Environ. Sci. Technol. 1998, 32, 3618-3621
Vanadium as an Internal Marker To Evaluate Microbial Degradation of Crude Oil TETSUYA SASAKI, HIDEAKI MAKI, MASAMI ISHIHARA, AND SHIGEAKI HARAYAMA* Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi City, Iwate 026-0001, Japan
17R,21β-Hopane is used as an internal marker to evaluate the biodegradation and/or weathering of petroleum products. In this study, vanadium, the most abundant heavy metal in crude oil, was investigated as an alternative internal marker to 17R,21β-hopane. It was demonstrated that the amounts of dichloromethane-extractable vanadium and 17R,21β-hopane were not significantly reduced even after an intensive biodegradation of crude oil. Thus, vanadium was considered to be a possible internal marker for the evaluation of the biodegradation and/or weathering of crude oil. This assumption was tested in beach-simulating tanks where crude oil was biodegraded under conditions that mimic a natural beach environment. Crude oil in the tanks was extracted at appropriate times after the subjection to biodegradation, and the concentrations of various crude oil components including those of vanadium and 17R,21β-hopane were determined. The concentration changes of the crude oil components determined on the basis of the vanadium concentration agreed well with those determined on the basis of the concentration of 17R,21β-hopane. It was concluded that vanadium could be used as an internal marker of crude oil for the estimation of biodegradation and/or weathering.
Introduction A large accidental oil spill such as the Exxon Valdez incident in the Prince William Sound in 1989 does not occur frequently; however, it can cause a significant destruction of ocean and shoreline environments (1). Techniques dealing with oil pollution include physical, chemical, and biological methods. The most familiar biological method, known as bioremediation, involves the addition of nutrients to encourage microbiological degradation of oil. For the bioremediation of polluted shorelines, it is important to monitor the extent of the biodegradation of spilled oil to evaluate its effectiveness. Changes in oil composition have been used to estimate the extent of biodegradation of spilled oil. Historically, this has been done by determining the weight ratio between readily biodegradable C17 and C18 n-alkanes and less biodegradable branched alkanes, pristane and phytane (2-6). However, this technique is only useful in early stages of the oil biodegradation before the branched alkanes are completely degraded (7). Then, 17R,21β-hopane, which is a trace oil component, was identified to be an excellent marker (8-10): compositional changes of several oil components relative to * Author for correspondence. Telephone: +81-193-26-6544; fax: +81-193-26-6592; e-mail:
[email protected]. 3618
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FIGURE 1. Beach simulating tank. Seawater is introduced into the reservoir where the temperature is controlled at 20 °C. Air-saturated seawater in the reservoir is subsequently introduced into the main tank filled with gravel. The seawater in the main tank is then introduced into the level controller from which excess seawater overflows. 17R,21β-hopane are believed to provide the extent of oil biodegradation since the latter compound is biodegraded with the rates extremely slow as compared with most other hydrocarbon components (7, 11). Nevertheless, 17R,21βhopane may not be the universal marker for the biodegradation of oil as the content of this compound in some oil samples exemplified by heavy oil is very low: the estimation of the degree of biodegradation based on the amount of 17R,21βhopane in such a sample may accompany a large error. Furthermore, there is some evidence that 17R,21β-hopane is biodegraded (12). Therefore, an additional marker to 17R,21β-hopane may be needed to ascertain the evaluation of biodegradation of oil. Vanadium is one of the most abundant metals in crude oil and is thought to exist largely as porphyrin complexes. It is mostly found in the heavier fraction of oil in a process of petroleum refining (13, 14). In this study, we examined the validity of vanadium as an internal marker of crude oil for the evaluation of biodegradation and/or weathering.
Experimental Section Materials. Arabian light crude oil heated to 230 °C, denoted simply as crude oil hereafter, was used in this study. A slowrelease solid granular nitrogen and phosphorus fertilizer, Super IB and Linstar, respectively, were purchased from Mitsubishi Chemicals. Super IB and Linstar contain 35% (w/w) nitrogen as isobutylidene diurea (IBDU) and 35% (w/ w) phosphorus as calcium and/or magnesium phosphate salts, respectively. These were essentially vanadium-free. Natural seawater was collected from the Kamaishi Bay at a depth of 15 m. All chemicals used were of the highest available purity and vanadium-free. Batch Cultures. For batch cultures, 500-mL “Sakaguchi” flasks (Iwaki Glass) containing 200 mL of nonsterilized seawater supplemented with 0.8 g of crude oil, 0.5 g of the nitrogen fertilizer, and 0.1 g of the phosphorus fertilizer were incubated at 20 °C for 45 days with shaking at 100 strokes/ min. In our previous experiments (15), it has been demonstrated that natural seawater contains oil-degrading microbial populations. Beach-Simulating Tanks. A pair of tanks that simulates the intertidal shore consists of a double-walled plastic tank, a reservoir, and a level-controlling device (Figure 1). The 10.1021/es980287o CCC: $15.00
1998 American Chemical Society Published on Web 09/19/1998
TABLE 1. Concentration of Heavy Metals in Crude Oil and Seawater elements
(mg/kg)a
oil seawater (mg/mL)c
V
Cr
Mn
21.61 NDd
NDb
NDb
NDd
NDd
Fe 5.99 0.016
Ni
Cu
Zn
2.91 NDd
NDb
NDb 0.022
NDd
a Measured by ICP-ES; mean values of duplicate determination. Under the detection limit that was typically under 0.008 mg/L. c Measured by atomic absorption spectrometry. d Under the detection limit that was typically under 0.005 mg/L. b
tanks whose internal size was 1 m large, 1 m wide, and 1.5 m high were filled with 1 m3 gravel. The gravel used was rubble of rocks (2-8 mm in diameter) and contained approximately 30% magnetite. Natural seawater filtered through layers of course and fine sand was continuously added to the reservoir at a flow rate of 60 L/h. Seawater in the reservoir was aerated by bubbling and temperaturecontrolled at 20 °C. The level of seawater in the tanks was controlled by the level controller. An outlet of seawater in the level controller moved between 40 and 120 cm above the bottom of the tanks realizing two cycles per day of the ebb and flow in the beach-simulating tanks (16). At the beginning of the experiment, 1 kg of crude oil was poured in each tank on the surface of seawater at a high tide level, and then 200 g of the nitrogen fertilizer, and 40 g of the phosphorus fertilizer were added in one (bioremediated) tank but not in the other (control) tank. Within 2 days, the crude oil uniformly spread on the surface of seawater and gradually adhered to the surface of gravel. Analysis of Crude Oil. Crude oil was extracted from cultural fluids with dichloromethane, and dichloromethane was evaporated at 50 °C. For the sampling of crude oil from the beach-simulating tanks, the surface area of gravel in the tanks was divided into several blocks. Gravel covered with crude oil was sampled from the upper gravel layers (0-15 cm) of randomly chosen blocks and subsequently subjected to Soxleht’s extraction by recycling dichloromethane. Resulting dichloromethane extracts were dehydrated by the addition of dehydrated sodium sulfate and concentrated by using a rotary evaporator. Concentrated samples were heated to 50 °C to evaporate solvent completely in an incubator overnight. The dried extracts were dissolved in a determined volume of dichloromethane and subsequently subjected to analyses by the following methods. Gravimetry. The weights of dried samples were determined gravimetrically. Thin-Layer Chromatography/Flame-Ionization Detection (TLC/FID). The analytical procedures and instrumentation for the TLC/FID analysis used in this study have been described elsewhere (17). Gas Chromatography/Mass Spectrometry (GC/MS). The GC/MS instrument was operated in the selective ion monitoring (SIM) mode. The column temperature was increased at 6 °C/min from 50 to 300 °C. The carrier gas was helium (1 mL/min).
Inductively Coupled Plasma Emission Spectrometry (ICP-ES). ICP-ES analysis was performed using ICPS-1000III (Shimadzu) fitted with a plasma-torch for organic solvents. A total of 0.5 g of sample oil was dissolved into 10 mL of xylene, and the solution was analyzed at the wavelength of 311.071 nm for vanadium, which was selected to avoid interference with other metals such as magnesium in the phosphorus fertilizer and seawater. Conostan S-21 (Conostan) was used as the standard sample for calibration. Atomic Absorption Spectrometry. Atomic absorption spectrometry analysis was done according to the method of JIS-K-0102 (18).
Results and Discussion Change in the Concentrations of Vanadium and 17r,21βHopane after the Biodegradation of Crude Oil. Table 1 shows the concentrations of heavy metals in crude oil. The oil contained vanadium most, followed by iron and nickel. This result is consistent with those in the previous works that showed vanadium to be most abundant in various oil samples (19). Table 1 also shows that the concentration of vanadium in natural seawater was much lower than that in crude oil. On the basis of these results, we examined the possibility of using vanadium as an internal marker of crude oil to evaluate the degree of its biodegradation and/or weathering. In batch culture experiments, 0.8 g of crude oil was put in each flask, and the flask was incubated for 45 days. In control cultures where no nutrient was added, neither the growth of bacteria nor the emulsification of crude oil was observed. From these cultures, 95% of crude oil initially added to the flasks was recovered. The 5% loss in weight of crude oil in the control cultures may be due neither to the biodegradation nor the evaporation of crude oil because no bacterial growth was observed in these cultures, and the crude oil was heated at 230 °C prior to use. Thus, we expected that the extraction efficiency of crude oil by the present method is around 95%. In the biodegraded cultures where fertilizers were added, the growth of bacteria and the emulsification of crude oil were observed. From the cultures, less than 50% of the initial weight of crude oil was recovered. The recovery of vanadium and 17R,21β-hopane, in contrast, was high, being more than 90% as compared to the recovery of these compounds from the control cultures (Table 2). Since the extraction of crude oil from emulsified samples was more difficult than nonemulsified samples, we interpreted that 90% recovery of vanadium and 17R,21β-hopane from the biodegraded cultures may mainly be due to the lower extraction efficiency, although the biodegradation to a small extent of 17R,21βhopane and ligands of vanadium was not ruled out. These observations suggested that both vanadium and 17R,21βhopane are essentially not degraded by naturally occurring oil-degrading microorganisms. The extent of the biodegradation of crude oil in the biodegraded cultures estimated on the basis of the vanadium and 17R,21β-hopane concentrations was 41% and 40%, respectively. Biodegradation of Crude Oil in Beach-Simulator Tanks Estimated on the Basis of the Vanadium and 17r,21β-
TABLE 2. Amount of Vanadium and 17r(H),21β(H)-Hopane per Flask in Batch Cultures
initial controlc biodegradedd
recovery of oil (%)a,b
vanadium (mg/flask)a
17r(H),21β(H)-hopane (peak area/flask)a
vanadium
100 94.0 48.7
17.40 16.46 ( 0.43 15.05 ( 0.41
940 226 889 076 ( 24 037 809 137 ( 29 060
100 94.6 86.5
recovery (%)b 17r(H),21β(H)-hopane 100 94.6 86.1
a Mean values of triplicate determination except for the initial oil sample. b Relative to the initial amount of oil. c No fertilizer was added to the flasks. d Nitrogen and phosphorus fertilizers were added to the flasks in addition to crude oil.
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FIGURE 2. Time-course of the changes in the amounts of vanadium and 17r,21β-hopane per fixed weight of crude oil in the control and bioremediated tanks. The initial concentrations of vanadium and 17r,21β-hopane were taken as 100%. Hopane Concentrations. We also examined the changes in the concentrations of vanadium and 17R,21β-hopane in crude oil retained in the beach-simulator tanks (Figure 2). The concentrations of vanadium and 17R,21β-hopane relative to the weight of crude oil increased by 70% in the bioremediated tank while these were almost constant in the control tank. From these data, it was demonstrated that the biological removal of vanadium from crude oil, if it occurs, is as slow as that of 17R,21β-hopane. It has been reported that vanadium at its high concentration in seawater is incorporated into oil (19). Under such circumstances, an overestimation of the extent of biodegradation would result. However, it is unlikely that the mass transfer of vanadium from seawater to crude oil occurred in the present experiments because the vanadium concentrations relative to the weight of crude oil were constant and parallel to the concentrations of 17R,21β-hopane in the control tank. The vanadium concentration in seawater is generally quite low being between 1 and 2 µg/L (20). The concentrations of GC/MS-detectable compounds of crude oil sampled from the bioremediated tank were normalized either by the concentrations of vanadium or those of 17R,21β-hopane (Figure 3). There was no significant difference between the values normalized by the vanadium concentrations and those by the 17R,21β-hopane concentrations. Figure 4 shows the results of TLC/FID analyses of crude oil sampled from the bioremediated tank. The biodegradation of the saturated and aromatic components of crude oil estimated by the vanadium normalization and that by the 17R,21β-hopane normalization were essentially identical. Both methods clearly indicated that the saturated components of crude oil were more susceptible to biodegradation than the aromatic components. The results obtained in this study demonstrated that a strong correlation between the concentrations of vanadium and those of 17R,21β-hopane in oil samples exist (r ) 0.99) and thus that the normalization by vanadium is as reliable as that by 17R,21β-hopane for the estimation of biodegradation and/or weathering of crude oil. It is known that vanadium tends to adsorb onto iron oxides (21). Such a property of the metal, however, may not influence its retainability in crude oil as the mass transfer of vanadium from crude oil to iron-rich gravel was not observed in the present study. 3620
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FIGURE 3. Time-course of the changes in the concentrations of GC/MS-detectable compounds in crude oil sampled from the bioremediated tank. The concentrations of GC/MS-detectable compounds were normalized either by the concentrations of vanadium or by those of 17r,21β-hopane. The normalized concentrations at day 0 were taken as 100%.
FIGURE 4. Time-course of the changes in the concentrations of saturates and aromatics in crude oil sampled from the bioremediated tank. The concentrations of saturates and aromatics were determined by TLC/FID, and these were normalized either by the concentrations of vanadium or by those of 17r,21β-hopane. The normalized concentrations at day 0 were taken as 100%. As mentioned earlier, vanadium is complexed with ligands in crude oil and fractionated in heavier fractions of oil. On the other hand, 17R,21β-hopane is fractionated in the saturated fraction of oil. The contents of vanadium and 17R,21β-hopane varied in different crude oils and in different petroleum products. The concentration of vanadium is generally high in crude oils derived from marine depositions, e.g., Arabian and North Sea oils (22), while the concentration
of 17R,21β-hopane was quite low in certain heavy oil samples (our unpublished data). Thus, vanadium may be an useful internal marker for many petroleum products containing a high concentration of vanadium and a low concentration of 17R,21β-hopane.
Acknowledgments We are thankful to Etsuro Sasaki for his technical help. This work was supported by the New Energy and Industrial Technology Development Organization.
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(9) Wang, Z.; Fingas, M.; Li, K. J. Chromatogr. Sci. 1994, 32, 367382. (10) Prince, R. C.; Elmendorf, K. L.; Lute, J. R.; Hsu, C. S.; Halth, C. E.; Senius, J. D.; Dechert, G. J.; Douglas, G. S.; Butler, E. L. Environ. Sci. Technol. 1994, 28, 142-145. (11) Bragg, J. R.; Prince, R. C.; Harner, E. J.; Atlas, R. M. Nature 1994, 368, 413-418. (12) Prince, R. C.; Drake, E. N.; Madden, P. C.; Douglas, G. S. In In situ and on-site bioremediation: volume 4; Alleman, B. C., Leeson, A., Eds.; Battelle Press: Columbus, OH, 1995; pp 205210. (13) Matar, S.; Hatch, L. F. In Chemistry of Petrochemical Process; Gulf Publishing Co.: Houston, TX, 1994; pp 18-19. (14) Leffler, W. L. In Petroleum Refining, 2nd ed.; Penn Well Publishing Co.: Tulsa, OK, 1985; pp 115-116. (15) Venkateswaran, K.; Harayama, S. Can. J. Microbiol. 1995, 41, 767-775. (16) Ishihara, M.; Sugiura, K.; Asaumi, M.; Goto, M.; Sasaki, E.; Harayama, S. In In situ and on-site bioremediation: volume 3; Alleman, B. C., Leeson, A., Eds.; Battelle Press: Columbus, OO, 1995; pp 101-116. (17) Goto, M.; Kato, M.; Asaumi, M.; Shirai, K.; Venkateswaran, K. J. Mar. Biotechnol. 1994, 2, 45-50. (18) Japanese Standard Association. Standard methods for the examination of chemicals; Japanese Standard Association: 1992. (19) Barnea, J.; Omana, R.; McDavid, R.; Yen, T. F. In 3rd International Conference Heavy Crude and Tar Sands; Long Beach, CA, July 1985; pp 393-402. (20) Pettine, M.; Mastroianni, D.; Camusso, M.; Guzzi, L.; Martinotti, W. Mar. Chem. 1997, 58, 335-349. (21) Trefry, J. H.; Metz, S. Nature (London) 1989, 342, 531-533. (22) Barwise, A. J. G. Energy Fuels 1990, 4, 647-652.
Received for review March 24, 1998. Revised manuscript received July 20, 1998. Accepted August 3, 1998. ES980287O
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