amounting to 2% and 5% of the initial weight were observed in the control samples for biodegradation and photooxidation, respectively, indicating that some evaporation of h-crude oil occurred during incubation for 8 and 4 weeks (Table 1). When the initial amount of each component in h-crude oil (the “h-crude oil” column in Table 2) was compared with that in the biodegradation control (Table 2) or that in the photooxidation control (Table 3), it became clear that the lower-molecular-weight components of h-crude oil were preferentially lost in these controls. The extent of loss was greater in the photooxidation control than

in the biodegradation control, probably because of the thinness of the oil layer in the photooxidation control and of the fact that the chamber for photooxidation was continuously ventilated to keep the temperature constant under the strong illumination. When h-crude oil which has been subjected to the photooxidation treatment was used for the biodegradation experiment, a very small decrease in weight (0.6%) was observed in the control sample (Table 1), indicating that the potentially volatile components in h-crude oil had mostly been evaporated during the photooxidation treatment. Gravimetric analyses show that 28% of h-crude oil had been biologically degraded in 8 weeks. This degree of the biodegradation was almost always observed using different seawater samples (29), and the reproducible biodegradation VOL. 34, NO. 8, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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was attained by the outgrowth of specific oil-degrading bacteria ubiquitously present in the seawater (20). The biodegradation transformed mainly the n-hexane and nhexane:benzene fractions, most of the biodegraded components being mineralized into CO2 and H2O (Table 1). Total ion chromatograms by GC indicated that the n-alkanes had disappeared after biodegradation, leaving a large hump or unresolved complex mixture consisting of highly branched alkanes, cyclic alkanes, T-branched alkanes, substituted/ unsubstituted aromatics, etc. (27, 31). GC-MS analyses in the SIM mode revealed almost complete depletion of alkanes and naphthalene derivatives and less extensive elimination of other components by biodegradation in the order of n-alkane > naphthalenes > branched alkanes > fluorenes > phenanthrenes > dibenzothiophenes. The aromatics with larger side chains were less susceptible to biodegradation (Table 2). Such an order of biodegradation has already been observed in previous studies (16). The photooxidation treatment gave a completely different result. The weight of the photooxidized oil increased slightly comparing to the weight of its control (Table 1). The increase by 1.5% of the photooxidized oil may partly be ascribed to the incorporation of oxygen atoms in the oxidized components of h-crude oil. This hypothesis was confirmed by FTIR analyses: a broad peak around 3000-3500 cm-1 most likely due to OH stretching vibrations appeared in the spectrum of photooxidized samples along with another major peak at 1711 cm-1 indicative of carbonyl function. A peak around 950-1200 cm-1 with a shoulder at 1030 cm-1 and a small peak around 600 cm-1 were also observed; they might be derived from sulfur containing compounds oxidized to sulfoxide, sulfone, and/or sulfate (32). Significant increases in the n-hexane-insoluble fraction (from 4.1% to 38.5% of the total amount) and in the methanol fraction (from 6.1% to 13.2%) were observed. These changes were mainly provoked by transformation of the n-hexane:benzene fraction whose content was reduced from 30% to 2.9% and of the n-hexane fraction (from 54% to 31.2%). It has long been believed that the n-hexane fraction only contains saturates. However, we found that one-third of its content was long side-chain alkylaromatics, the majority of these alkylaromatics being converted to polar fraction materials by photooxidation (manuscript in preparation). Thus, our observations were in agreement with those of Garrett et al. (12) who showed the resistance of saturates and the susceptibility of aromatics to photooxidation. After the photooxidation treatment, an increase in the concentrations of C14 to C24 n-alkanes, pristane, and phytane was observed with respect to the photooxidation control (Table 3). The standard errors of the data in two independent experiments were at a range of 5% or less, thus the increase in the concentrations of alkanes of chain length between 14 and 19 was significant. These alkanes may have been generated by photooxidative cleavage of the alkyl side chains of crude oil components as has been demonstrated by the photooxidation of nonylbenzene (23). The presence of aromatic compounds possessing a long alkyl side chain(s) has been reported in various crude oil samples (33-36) and more specifically in an UCM (27). These alkanes may have also been generated by the photooxidative decarboxylation of n-alkanoic acids or by the Norrish type I rearrangement via an alkyl radical (8) of ketones, which can be generated by the photooxidation of n-alkanes. GC-MS data (Table 3) also indicated an increase in the fraction of aromatics possessing a short alkyl side chain (see C0- and C1-dibenzothiophene and C0-phenanthrene). This increase was more apparent with a shorter incubation period (data not shown), indicating evaporative loss of these photoproducts with a longer incubation period. The mechanisms of the generation of these photoproducts are not 1504

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TABLE 4. Average Molecular Parameters of Crude Oil Samples Obtained by NMR Analysisa 1H

h-crude oil biodegraded h-crude oil photooxidized h-crude oil photooxidized and biodegraded h-crude oil

13C

NMR

NMR

Hali

Haro

Hali:Haro

Cali

Caro

0.948 0.940 0.972 0.965

0.052 0.060 0.028 0.035

18.2:1 15.7:1 34.7:1 27.6:1

0.845 0.824 0.926 0.910

0.155 0.176 0.074 0.090

a H , ratio of aliphatic hydrogen; H ali aro, ratio of aromatic hydrogen: Cali, ratio of aliphatic carbon; and Caro, ratio of aromatic carbon.

FIGURE 2. UV-visible absorption spectra of h-crude oil (A), biodegraded h-crude oil (B), photooxidized h-crude oil (C), and photooxidized and biodegraded h-crude oil (D). The concentration of oil components in each sample was 20 µg/mL. known. However, the photochemical oxidation of dimethylnaphthalene to its alcohol, aldehyde, and carboxylate derivatives has previously been reported (37). On the other hand, the photocatalytic decarboxylation of n-alkanoates has also been reported (8, 38). Therefore, the photooxidation of methylphenanthrene or methyldibenzothiophene to its carboxylate derivative followed by the photocatalytic decarboxylation may be one of the possible mechanisms for the photochemical generation of phenanthrene or dibenzothiophene. When the photooxidized oil was subjected to biodegradation, 36% in weight of its components was eliminated (Table 1). Continuation of the biodegradation process for a further 8 weeks brought about an additional reduction of 2-3% in weight, indicating that most of the susceptible components had been biodegraded in the first 8 weeks. The biodegradation of the photooxidized h-crude oil resulted in a major reduction in the n-hexane fraction and the n-hexaneinsoluble fraction. The results of the 1H and 13C NMR analyses are summarized in Table 4. The decrease in the ratio of aliphatic to aromatic protons in the biodegraded sample indicates the preferential biodegradation of saturates. On the contrary, the increase in this ratio in the photooxidized sample may mainly have been due to photooxidative cleavage of the aromatic ring. The 13C NMR results supported such assumptions. A sharp decrease in the aromaticity by the photooxidation treatment is also verified by the UV-visible spectra shown in Figure 2. The light absorption by h-crude oil at a wavelength of 300 nm, which is primarily due to conjugated aromatic molecules, was reduced in the photooxidized samples. In summary, it was observed that the biodegradation of h-crude oil led to the mineralization of more than 40% of the n-hexane fraction and to about one-third of the hexane: benzene fraction, while the photooxidation treatment transformed most of the aromatics to polar fractions. On the other hand, photooxidation increased the biodegradative potential, leading to 36% of biological mineralization in contrast to a

28% figure without photooxidation. Thus, in a natural marine environment, biological and photochemical reactions would mutually stimulate the degradation of crude oil components. Further studies are required to learn the fate of the more recalcitrant components of crude oil in a natural marine environment.

Acknowledgments This work was supported by New Energy and Industrial Technology Development Organization (NEDO).

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Received for review September 15, 1999. Revised manuscript received January 3, 2000. Accepted January 7, 2000. ES991063O

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