Marine Pollution Bulletin xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Studies on crude oil removal from pebbles by the application of biodiesel Wen-xiang Xia a, Yan Xia a, Jin-cheng Li a,⇑, Dan-feng Zhang a, Qing Zhou b, Xin-ping Wang b a
College of Environmental and Municipal Engineering, Qingdao Technological University, Qingdao 266033, China Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, North China Sea Environmental Monitoring Center of State Oceanic Administration, Shandong, Qingdao 266033, China b
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Bioremediation Petroleum contamination Pebble Biodiesel Nutrients
a b s t r a c t Oil residues along shorelines are hard to remove after an oil spill. The effect of biodiesel to eliminate crude oil from pebbles alone and in combination with petroleum degrading bacteria was investigated in simulated systems. Adding biodiesel made oil detach from pebbles and formed oil–biodiesel mixtures, most of which remained on top of seawater. The total petroleum hydrocarbon (TPH) removal efficiency increased with biodiesel quantities but the magnitude of augment decreased gradually. When used with petroleum degrading bacteria, the addition of biodiesel (BD), nutrients (NUT) and BD + NUT increased the dehydrogenase activity and decreased the biodegradation half lives. When BD and NUT were replenished at the same time, the TPH removal efficiency was 7.4% higher compared to the total improvement of efficiency when BD and NUT was added separately, indicating an additive effect of biodiesel and nutrients on oil biodegradation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Oil spills are one of the major reasons to ocean pollution, causing dramatic damages to the sea and shorelines. The spilled oil may persist in coastal areas for years, transport through the food chains (Lee, 1976; Teal et al., 1992) and exhibit long-term toxicity (Southward, 1982). Oil stranded on rocky shorelines is extremely difficult to remove especially since the shorelines are lack of seawater washing. A layer of fuel oil may stick firmly to the surface of rocks or pebbles due to the accumulation of the recalcitrant components and act as a slow-release source of contamination. High pressure flushing with hot water has been used in shorelines affected by the Prestige oil spill. Nevertheless, it is labor intensive and time consuming, and a large amount of wastes was produced with high energy consumption (Fernández-Álvarez et al., 2006). Bioremediation has the advantage of low cost and high efficiency under favorable conditions without causing environmental damage (Beolchini et al., 2010; Mohajeri et al., 2010; Pritchard and Costa, 1991; Venosa and Zhu, 2003; Wang et al., 1998). It is an efficient alternative for the removal of oil from rocks or pebbles by physical means. Petroleum degrading bacteria (PDB) are ubiquitous in the marine environment (Atlas, 1993). They require not only carbon
⇑ Corresponding author. Tel.: +86 532 85071262; fax: +86 532 85015375. E-mail address: [email protected] (J.-c. Li).
but also nitrogen and phosphorus for incorporation of biomass. The application of nutrients has proven to be an effective way to stimulate oil biodegradation in the bioremediation of petroleum contaminated shorelines (Santas et al., 1999; Venosa et al., 1996). However, mineral oil attached to the pebbles has low water solubility, leading to difficulties for microbes to utilize it as an energy source. Therefore, the bioavailability of petroleum hydrocarbons to oil degraders is a major factor influencing the bioremediation effect. Biodiesel is composed primarily of fatty acid methyl esters. It is an environmentally benign addition since it is readily degraded by microorganisms (Makareviciene and Janulis, 2003; DeMello et al., 2007) and less toxic than mineral oils (Birchall et al., 1995). In recent years, biodiesel has been used to eliminate petroleum contamination in laboratory and field tests. For example, Taylor and Jones (2001) reported that the addition of biodiesel to soil containing coal tar increased the degradation of coal tar polycyclic aromatic hydrocarbons (PAHs), which was ascribed to tar solubilization and dispersion thereby increasing the PAHs bioavailability. Fernández-Álvarez et al. (2006) applied biodiesel for removal of crude oil that contaminated rocks and sand after the Prestige oil spill. They found it not only accelerated the clean-up of the polluted surface but also enhanced the degradation of the residual oil. However, neither the added microorganisms nor the nutrients accelerated the degradation rate of the residual oil in the affected shorelines in their studies, which was quite different from other reports (Hozumi et al., 2000; Oh et al., 2001).
http://dx.doi.org/10.1016/j.marpolbul.2014.11.026 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
2
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
On the whole, the removal of petroleum contamination by the addition of biodiesel is both a physicochemical and a biochemical process. These dual actions were discussed in previous studies (Taylor and Jones, 2001; Fernández-Álvarez et al., 2006). However, the role of biodiesel played alone on the removal of fuel oil was unclear. Furthermore, when biodiesel was applied together with nutrients, their combined influence on the hydrocarbon degrading microorganisms need to be further explored. Laboratory studies are valuable in assessing the effectiveness of a shoreline cleaning strategy since it is not practical to replicate the true conditions of the shoreline environment (Mearns, 1997; Santas et al., 1999). In this study, we use a simulated system to explore the effect and mechanism of biodiesel on removing oil from shorelines. First, biodiesel (BD) was applied to crude oil contaminated pebbles in a sterilization system without inoculum (INO) in order to study the physiochemical effects of biodiesel alone. Secondly, with INO present in the system, BD, nutrients (NUT) and BD + NUT were replenished respectively to study the biochemical effects of biodiesel in combination with oil degrading bacteria. This article describes the results of our experimental studies. 2. Materials and methods 2.1. Samples Clean seawater collected from Jinsha Beach near Qingdao, China was filtered through 0.22 lm membrane to eliminate the impact of micro-organisms. Pebbles (diameter 4–5 cm) collected from Qingdao Huiquan Bay were soaked with hydrochloric acid and sterilized before use. Crude oil samples were purchased from Huangdao oil depot. The density, kinetic viscosity and API value of the oil at 20 °C was 0.987 (g/cm3), 86.55 (mm2/s) and 11.9 respectively. In order to avoid the influence of oil volatilization, the crude oil was weathered for 4 weeks in a fume hood before use, losing 10.8% of its initial weight. Biodiesel in the form of rapeseed oil methyl esters was purchased from local suppliers in Qingdao, China. The density and kinetic viscosity at 20 °C was 0.836 (g/cm3) and 5.6 (mm2/s) respectively. 2.2. Microorganism acclimatization Since no single strain of bacteria has the metabolic capacity to degrade all components found in crude oil, a mixture of bacterial suspension was used in the experiment. A medium containing 1 g/L NH4NO3, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.2 g/L MgSO47H2O, 0.05 g/L FeCl3, and 0.02 g/L CaCl2 was used to culture bacteria (Dutta and Harayama, 2000; Mohajeri et al., 2010). Oil-polluted seawater containing oil degrading bacteria was collected from Dagang Wharf No. 6 in Jiaozhou Bay was added to 100 mL of medium and incubated on a rotary shaker at 25 °C, 200 r/min for 2 weeks. Then enriched solution was added to fresh medium and incubated at 25 °C for 1 week. To obtain standard inoculum, the mixed cells were harvested by centrifugation, rinsed three times in sterile saline before being re-suspended in sterile liquid basal medium to yield an absorbance reading of 0.5 at 540 nm.
with a flow control device was inserted through holes around the containers at a height of 0, 5 and 10 cm from the top of the pebbles to allow seawater to be drained through the system and analyzed (Fig. 1). The containers were placed indoor and the temperatures is 20 °C. In order to mimic the washing of the wave and replacement effect of seawater, fish pump with 4 outlets was installed under the bottom of the pebbles, and seawater (800 mL) was flushed and drained through the holes with the same amount of fresh seawater replenished every day after the seawater samples were taken. 2.3.1. Effects of biodiesel on residual oil clean-up without hydrocarbon degraders Four containers were used in each group, one for control, and three for experiments which were treated with biodiesel. To each of the containers, 20 g weathered crude oil was applied to the surface of the pebbles as uniformly as possible to simulate pollution density occurred along Qingdao shorelines in 2013. Results from our previous studies (dada not shown) indicated the earlier the biodiesel was added, the higher the oil removal efficiency. Since it needs some time for the response action to take place when large area of pollution occurs in real situations, 3 h is chosen in our experiment to mimic real situations and biodiesel (6, 12 and 18 mL) was sprayed respectively in 3 experimental tests. The washing effect of the tide was simulated by filling the container with seawater until it submerged the pebbles with fish pump switching on and off. No biodiesel was added in the control. Seawater samples (20 mL) containing the biodiesel-oil mixture was taken at different position from sample outlets in 1, 2, 3, 4, and 7 days after the addition of the biodiesel, and 4 samples at the same height were mixed and treated to analyze the total petroleum hydrocarbon (TPH) content. Seawater 240 mL was replenished in the container after sampling. When the experiment ended, oil remaining on pebbles was analyzed. Another two groups were used as parallel experiments and the above process was repeated three times. 2.3.2. Effects of biodiesel on residual oil biodegradation in the presence of hydrocarbon degraders In order to study the subsequent biodegradation of oil detached from pebbles after the addition of biodiesel, five containers were prepared as a group, 1 control and 4 experimental. The arrangements are shown in Table 1. Crude oil contaminated pebbles are prepared as previously mentioned. One control test was set up with only seawater added, and 4 experimental tests were prepared with the addition of inoculum (INO) as well as biodiesel (BD), nutrients (NUT) or BD + NUT (Table 1). NO3–N and PO4–P were amended according to the results of former study (Xia et al., 2005), and the N:P ratio is 10:1. Oxygen was supplied by fish pump in order to simulate the aerobic condition along shoreline and make inoculum distribute evenly, and the dissolved oxygen (DO) of the seawater varied between 3.8 and 4.5. Seawater samples (20 mL) in water surface were obtained from each outlets on day 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 following the addition of the biodiesel, and 4 seawater samples from different positions were mixed and prepared to analyze TPH, petroleum degrading bacteria (PDB), dehydrogenase activity (DHA) and DO. When the experiment ended, the residual oil still attached to the pebbles was analyzed. Another two groups were used as parallel experiments and the above process was repeated three times.
2.3. Experimental design 2.4. Sample analysis Plastic containers (400 260 200 mm) were prepared and sterilized pebbles were put into them, and seawater was poured into the container to attain a height of 150 mm which is about 10 mm higher than the top of pebbles. Twelve plastic tubes fitted
2.4.1. Total petroleum hydrocarbon (TPH) in seawater The mixture of biodiesel and crude oil in the seawater were extracted using carbon tetrachloride. The biodiesel–oil in carbon
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
3
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Fig. 1. Schematic diagram of experimental set-up.
Table 1 Experimental design for biodegradation tests.
Control INO INO + BD INO + NUT INO + BD + NUT
Crude oil (g)
Inoculum (mL)
Biodiesel (mL)
Nutrients (lmol/L)
20 20 20 20 20
0 100 100 100 100
0 0 12 0 12
0 0 0 500 NO3–N + 50 PO4–P 500 NO3–N + 50 PO4–P
tetrachloride was then passed through adsorption column loaded with magnesium silicate. The hydrocarbons of the crude oil passed through the adsorption column whilst the biodiesel was retained. Then total petroleum hydrocarbon (TPH) in crude oil was determined by Oil Analyzer of infrared spectrometer (Jilin BeiGuang Optical Instrument Factory, China). 2.4.2. Removal efficiency (%) of TPH from pebbles At the end of the experiment, all pebbles were put into a beaker containing carbon tetrachloride and extracted twice. The extracts were combined and diluted for gravimetric analysis of TPH as previously stated. TPH removal efficiency (%) from pebbles was calculated as follows:
TPH removal efficiencyð%Þ ¼ ðTPHcont TPHres Þ=TPHcont 100% where TPHcont – TPH in control group; TPHres – TPH residues in test group. 2.4.3. Microbial enumeration The most probable number (MPN) technique was used to estimate the number of petroleum degrading bacteria (PDB) in seawater in all treatments. Bushnell–Hass medium supplemented with 2% (w/w) NaCl was used as the growth medium for PDB. Tubes were inoculated with 1 mL of serially diluted samples and then incubated for enumeration of PDB for two weeks at room temperature before being scored for positive growth with the Sheen Screen test for crude oil degraders. A corrected most probable number table was used to estimate microbial numbers (Xia et al., 2012). 2.4.4. Dehydrogenases activity (DHA) measurement Triphenyl tetrazolium chloride (TTC) is an artificial electron acceptor for several dehydrogenases. It is water soluble and nearly all microorganisms reduce it to TPF, which can be colorimetrically estimated (Alef, 1995). DHA was measured using UV–VIS spectrophotometer (UV759S, Shanghai Jingke Instrument Company, Ltd.) as follows: Samples from seawater were added to 10 mL centrifuge tubes containing a 0.1% TTC aqueous solution, and then incubated for 48 h at 30 ± 1 °C in the dark. The hydrolysis reaction product (TPF) was extracted for 2 h at 30 °C with acetone and absorbance
at 482 nm was recorded (Rogers and Li, 1985). Data are expressed as lg TPF/mL/h. 2.4.5. Other chemical analysis DO and pH were measured by HACH sension™ 156 Multiparameter Meters. NO3–N and PO4–P was measured using cadmium–copper reduction method and phosphomolybdenum blue method (State Oceanic Administration of China, 1998) respectively. 2.5. Statistical analysis The results were subjected to one way analysis of variance (ANOVA) to determine if mean values of TPH removal efficiency, PDB as well as DHA in the controls and experimental tests differed significantly. Data were considered to be significantly different between the three treatments if P < 0.05. All statistical analyses were performed using Excel 2003. 3. Results and discussion 3.1. The distribution of TPH in different heights of seawater with varied amount of biodiesel Biodiesel was added in 0 (control), 6, 12 and 18 mL quantities on oil polluted pebbles with no hydrocarbon degraders in the system. The variations of TPH on day one in different height of the seawater is shown in Fig. 2 (the trend in other days were the same and the data were not shown). The addition of biodiesel increased the concentration of TPH in seawater more than 10 times compared to control (Fig. 2), and the differences between control and other treatments are statistically significant (P < 0.05). For example, the concentration of TPH at the surface seawater increased as high as 13.23–19.27 mg/L, while in control test this value was only 1.18 mg/L indicating biodiesel can make large amounts of residual oil detach from pebbles. Results also indicated the lower part in seawater contained less TPH than the surface water regardless how much biodiesel was added in the container (Fig. 2). For example, when 18 mL of biodiesel was added, the TPH in surface seawater was 19.27 mg/L while
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
4
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Fig. 2. The average value of TPH (mg/L) in different height of the seawater on day one with varied biodiesel addition.
the value was 3.88 and 2.25 mg/L respectively beneath the surface at 5 cm and 10 cm. The same results were reported by Pereira and Mudge (2004) that only small amounts of crude oil and biodiesel (3–14.5%) accumulated at depths below 5 cm. This is because biodiesel has lower viscosity and higher buoyancy than crude oil. When biodiesel was added to the oil-polluted pebbles, crude oil was released from pebbles and dissolved into biodiesel. The oil– biodiesel mixture had higher mobility and most of them rose to the surface of seawater. Because of this, only TPH in surface seawater was investigated in later experiments in this study. In the experimental tests, the addition of 6 mL biodiesel resulted in 13.23 mg/L of TPH in surface seawater, which was remarkably less than the addition of 12 mL and 18 mL of biodiesel where TPH values was 18.21 mg/L and 19.27 mg/L respectively. These experiments indicated that the more the biodiesel was added to the system, the more oil detached from pebbles, with 18 mL of biodiesel giving the best result. Results from the above treatments showed that when no petroleum degrading bacteria were present, biodiesel had a considerable capacity to detach crude oil from pebbles and form an oil–biodiesel mixture which can eventually be removed by water with little remained on the pebbles. Similar results were found in other studies (Wedel, 2000; Miller and Mudge, 1997; Fernández-Álvarez et al., 2007) in their studies. However, the relationship between the BD addition and the clean-up efficiency of crude oil from pebbles need to be further explored. 3.2. Influence of the biodiesel volume on the removal of residual oil from pebbles After 7 days, seawater in the containers was emptied and the crude oil remained on pebbles was extracted and analyzed after flushing the oil slick. Oil removal efficiency was calculated in
Fig. 3. Comparison of TPH removal efficiency (%) from pebbles with varied biodiesel volume after day 7. Error bars represent mean ± one standard deviation (n = 3).
Fig. 4. The variations of TPH in surface seawater. Error bars represent mean ± one standard deviation (n = 3).
accordance with the biodiesel volume and the results were shown in Fig. 3. The treatments significantly differed from the control (P < 0.01) and the removal effect of biodiesel to eliminate residual oil from pebbles appeared to be dependent on the volume of biodiesel sprayed. In the experiment, 6 mL biodiesel (biodiesel/oil = 30%, v/v) significantly increased the TPH removal efficiency to 30.1% compared to the control where no biodiesel was used (P = 0.005). When additional 6 mL biodiesel was applied in the system, the increase of TPH removal efficiency is 13.1% higher than the 6 mL treatment (P = 0.005). When 18 mL biodiesel was used, the increase of TPH removal efficiency is only 4.5% higher compared to the 12 mL treatment (P = 0.004). These results indicate that higher ratio of biodiesel to crude oil resulted in greater removal rate of oil from pebbles but the increase rate slowed down quickly. Adding more biodiesel to the system may reach a plateau in TPH removal efficiency. In practice, the volume of biodiesel applied to clean oil from pebbles depends on the pollution intensity and economic feasibility. More work need to be done to determine the optimal application amount of biodiesel to remove residual oil for a particular shoreline situation. 3.3. Variations of chemical and microbial parameters during biodegradation In the first experiment, most of the oil on the pebbles detached from pebbles after the application of biodiesel and formed oil– biodiesel mixture floating in seawater. Since petroleum degrading bacteria are ubiquitous along shorelines (Atlas, 1993), the elimination of residual oil rely largely on the biodegradation process. Spilled oil constitutes a carbon source and by adding N and P nutrients can promote the biodegradation of petroleum (Bragg et al., 1994; Swannell et al., 1996; Xia et al., 2005). In the next experiment, petroleum degrading bacteria expressed as inoculum (INO) were applied in four experimental groups besides the control group. Then biodiesel (BD), nutrient (NUT) and biodiesel + nutrient (BD + NUT) were added respectively in the systems in order to explore their effect on oil biodegradation. 3.3.1. The variations of TPH Results showed that in the three tests (Control, INO and INO + NUT) where no biodiesel was added, about 8 mg/L TPH was obtained on day 5 in seawater (Fig. 4). However, this value increased approximately to 27 mg/L in the two tests (INO + BD and INO + BD + NUT) on day 5 where biodiesel was added. This may be due to the fact that most of the crude oil was still attached to the pebbles when no biodiesel was applied. TPH in INO and INO + NUT groups began to decrease after day 5, but the changes
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
5
in INO group were not apparent as that in INO + NUT group. On day 21, the TPH in INO + NUT test where nutrients had been added was 51.6% lower than that in INO treatment. TPH in the control group changed little and the variation of TPH might result from the volatilization of petroleum hydrocarbon. It is assumed that the change of TPH in seawater before day 5 was mostly a physicochemical process where oil was released from pebbles by biodiesel addition. As the experiment went on, the petroleum degraders presented in the system gradually adapted to the toxicity of crude oil and the biochemical process began to play an important role. In order to confirm the assumption, the variations of PDB (Fig. 5) and DHA (Fig. 6) with time were analyzed. 3.3.2. Variations of PDB in surface seawater The original value of PDB was 1.0E+6 mL1 at t0 in each experimental group. Decreased numbers of PDB were observed in all the treatments on day 1 which may result from the toxic effects of crude oil on oil degrading bacteria. PDB in INO + NUT treatment declined slowly compared to others because TPH in this group was relatively low (Fig. 4) with no biodiesel addition and PDB was easily adaptable. Furthermore, sufficient nutrients in INO + NUT were more suitable to PDB for growth than the INO treatment. PDB in each group increased slowly on day 5, but none of them recovered to the original level on that day. PDB in each experimental group began to increase after day 5 compared to t0. The two treatments where biodiesel was added were the fastest growing groups which indicate the application of biodiesel enhanced the multiplication of PDB. In the group where biodiesel and nutrients were added simultaneously (INO + BD + NUT), PDB grew more quickly than in other groups. For example, the PDB numbers in the INO + BD + NUT treatment exceeded those in INO + BD, INO + NUT and INO treatments by 3.7, 10.5, and 50.8 times (P = 0.005, 0.004 and 0.005) respectively on day 17 (Fig. 6). The long-term abundance of PDB in INO + BD + NUT treatments indicated that biodiesel might be used as more easily degraded carbon source and the supply of N and P was helpful for nutrition balances in the growth of PDB. PDB in INO treatment was consistently low during the experiment probably because the nutrients were insufficient. PDB in INO + NUT was higher compared to the INO treatment, which indicated the addition of N and P sources was beneficial for the growth of PDB, therefore TPH in seawater declined quicker than the INO treatment (Fig. 4). However, since most of the crude oil was still attached to the pebbles in INO + NUT test, the lack of carbon source resulted in lower PDB than those groups where biodiesel were added (INO + BD + NUT and INO + BD). Overall, the variations of PDB are in correspondence with the changes of TPH in Fig. 4. None of PDB was detected in control group (data not shown) indicating the decrease of petroleum hydrocarbon was due to their volatilization.
Fig. 5. Population dynamics of PDB in seawater measured by the MPN method. Error bars represent mean ± one standard deviation (n = 3).
Fig. 6. The variations of Dehydrogenase activity in the system. Error bars represent mean ± one standard deviation (n = 3).
3.3.3. The variations of dehydrogenase activity in biodegradation tests Dehydrogenase activity (DHA) represents active bacterial cells in a system (Sherr et al., 1999). It provides information on biodegradation efficiency (Oh et al., 2001). In our experiments, microbial DHA in the control was absent (data not shown), meaning any petroleum hydrocarbon loss was solely due to abiotic losses. The variation of DHA is shown in Fig. 6. In the 2 groups where biodiesel were applied, DHA exceeded the original value on the first day. In comparison, DHA in the 2 groups with no biodiesel addition decreased on day 1 and then increased slowly. The average DHA in groups amended with biodiesel had higher values. For example, DHA in the INO + BD + NUT group was 3.2 times higher than that in the INO + NUT group. In the INO + BD group the average DHA was 6.3 times higher that in the INO treatment. It shows that the application of nutrients increased the value of DHA and this trend lasted for a longer time (Fig. 6). For instance, the average DHA in INO + BD + NUT group was 26.7% higher than that in INO + BD group and DHA in the latter began to decrease remarkably on day 13 while in the former the DHA did not change. Similar results were observed between the INO + NUT and INO groups. On the whole, petroleum degraders in the system reduced the concentration of residual oil in seawater in comparison to abiotic control. Furthermore, the application of biodiesel and nutrients increased the amount of PDB and raised their dehydrogenase activity.
3.4. Analysis of biodegradation kinetics Kinetic analysis is essential to determine the speed and governing factors of biodegradation process in the bioremediation of oil contaminated shorelines. According to the variations of TPH, the amount of petroleum degraders and DHA in seawater, the primary process occurred in the systems before day 5 was the detachment of residual oil from pebbles due to the solving nature of biodiesel. The biodegradation process took the main role afterwards. Therefore, the analysis of biodegradation kinetics was carried out from day 5. The first-order degradation rate constant (k) was determined according to the equation Ct = C0 ⁄ ekt, where Ct (mg/L) is the petroleum hydrocarbon concentration at time t and was calculated from the initial petroleum hydrocarbon concentration C0 (mg/L) on day 5 and the degradation data (Sinkkonen and Paasivirta, 2000; Matthies et al., 2008; Onwurah and Alumanah, 2005). The effects with the addition of inoculum, biodiesel or nutrients were evaluated and analyzed in INO, INO + BD, INO + NUT, and INO + BD + NUT groups respectively (see Table 2). The fit to the data in all four treatments after day 5 was relatively high and the correlation coefficient R2 were all above 0.9
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
6
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Table 2 Kinetic equation and half-life times for different treatments. Treatment
Regression equation
R2
Rate constant K (1/d)
Half-life times t1/2 (d)
INO + BD + NUT INO + NUT INO + BD INO
y = 0.107x 0.732 y = 0.082x 0.548 y = 0.054x 0.391 y = 0.035x 0.229
0.923 0.974 0.938 0.962
0.107 0.082 0.054 0.035
9.3 12.2 18.5 28.6
resulting in the increase of multiplication capacity and microbial activity. 4. Conclusions
Fig. 7. The removal efficiency (%) of residual oil from pebbles on day 21. Error bars represent mean ± one standard deviation (n = 3).
indicating the biodegradation process can be described by first order kinetics equation. The highest t1/2 of 28.6 days observed for INO treatment was reduced to 18.5 days, 12.2 days and 9.3 days for INO + BD, INO + NUT and INO + BD + NUT respectively, showing the positive effect of nutrient supplementation and biodiesel addition. The rate constant in the INO + BD + NUT treatment was 0.107 d1 which was significantly (P < 0.05) higher and exceeded the rate in the INO treatment by a factor of 2.1. This suggests that the addition of biodiesel and nutrients considerably accelerated the biodegradation rate of crude oil.
3.5. The removal efficiency (%) of TPH from pebbles When the biodegradation experiment ended on day 21, oil remaining on the pebbles was extracted and the oil removal efficiency (%) from pebbles was calculated (Fig. 7). It was found that TPH removal efficiency in four treatments was significantly (P < 0.05) higher than in the control. For example, it rose from 4.5% in abiotic control to 31.3% in INO treatment demonstrating PDB added in the system had great effect on residual oil clean-up (Fig. 7). When nutrients or biodiesel was added respectively in the system, the removal efficiency improved to 33.8% (INO + BD) and 22.9% (INO + NUT) respectively in comparison to the control, suggesting the combined nutrition and detachment effects of biodiesel promoted residual oil clean-up. When biodiesel and nutrients were added at the same time, the TPH removal efficiency in INO + BD + NUT increased 64.1% which is 7.4% higher (P < 0.05) than the total removal efficiency improvement of INO + BD and INO + NUT, indicating an additive effect of biodiesel and nutrients. In combination with the previous results from Figs. 5 and 6, it was clear that the supplement of nutrients and biodiesel at the same time increased the values of PDB and DHA dramatically resulting in higher removal efficiency of residual oil clean-up. One reason may be that biodiesel detached oil from pebbles and then formed oil–biodiesel mixtures. These mixtures were easily biodegraded by petroleum degraders and had larger contact area with PDB. Therefore they were more easily utilized compared to the original residual oil on pebbles. Another reason may be the supplement of nutrients provided the petroleum degraders sufficient N and P when they utilized large amount of C source,
The effects of biodiesel to eliminate residual oil from pebbles were investigated in the study using simulated systems. When hydrocarbon degraders was absent in the system, the application of biodiesel to crude oil-contaminated pebbles was effective in detaching the residual oil from pebbles. Oil–biodiesel mixture was formed quickly, most of which remained in the surface of seawater. The use of larger volumes of biodiesel resulted in a more effective cleaning of the residual oil. However, the optimal biodiesel to crude oil ratio need to be determined based on the pollution intensity and economic considerations. When hydrocarbon degraders were present in the system, the replenishment of biodiesel or nutrients increased the amount of petroleum degraders and microbial activity in seawater and decreased the half life of oil biodegradation. Particularly, when biodiesel and nutrients were added together, their additive effect increased the removal efficiency of residual oil from pebbles dramatically. More tests need to be done in field conditions in future studies because many variables exist in the natural environment. Considering that the bioremediation effects of marine oil spills are limited by the dilution of nutrients and hydrocarbon degraders, inexpensive materials have been used as carriers of petroleum degrading bacteria in order to alleviate the washing and dilution effects of wave and tide, and results showed that hydrocarbon degradation would continue to occur wherever the carriers were carried to by wave action (Simons et al., 2012). It is suggested that immobilized petroleum degraders, optimal nutrients and biodiesel are applied simultaneously in order to provide an economically, environmentally and socially acceptable alternative to remove spilled oil from shorelines. Acknowledgements This study was supported by ‘‘The National Natural Science Foundation of China (No. 50979038)’’, ‘‘The Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of State Oceanic Administration (201403)’’, and ‘‘Taishan Scholar Fund of Shandong Province (No. 2010016010)’’. References Alef, K., 1995. Dehydrogenase activity. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, New York, pp. 228–229. Atlas, R.M., 1993. Bacteria and bioremediation of marine oil spills. Oceanus 36 (2), 71. Beolchini, F., Rocchetti, L., Regoli, F., Dell’Anno, A., 2010. Bioremediation of marine sediments contaminated by hydrocarbons: experimental analysis and kinetic modeling. J. Hazard. Mater. 182, 403–407. Birchall, C., Newman, J.R., Greaves, M.P., 1995. Degradation and phototoxicity of biodiesel oil. Institute of Arable Crops Research, Long Ashton Research Station, Bristol, p. 50.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx Bragg, J.R., Prince, R.C., Harner, E.J., et al., 1994. Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature 368, 413–418. DeMello, J.A., Carmichael, C.A., Peacock, E.E., et al., 2007. Biodegradation and environmental behavior of biodiesel mixtures in the sea: an initial study. Mar. Pollut. Bull. 54, 894–904. Dutta, T.K., Harayama, S., 2000. Fate of crude oil by the combination of photo oxidation and biodegradation. Environ. Sci. Technol. 34, 1500–1505. Ferna´ndez-A´lvarez, P., Vila, J., Garrido-Ferna´ndez, J.M., Grifoll, M., Lema, J.M., 2006. Trials of bioremediation on a beach affected by the heavy oil spill of the Prestige. J. Hazard. Mater. B137, 1523–1531. Fernández-Álvarez, P., Vila, J., Garrido, J.M., Grifoll, M., Feijoo, G., Lema, J.M., 2007. Evaluation of biodiesel as bioremediation agent for the treatment of the shore affected by the heavy oil spill of the prestige. J. Hazard. Mater. 147, 914–922. Hozumi, T., Tsutsumi, H., Kono, M., 2000. Bioremediation on the shore after an oil spill from the Nakhodka in the Sea of Japan. I. Chemistry and characteristics of heavy oil loaded on the Nakhodka and biodegradation tests by a bioremediation agent with microbiological cultures in the laboratory. Mar. Pollut. Bull. 40 (4), 308–314. Lee, R.F., 1976. Accumulation and turnover of petroleum hydrocarbons in marine organisms. In: Wolfe, D.A. (Ed.), Fate and Effects of Petroleum Hydrocarbons in Marine Ecosystems and Organisms. Pergamon Press, Oxford, pp. 60–70. Makareviciene, V., Janulis, P., 2003. Environmental effect of rapeseed oil ethyl ester. Renew. Energy 28, 2395–2403. Matthies, M., Witt, J., Klasmeier, J., 2008. Determination of soil biodegradation half lives from simulation testing under aerobic laboratory conditions: a kinetic model approach. Environ. Pollut. 156, 99–105. Mearns, A.J., 1997. Cleaning oiled shores: putting bioremediation to the test. Spill Sci. Technol. Bull. 4 (4), 209–217. Miller, N.J., Mudge, S.M., 1997. The effect of biodiesel on the rate of removal and weathering characteristics of crude oil within artificial sand columns. Spill Sci. Technol. Bull. 4, 17–33. Mohajeri, L., Aziz, H.A., Isa, M.H., Zahed, M.A., 2010. A statistical experiment design approach for optimizing biodegradation of weathered crude oil in coastal sediments. Bioresour. Technol. 101, 893–900. Oh, Y.S., Sim, D.S., Kim, S.J., 2001. Effects of nutrients on crude oil biodegradation in the upper intertidal zone. Mar. Poll. Bull. 42, 1367–1372. Onwurah, I.N.E., Alumanah, E.E., 2005. Integration of biodegradation half-life model and oil toxicity model into a diagnostic tool for assessing bioremediation technology. Ind. Biotechnol. 1, 292–296. Pereira, M.G., Mudge, S.M., 2004. Cleaning oiled shores: laboratory experiments testing the potential use of vegetable oil biodiesels. Chemosphere 54, 297–304. Pritchard, P.H., Costa, C.F., 1991. EPA’s Alaska oil spill bioremediation project. Environ. Sci. Technol. 25, 372–379.
7
Rogers, J.E., Li, S.W., 1985. Effects of metals and other inorganic ions on soil microbial activity: soil DH assay as a simple toxicity test. Bull. Environ. Contam. Toxicol. 34, 858–865. Santas, R., Korda, A., Tenente, A., Buchholz, K., Santas, P.H., 1999. Mesocosm assays of oil spill bioremediation with oleophilic fertilizers. Mar. Pollut. Bull. 38 (1), 44–48. Sherr, B.F., Giorgio, D.P., Sherr, E.B., 1999. Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC. Aquat. Microb. Ecol. 18, 117–131. Simons, K.L., Ansar, A., Krishna, K., et al., 2012. Investigating the effectiveness of economically sustainable carrier material complexes for marine oil remediation. Bioresour. Technol. 126, 202–207. Sinkkonen, S., Paasivirta, J., 2000. Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling. Chemosphere 40, 943–949. Southward, A.J., 1982. An ecologist’s view if the implications of the observed physiological and biochemical effects of petroleum compounds on marine organisms and ecosystems. Philos. Trans. R. Soc. London 297 (B), 241–255. State Oceanic Administration of China, 1998. Specifications of Oceanographic Survey. Ocean press, Beijing, pp. 221–223. Swannell, R.P.J., Lee, K., Mcdonagh, M., 1996. Field evaluations of marine oil spill bioremediation. Microbiol. Rev. 60, 342–365. Taylor, L.T., Jones, D.M., 2001. Bioremediation of coal tar PAH in soils using biodiesel. Chemosphere 44, 1131–1136. Teal, J.M., Farrington, J.W., Burns, K.A., et al., 1992. The west Falmouth oil spill after 30 years: fate of fuel oil compounds and effects on animals. Mar. Pollut. Bull. 24, 607–614. Venosa, A.D., Zhu, X., 2003. Biodegradation of crude oil contaminating marine shorelines and freshwater wetlands. Spill Sci. Technol. Bull. 8, 163–178. Venosa, A.D., Suidan, M.T., Wrenn, B.A., et al., 1996. Bioremediation of an experimental oil spill on the shoreline of Delaware Bay. Environ. Sci. Technol. 30, 1764–1775. Wang, Z.D., Fingas, M., Blenkinsopp, S., et al., 1998. Comparison of oil composition changes due to biodegradation and physical weathering in different oils. J. Chromatogr. A 809, 89–107. Randall von Wedel, 2000. Cytosol-cleaning oiled shorelines with a vegetable oil biosolvent. Spill Sci Technol. Bull. 6, 357–359. Xia, W., Zheng, X., Li, J., et al., 2005. Degradation of crude oil by indigenous microorganisms supplemented with nutrients. J. Environ. Sci. 17 (4), 659–661. Xia, W., Li, J., Xia, Y., et al., 2012. Optimization of diesel oil biodegradation in seawater using statistical experimental methodology. Water Sci. Technol. 66 (6), 1301–1309.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Studies on crude oil removal from pebbles by the application of biodiesel Wen-xiang Xia a, Yan Xia a, Jin-cheng Li a,⇑, Dan-feng Zhang a, Qing Zhou b, Xin-ping Wang b a
College of Environmental and Municipal Engineering, Qingdao Technological University, Qingdao 266033, China Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology, North China Sea Environmental Monitoring Center of State Oceanic Administration, Shandong, Qingdao 266033, China b
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Bioremediation Petroleum contamination Pebble Biodiesel Nutrients
a b s t r a c t Oil residues along shorelines are hard to remove after an oil spill. The effect of biodiesel to eliminate crude oil from pebbles alone and in combination with petroleum degrading bacteria was investigated in simulated systems. Adding biodiesel made oil detach from pebbles and formed oil–biodiesel mixtures, most of which remained on top of seawater. The total petroleum hydrocarbon (TPH) removal efficiency increased with biodiesel quantities but the magnitude of augment decreased gradually. When used with petroleum degrading bacteria, the addition of biodiesel (BD), nutrients (NUT) and BD + NUT increased the dehydrogenase activity and decreased the biodegradation half lives. When BD and NUT were replenished at the same time, the TPH removal efficiency was 7.4% higher compared to the total improvement of efficiency when BD and NUT was added separately, indicating an additive effect of biodiesel and nutrients on oil biodegradation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Oil spills are one of the major reasons to ocean pollution, causing dramatic damages to the sea and shorelines. The spilled oil may persist in coastal areas for years, transport through the food chains (Lee, 1976; Teal et al., 1992) and exhibit long-term toxicity (Southward, 1982). Oil stranded on rocky shorelines is extremely difficult to remove especially since the shorelines are lack of seawater washing. A layer of fuel oil may stick firmly to the surface of rocks or pebbles due to the accumulation of the recalcitrant components and act as a slow-release source of contamination. High pressure flushing with hot water has been used in shorelines affected by the Prestige oil spill. Nevertheless, it is labor intensive and time consuming, and a large amount of wastes was produced with high energy consumption (Fernández-Álvarez et al., 2006). Bioremediation has the advantage of low cost and high efficiency under favorable conditions without causing environmental damage (Beolchini et al., 2010; Mohajeri et al., 2010; Pritchard and Costa, 1991; Venosa and Zhu, 2003; Wang et al., 1998). It is an efficient alternative for the removal of oil from rocks or pebbles by physical means. Petroleum degrading bacteria (PDB) are ubiquitous in the marine environment (Atlas, 1993). They require not only carbon
⇑ Corresponding author. Tel.: +86 532 85071262; fax: +86 532 85015375. E-mail address: [email protected] (J.-c. Li).
but also nitrogen and phosphorus for incorporation of biomass. The application of nutrients has proven to be an effective way to stimulate oil biodegradation in the bioremediation of petroleum contaminated shorelines (Santas et al., 1999; Venosa et al., 1996). However, mineral oil attached to the pebbles has low water solubility, leading to difficulties for microbes to utilize it as an energy source. Therefore, the bioavailability of petroleum hydrocarbons to oil degraders is a major factor influencing the bioremediation effect. Biodiesel is composed primarily of fatty acid methyl esters. It is an environmentally benign addition since it is readily degraded by microorganisms (Makareviciene and Janulis, 2003; DeMello et al., 2007) and less toxic than mineral oils (Birchall et al., 1995). In recent years, biodiesel has been used to eliminate petroleum contamination in laboratory and field tests. For example, Taylor and Jones (2001) reported that the addition of biodiesel to soil containing coal tar increased the degradation of coal tar polycyclic aromatic hydrocarbons (PAHs), which was ascribed to tar solubilization and dispersion thereby increasing the PAHs bioavailability. Fernández-Álvarez et al. (2006) applied biodiesel for removal of crude oil that contaminated rocks and sand after the Prestige oil spill. They found it not only accelerated the clean-up of the polluted surface but also enhanced the degradation of the residual oil. However, neither the added microorganisms nor the nutrients accelerated the degradation rate of the residual oil in the affected shorelines in their studies, which was quite different from other reports (Hozumi et al., 2000; Oh et al., 2001).
http://dx.doi.org/10.1016/j.marpolbul.2014.11.026 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
2
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
On the whole, the removal of petroleum contamination by the addition of biodiesel is both a physicochemical and a biochemical process. These dual actions were discussed in previous studies (Taylor and Jones, 2001; Fernández-Álvarez et al., 2006). However, the role of biodiesel played alone on the removal of fuel oil was unclear. Furthermore, when biodiesel was applied together with nutrients, their combined influence on the hydrocarbon degrading microorganisms need to be further explored. Laboratory studies are valuable in assessing the effectiveness of a shoreline cleaning strategy since it is not practical to replicate the true conditions of the shoreline environment (Mearns, 1997; Santas et al., 1999). In this study, we use a simulated system to explore the effect and mechanism of biodiesel on removing oil from shorelines. First, biodiesel (BD) was applied to crude oil contaminated pebbles in a sterilization system without inoculum (INO) in order to study the physiochemical effects of biodiesel alone. Secondly, with INO present in the system, BD, nutrients (NUT) and BD + NUT were replenished respectively to study the biochemical effects of biodiesel in combination with oil degrading bacteria. This article describes the results of our experimental studies. 2. Materials and methods 2.1. Samples Clean seawater collected from Jinsha Beach near Qingdao, China was filtered through 0.22 lm membrane to eliminate the impact of micro-organisms. Pebbles (diameter 4–5 cm) collected from Qingdao Huiquan Bay were soaked with hydrochloric acid and sterilized before use. Crude oil samples were purchased from Huangdao oil depot. The density, kinetic viscosity and API value of the oil at 20 °C was 0.987 (g/cm3), 86.55 (mm2/s) and 11.9 respectively. In order to avoid the influence of oil volatilization, the crude oil was weathered for 4 weeks in a fume hood before use, losing 10.8% of its initial weight. Biodiesel in the form of rapeseed oil methyl esters was purchased from local suppliers in Qingdao, China. The density and kinetic viscosity at 20 °C was 0.836 (g/cm3) and 5.6 (mm2/s) respectively. 2.2. Microorganism acclimatization Since no single strain of bacteria has the metabolic capacity to degrade all components found in crude oil, a mixture of bacterial suspension was used in the experiment. A medium containing 1 g/L NH4NO3, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.2 g/L MgSO47H2O, 0.05 g/L FeCl3, and 0.02 g/L CaCl2 was used to culture bacteria (Dutta and Harayama, 2000; Mohajeri et al., 2010). Oil-polluted seawater containing oil degrading bacteria was collected from Dagang Wharf No. 6 in Jiaozhou Bay was added to 100 mL of medium and incubated on a rotary shaker at 25 °C, 200 r/min for 2 weeks. Then enriched solution was added to fresh medium and incubated at 25 °C for 1 week. To obtain standard inoculum, the mixed cells were harvested by centrifugation, rinsed three times in sterile saline before being re-suspended in sterile liquid basal medium to yield an absorbance reading of 0.5 at 540 nm.
with a flow control device was inserted through holes around the containers at a height of 0, 5 and 10 cm from the top of the pebbles to allow seawater to be drained through the system and analyzed (Fig. 1). The containers were placed indoor and the temperatures is 20 °C. In order to mimic the washing of the wave and replacement effect of seawater, fish pump with 4 outlets was installed under the bottom of the pebbles, and seawater (800 mL) was flushed and drained through the holes with the same amount of fresh seawater replenished every day after the seawater samples were taken. 2.3.1. Effects of biodiesel on residual oil clean-up without hydrocarbon degraders Four containers were used in each group, one for control, and three for experiments which were treated with biodiesel. To each of the containers, 20 g weathered crude oil was applied to the surface of the pebbles as uniformly as possible to simulate pollution density occurred along Qingdao shorelines in 2013. Results from our previous studies (dada not shown) indicated the earlier the biodiesel was added, the higher the oil removal efficiency. Since it needs some time for the response action to take place when large area of pollution occurs in real situations, 3 h is chosen in our experiment to mimic real situations and biodiesel (6, 12 and 18 mL) was sprayed respectively in 3 experimental tests. The washing effect of the tide was simulated by filling the container with seawater until it submerged the pebbles with fish pump switching on and off. No biodiesel was added in the control. Seawater samples (20 mL) containing the biodiesel-oil mixture was taken at different position from sample outlets in 1, 2, 3, 4, and 7 days after the addition of the biodiesel, and 4 samples at the same height were mixed and treated to analyze the total petroleum hydrocarbon (TPH) content. Seawater 240 mL was replenished in the container after sampling. When the experiment ended, oil remaining on pebbles was analyzed. Another two groups were used as parallel experiments and the above process was repeated three times. 2.3.2. Effects of biodiesel on residual oil biodegradation in the presence of hydrocarbon degraders In order to study the subsequent biodegradation of oil detached from pebbles after the addition of biodiesel, five containers were prepared as a group, 1 control and 4 experimental. The arrangements are shown in Table 1. Crude oil contaminated pebbles are prepared as previously mentioned. One control test was set up with only seawater added, and 4 experimental tests were prepared with the addition of inoculum (INO) as well as biodiesel (BD), nutrients (NUT) or BD + NUT (Table 1). NO3–N and PO4–P were amended according to the results of former study (Xia et al., 2005), and the N:P ratio is 10:1. Oxygen was supplied by fish pump in order to simulate the aerobic condition along shoreline and make inoculum distribute evenly, and the dissolved oxygen (DO) of the seawater varied between 3.8 and 4.5. Seawater samples (20 mL) in water surface were obtained from each outlets on day 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 following the addition of the biodiesel, and 4 seawater samples from different positions were mixed and prepared to analyze TPH, petroleum degrading bacteria (PDB), dehydrogenase activity (DHA) and DO. When the experiment ended, the residual oil still attached to the pebbles was analyzed. Another two groups were used as parallel experiments and the above process was repeated three times.
2.3. Experimental design 2.4. Sample analysis Plastic containers (400 260 200 mm) were prepared and sterilized pebbles were put into them, and seawater was poured into the container to attain a height of 150 mm which is about 10 mm higher than the top of pebbles. Twelve plastic tubes fitted
2.4.1. Total petroleum hydrocarbon (TPH) in seawater The mixture of biodiesel and crude oil in the seawater were extracted using carbon tetrachloride. The biodiesel–oil in carbon
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
3
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Fig. 1. Schematic diagram of experimental set-up.
Table 1 Experimental design for biodegradation tests.
Control INO INO + BD INO + NUT INO + BD + NUT
Crude oil (g)
Inoculum (mL)
Biodiesel (mL)
Nutrients (lmol/L)
20 20 20 20 20
0 100 100 100 100
0 0 12 0 12
0 0 0 500 NO3–N + 50 PO4–P 500 NO3–N + 50 PO4–P
tetrachloride was then passed through adsorption column loaded with magnesium silicate. The hydrocarbons of the crude oil passed through the adsorption column whilst the biodiesel was retained. Then total petroleum hydrocarbon (TPH) in crude oil was determined by Oil Analyzer of infrared spectrometer (Jilin BeiGuang Optical Instrument Factory, China). 2.4.2. Removal efficiency (%) of TPH from pebbles At the end of the experiment, all pebbles were put into a beaker containing carbon tetrachloride and extracted twice. The extracts were combined and diluted for gravimetric analysis of TPH as previously stated. TPH removal efficiency (%) from pebbles was calculated as follows:
TPH removal efficiencyð%Þ ¼ ðTPHcont TPHres Þ=TPHcont 100% where TPHcont – TPH in control group; TPHres – TPH residues in test group. 2.4.3. Microbial enumeration The most probable number (MPN) technique was used to estimate the number of petroleum degrading bacteria (PDB) in seawater in all treatments. Bushnell–Hass medium supplemented with 2% (w/w) NaCl was used as the growth medium for PDB. Tubes were inoculated with 1 mL of serially diluted samples and then incubated for enumeration of PDB for two weeks at room temperature before being scored for positive growth with the Sheen Screen test for crude oil degraders. A corrected most probable number table was used to estimate microbial numbers (Xia et al., 2012). 2.4.4. Dehydrogenases activity (DHA) measurement Triphenyl tetrazolium chloride (TTC) is an artificial electron acceptor for several dehydrogenases. It is water soluble and nearly all microorganisms reduce it to TPF, which can be colorimetrically estimated (Alef, 1995). DHA was measured using UV–VIS spectrophotometer (UV759S, Shanghai Jingke Instrument Company, Ltd.) as follows: Samples from seawater were added to 10 mL centrifuge tubes containing a 0.1% TTC aqueous solution, and then incubated for 48 h at 30 ± 1 °C in the dark. The hydrolysis reaction product (TPF) was extracted for 2 h at 30 °C with acetone and absorbance
at 482 nm was recorded (Rogers and Li, 1985). Data are expressed as lg TPF/mL/h. 2.4.5. Other chemical analysis DO and pH were measured by HACH sension™ 156 Multiparameter Meters. NO3–N and PO4–P was measured using cadmium–copper reduction method and phosphomolybdenum blue method (State Oceanic Administration of China, 1998) respectively. 2.5. Statistical analysis The results were subjected to one way analysis of variance (ANOVA) to determine if mean values of TPH removal efficiency, PDB as well as DHA in the controls and experimental tests differed significantly. Data were considered to be significantly different between the three treatments if P < 0.05. All statistical analyses were performed using Excel 2003. 3. Results and discussion 3.1. The distribution of TPH in different heights of seawater with varied amount of biodiesel Biodiesel was added in 0 (control), 6, 12 and 18 mL quantities on oil polluted pebbles with no hydrocarbon degraders in the system. The variations of TPH on day one in different height of the seawater is shown in Fig. 2 (the trend in other days were the same and the data were not shown). The addition of biodiesel increased the concentration of TPH in seawater more than 10 times compared to control (Fig. 2), and the differences between control and other treatments are statistically significant (P < 0.05). For example, the concentration of TPH at the surface seawater increased as high as 13.23–19.27 mg/L, while in control test this value was only 1.18 mg/L indicating biodiesel can make large amounts of residual oil detach from pebbles. Results also indicated the lower part in seawater contained less TPH than the surface water regardless how much biodiesel was added in the container (Fig. 2). For example, when 18 mL of biodiesel was added, the TPH in surface seawater was 19.27 mg/L while
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
4
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Fig. 2. The average value of TPH (mg/L) in different height of the seawater on day one with varied biodiesel addition.
the value was 3.88 and 2.25 mg/L respectively beneath the surface at 5 cm and 10 cm. The same results were reported by Pereira and Mudge (2004) that only small amounts of crude oil and biodiesel (3–14.5%) accumulated at depths below 5 cm. This is because biodiesel has lower viscosity and higher buoyancy than crude oil. When biodiesel was added to the oil-polluted pebbles, crude oil was released from pebbles and dissolved into biodiesel. The oil– biodiesel mixture had higher mobility and most of them rose to the surface of seawater. Because of this, only TPH in surface seawater was investigated in later experiments in this study. In the experimental tests, the addition of 6 mL biodiesel resulted in 13.23 mg/L of TPH in surface seawater, which was remarkably less than the addition of 12 mL and 18 mL of biodiesel where TPH values was 18.21 mg/L and 19.27 mg/L respectively. These experiments indicated that the more the biodiesel was added to the system, the more oil detached from pebbles, with 18 mL of biodiesel giving the best result. Results from the above treatments showed that when no petroleum degrading bacteria were present, biodiesel had a considerable capacity to detach crude oil from pebbles and form an oil–biodiesel mixture which can eventually be removed by water with little remained on the pebbles. Similar results were found in other studies (Wedel, 2000; Miller and Mudge, 1997; Fernández-Álvarez et al., 2007) in their studies. However, the relationship between the BD addition and the clean-up efficiency of crude oil from pebbles need to be further explored. 3.2. Influence of the biodiesel volume on the removal of residual oil from pebbles After 7 days, seawater in the containers was emptied and the crude oil remained on pebbles was extracted and analyzed after flushing the oil slick. Oil removal efficiency was calculated in
Fig. 3. Comparison of TPH removal efficiency (%) from pebbles with varied biodiesel volume after day 7. Error bars represent mean ± one standard deviation (n = 3).
Fig. 4. The variations of TPH in surface seawater. Error bars represent mean ± one standard deviation (n = 3).
accordance with the biodiesel volume and the results were shown in Fig. 3. The treatments significantly differed from the control (P < 0.01) and the removal effect of biodiesel to eliminate residual oil from pebbles appeared to be dependent on the volume of biodiesel sprayed. In the experiment, 6 mL biodiesel (biodiesel/oil = 30%, v/v) significantly increased the TPH removal efficiency to 30.1% compared to the control where no biodiesel was used (P = 0.005). When additional 6 mL biodiesel was applied in the system, the increase of TPH removal efficiency is 13.1% higher than the 6 mL treatment (P = 0.005). When 18 mL biodiesel was used, the increase of TPH removal efficiency is only 4.5% higher compared to the 12 mL treatment (P = 0.004). These results indicate that higher ratio of biodiesel to crude oil resulted in greater removal rate of oil from pebbles but the increase rate slowed down quickly. Adding more biodiesel to the system may reach a plateau in TPH removal efficiency. In practice, the volume of biodiesel applied to clean oil from pebbles depends on the pollution intensity and economic feasibility. More work need to be done to determine the optimal application amount of biodiesel to remove residual oil for a particular shoreline situation. 3.3. Variations of chemical and microbial parameters during biodegradation In the first experiment, most of the oil on the pebbles detached from pebbles after the application of biodiesel and formed oil– biodiesel mixture floating in seawater. Since petroleum degrading bacteria are ubiquitous along shorelines (Atlas, 1993), the elimination of residual oil rely largely on the biodegradation process. Spilled oil constitutes a carbon source and by adding N and P nutrients can promote the biodegradation of petroleum (Bragg et al., 1994; Swannell et al., 1996; Xia et al., 2005). In the next experiment, petroleum degrading bacteria expressed as inoculum (INO) were applied in four experimental groups besides the control group. Then biodiesel (BD), nutrient (NUT) and biodiesel + nutrient (BD + NUT) were added respectively in the systems in order to explore their effect on oil biodegradation. 3.3.1. The variations of TPH Results showed that in the three tests (Control, INO and INO + NUT) where no biodiesel was added, about 8 mg/L TPH was obtained on day 5 in seawater (Fig. 4). However, this value increased approximately to 27 mg/L in the two tests (INO + BD and INO + BD + NUT) on day 5 where biodiesel was added. This may be due to the fact that most of the crude oil was still attached to the pebbles when no biodiesel was applied. TPH in INO and INO + NUT groups began to decrease after day 5, but the changes
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
5
in INO group were not apparent as that in INO + NUT group. On day 21, the TPH in INO + NUT test where nutrients had been added was 51.6% lower than that in INO treatment. TPH in the control group changed little and the variation of TPH might result from the volatilization of petroleum hydrocarbon. It is assumed that the change of TPH in seawater before day 5 was mostly a physicochemical process where oil was released from pebbles by biodiesel addition. As the experiment went on, the petroleum degraders presented in the system gradually adapted to the toxicity of crude oil and the biochemical process began to play an important role. In order to confirm the assumption, the variations of PDB (Fig. 5) and DHA (Fig. 6) with time were analyzed. 3.3.2. Variations of PDB in surface seawater The original value of PDB was 1.0E+6 mL1 at t0 in each experimental group. Decreased numbers of PDB were observed in all the treatments on day 1 which may result from the toxic effects of crude oil on oil degrading bacteria. PDB in INO + NUT treatment declined slowly compared to others because TPH in this group was relatively low (Fig. 4) with no biodiesel addition and PDB was easily adaptable. Furthermore, sufficient nutrients in INO + NUT were more suitable to PDB for growth than the INO treatment. PDB in each group increased slowly on day 5, but none of them recovered to the original level on that day. PDB in each experimental group began to increase after day 5 compared to t0. The two treatments where biodiesel was added were the fastest growing groups which indicate the application of biodiesel enhanced the multiplication of PDB. In the group where biodiesel and nutrients were added simultaneously (INO + BD + NUT), PDB grew more quickly than in other groups. For example, the PDB numbers in the INO + BD + NUT treatment exceeded those in INO + BD, INO + NUT and INO treatments by 3.7, 10.5, and 50.8 times (P = 0.005, 0.004 and 0.005) respectively on day 17 (Fig. 6). The long-term abundance of PDB in INO + BD + NUT treatments indicated that biodiesel might be used as more easily degraded carbon source and the supply of N and P was helpful for nutrition balances in the growth of PDB. PDB in INO treatment was consistently low during the experiment probably because the nutrients were insufficient. PDB in INO + NUT was higher compared to the INO treatment, which indicated the addition of N and P sources was beneficial for the growth of PDB, therefore TPH in seawater declined quicker than the INO treatment (Fig. 4). However, since most of the crude oil was still attached to the pebbles in INO + NUT test, the lack of carbon source resulted in lower PDB than those groups where biodiesel were added (INO + BD + NUT and INO + BD). Overall, the variations of PDB are in correspondence with the changes of TPH in Fig. 4. None of PDB was detected in control group (data not shown) indicating the decrease of petroleum hydrocarbon was due to their volatilization.
Fig. 5. Population dynamics of PDB in seawater measured by the MPN method. Error bars represent mean ± one standard deviation (n = 3).
Fig. 6. The variations of Dehydrogenase activity in the system. Error bars represent mean ± one standard deviation (n = 3).
3.3.3. The variations of dehydrogenase activity in biodegradation tests Dehydrogenase activity (DHA) represents active bacterial cells in a system (Sherr et al., 1999). It provides information on biodegradation efficiency (Oh et al., 2001). In our experiments, microbial DHA in the control was absent (data not shown), meaning any petroleum hydrocarbon loss was solely due to abiotic losses. The variation of DHA is shown in Fig. 6. In the 2 groups where biodiesel were applied, DHA exceeded the original value on the first day. In comparison, DHA in the 2 groups with no biodiesel addition decreased on day 1 and then increased slowly. The average DHA in groups amended with biodiesel had higher values. For example, DHA in the INO + BD + NUT group was 3.2 times higher than that in the INO + NUT group. In the INO + BD group the average DHA was 6.3 times higher that in the INO treatment. It shows that the application of nutrients increased the value of DHA and this trend lasted for a longer time (Fig. 6). For instance, the average DHA in INO + BD + NUT group was 26.7% higher than that in INO + BD group and DHA in the latter began to decrease remarkably on day 13 while in the former the DHA did not change. Similar results were observed between the INO + NUT and INO groups. On the whole, petroleum degraders in the system reduced the concentration of residual oil in seawater in comparison to abiotic control. Furthermore, the application of biodiesel and nutrients increased the amount of PDB and raised their dehydrogenase activity.
3.4. Analysis of biodegradation kinetics Kinetic analysis is essential to determine the speed and governing factors of biodegradation process in the bioremediation of oil contaminated shorelines. According to the variations of TPH, the amount of petroleum degraders and DHA in seawater, the primary process occurred in the systems before day 5 was the detachment of residual oil from pebbles due to the solving nature of biodiesel. The biodegradation process took the main role afterwards. Therefore, the analysis of biodegradation kinetics was carried out from day 5. The first-order degradation rate constant (k) was determined according to the equation Ct = C0 ⁄ ekt, where Ct (mg/L) is the petroleum hydrocarbon concentration at time t and was calculated from the initial petroleum hydrocarbon concentration C0 (mg/L) on day 5 and the degradation data (Sinkkonen and Paasivirta, 2000; Matthies et al., 2008; Onwurah and Alumanah, 2005). The effects with the addition of inoculum, biodiesel or nutrients were evaluated and analyzed in INO, INO + BD, INO + NUT, and INO + BD + NUT groups respectively (see Table 2). The fit to the data in all four treatments after day 5 was relatively high and the correlation coefficient R2 were all above 0.9
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
6
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
Table 2 Kinetic equation and half-life times for different treatments. Treatment
Regression equation
R2
Rate constant K (1/d)
Half-life times t1/2 (d)
INO + BD + NUT INO + NUT INO + BD INO
y = 0.107x 0.732 y = 0.082x 0.548 y = 0.054x 0.391 y = 0.035x 0.229
0.923 0.974 0.938 0.962
0.107 0.082 0.054 0.035
9.3 12.2 18.5 28.6
resulting in the increase of multiplication capacity and microbial activity. 4. Conclusions
Fig. 7. The removal efficiency (%) of residual oil from pebbles on day 21. Error bars represent mean ± one standard deviation (n = 3).
indicating the biodegradation process can be described by first order kinetics equation. The highest t1/2 of 28.6 days observed for INO treatment was reduced to 18.5 days, 12.2 days and 9.3 days for INO + BD, INO + NUT and INO + BD + NUT respectively, showing the positive effect of nutrient supplementation and biodiesel addition. The rate constant in the INO + BD + NUT treatment was 0.107 d1 which was significantly (P < 0.05) higher and exceeded the rate in the INO treatment by a factor of 2.1. This suggests that the addition of biodiesel and nutrients considerably accelerated the biodegradation rate of crude oil.
3.5. The removal efficiency (%) of TPH from pebbles When the biodegradation experiment ended on day 21, oil remaining on the pebbles was extracted and the oil removal efficiency (%) from pebbles was calculated (Fig. 7). It was found that TPH removal efficiency in four treatments was significantly (P < 0.05) higher than in the control. For example, it rose from 4.5% in abiotic control to 31.3% in INO treatment demonstrating PDB added in the system had great effect on residual oil clean-up (Fig. 7). When nutrients or biodiesel was added respectively in the system, the removal efficiency improved to 33.8% (INO + BD) and 22.9% (INO + NUT) respectively in comparison to the control, suggesting the combined nutrition and detachment effects of biodiesel promoted residual oil clean-up. When biodiesel and nutrients were added at the same time, the TPH removal efficiency in INO + BD + NUT increased 64.1% which is 7.4% higher (P < 0.05) than the total removal efficiency improvement of INO + BD and INO + NUT, indicating an additive effect of biodiesel and nutrients. In combination with the previous results from Figs. 5 and 6, it was clear that the supplement of nutrients and biodiesel at the same time increased the values of PDB and DHA dramatically resulting in higher removal efficiency of residual oil clean-up. One reason may be that biodiesel detached oil from pebbles and then formed oil–biodiesel mixtures. These mixtures were easily biodegraded by petroleum degraders and had larger contact area with PDB. Therefore they were more easily utilized compared to the original residual oil on pebbles. Another reason may be the supplement of nutrients provided the petroleum degraders sufficient N and P when they utilized large amount of C source,
The effects of biodiesel to eliminate residual oil from pebbles were investigated in the study using simulated systems. When hydrocarbon degraders was absent in the system, the application of biodiesel to crude oil-contaminated pebbles was effective in detaching the residual oil from pebbles. Oil–biodiesel mixture was formed quickly, most of which remained in the surface of seawater. The use of larger volumes of biodiesel resulted in a more effective cleaning of the residual oil. However, the optimal biodiesel to crude oil ratio need to be determined based on the pollution intensity and economic considerations. When hydrocarbon degraders were present in the system, the replenishment of biodiesel or nutrients increased the amount of petroleum degraders and microbial activity in seawater and decreased the half life of oil biodegradation. Particularly, when biodiesel and nutrients were added together, their additive effect increased the removal efficiency of residual oil from pebbles dramatically. More tests need to be done in field conditions in future studies because many variables exist in the natural environment. Considering that the bioremediation effects of marine oil spills are limited by the dilution of nutrients and hydrocarbon degraders, inexpensive materials have been used as carriers of petroleum degrading bacteria in order to alleviate the washing and dilution effects of wave and tide, and results showed that hydrocarbon degradation would continue to occur wherever the carriers were carried to by wave action (Simons et al., 2012). It is suggested that immobilized petroleum degraders, optimal nutrients and biodiesel are applied simultaneously in order to provide an economically, environmentally and socially acceptable alternative to remove spilled oil from shorelines. Acknowledgements This study was supported by ‘‘The National Natural Science Foundation of China (No. 50979038)’’, ‘‘The Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of State Oceanic Administration (201403)’’, and ‘‘Taishan Scholar Fund of Shandong Province (No. 2010016010)’’. References Alef, K., 1995. Dehydrogenase activity. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, New York, pp. 228–229. Atlas, R.M., 1993. Bacteria and bioremediation of marine oil spills. Oceanus 36 (2), 71. Beolchini, F., Rocchetti, L., Regoli, F., Dell’Anno, A., 2010. Bioremediation of marine sediments contaminated by hydrocarbons: experimental analysis and kinetic modeling. J. Hazard. Mater. 182, 403–407. Birchall, C., Newman, J.R., Greaves, M.P., 1995. Degradation and phototoxicity of biodiesel oil. Institute of Arable Crops Research, Long Ashton Research Station, Bristol, p. 50.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
W.-x. Xia et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx Bragg, J.R., Prince, R.C., Harner, E.J., et al., 1994. Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature 368, 413–418. DeMello, J.A., Carmichael, C.A., Peacock, E.E., et al., 2007. Biodegradation and environmental behavior of biodiesel mixtures in the sea: an initial study. Mar. Pollut. Bull. 54, 894–904. Dutta, T.K., Harayama, S., 2000. Fate of crude oil by the combination of photo oxidation and biodegradation. Environ. Sci. Technol. 34, 1500–1505. Ferna´ndez-A´lvarez, P., Vila, J., Garrido-Ferna´ndez, J.M., Grifoll, M., Lema, J.M., 2006. Trials of bioremediation on a beach affected by the heavy oil spill of the Prestige. J. Hazard. Mater. B137, 1523–1531. Fernández-Álvarez, P., Vila, J., Garrido, J.M., Grifoll, M., Feijoo, G., Lema, J.M., 2007. Evaluation of biodiesel as bioremediation agent for the treatment of the shore affected by the heavy oil spill of the prestige. J. Hazard. Mater. 147, 914–922. Hozumi, T., Tsutsumi, H., Kono, M., 2000. Bioremediation on the shore after an oil spill from the Nakhodka in the Sea of Japan. I. Chemistry and characteristics of heavy oil loaded on the Nakhodka and biodegradation tests by a bioremediation agent with microbiological cultures in the laboratory. Mar. Pollut. Bull. 40 (4), 308–314. Lee, R.F., 1976. Accumulation and turnover of petroleum hydrocarbons in marine organisms. In: Wolfe, D.A. (Ed.), Fate and Effects of Petroleum Hydrocarbons in Marine Ecosystems and Organisms. Pergamon Press, Oxford, pp. 60–70. Makareviciene, V., Janulis, P., 2003. Environmental effect of rapeseed oil ethyl ester. Renew. Energy 28, 2395–2403. Matthies, M., Witt, J., Klasmeier, J., 2008. Determination of soil biodegradation half lives from simulation testing under aerobic laboratory conditions: a kinetic model approach. Environ. Pollut. 156, 99–105. Mearns, A.J., 1997. Cleaning oiled shores: putting bioremediation to the test. Spill Sci. Technol. Bull. 4 (4), 209–217. Miller, N.J., Mudge, S.M., 1997. The effect of biodiesel on the rate of removal and weathering characteristics of crude oil within artificial sand columns. Spill Sci. Technol. Bull. 4, 17–33. Mohajeri, L., Aziz, H.A., Isa, M.H., Zahed, M.A., 2010. A statistical experiment design approach for optimizing biodegradation of weathered crude oil in coastal sediments. Bioresour. Technol. 101, 893–900. Oh, Y.S., Sim, D.S., Kim, S.J., 2001. Effects of nutrients on crude oil biodegradation in the upper intertidal zone. Mar. Poll. Bull. 42, 1367–1372. Onwurah, I.N.E., Alumanah, E.E., 2005. Integration of biodegradation half-life model and oil toxicity model into a diagnostic tool for assessing bioremediation technology. Ind. Biotechnol. 1, 292–296. Pereira, M.G., Mudge, S.M., 2004. Cleaning oiled shores: laboratory experiments testing the potential use of vegetable oil biodiesels. Chemosphere 54, 297–304. Pritchard, P.H., Costa, C.F., 1991. EPA’s Alaska oil spill bioremediation project. Environ. Sci. Technol. 25, 372–379.
7
Rogers, J.E., Li, S.W., 1985. Effects of metals and other inorganic ions on soil microbial activity: soil DH assay as a simple toxicity test. Bull. Environ. Contam. Toxicol. 34, 858–865. Santas, R., Korda, A., Tenente, A., Buchholz, K., Santas, P.H., 1999. Mesocosm assays of oil spill bioremediation with oleophilic fertilizers. Mar. Pollut. Bull. 38 (1), 44–48. Sherr, B.F., Giorgio, D.P., Sherr, E.B., 1999. Estimating abundance and single-cell characteristics of respiring bacteria via the redox dye CTC. Aquat. Microb. Ecol. 18, 117–131. Simons, K.L., Ansar, A., Krishna, K., et al., 2012. Investigating the effectiveness of economically sustainable carrier material complexes for marine oil remediation. Bioresour. Technol. 126, 202–207. Sinkkonen, S., Paasivirta, J., 2000. Degradation half-life times of PCDDs, PCDFs and PCBs for environmental fate modeling. Chemosphere 40, 943–949. Southward, A.J., 1982. An ecologist’s view if the implications of the observed physiological and biochemical effects of petroleum compounds on marine organisms and ecosystems. Philos. Trans. R. Soc. London 297 (B), 241–255. State Oceanic Administration of China, 1998. Specifications of Oceanographic Survey. Ocean press, Beijing, pp. 221–223. Swannell, R.P.J., Lee, K., Mcdonagh, M., 1996. Field evaluations of marine oil spill bioremediation. Microbiol. Rev. 60, 342–365. Taylor, L.T., Jones, D.M., 2001. Bioremediation of coal tar PAH in soils using biodiesel. Chemosphere 44, 1131–1136. Teal, J.M., Farrington, J.W., Burns, K.A., et al., 1992. The west Falmouth oil spill after 30 years: fate of fuel oil compounds and effects on animals. Mar. Pollut. Bull. 24, 607–614. Venosa, A.D., Zhu, X., 2003. Biodegradation of crude oil contaminating marine shorelines and freshwater wetlands. Spill Sci. Technol. Bull. 8, 163–178. Venosa, A.D., Suidan, M.T., Wrenn, B.A., et al., 1996. Bioremediation of an experimental oil spill on the shoreline of Delaware Bay. Environ. Sci. Technol. 30, 1764–1775. Wang, Z.D., Fingas, M., Blenkinsopp, S., et al., 1998. Comparison of oil composition changes due to biodegradation and physical weathering in different oils. J. Chromatogr. A 809, 89–107. Randall von Wedel, 2000. Cytosol-cleaning oiled shorelines with a vegetable oil biosolvent. Spill Sci Technol. Bull. 6, 357–359. Xia, W., Zheng, X., Li, J., et al., 2005. Degradation of crude oil by indigenous microorganisms supplemented with nutrients. J. Environ. Sci. 17 (4), 659–661. Xia, W., Li, J., Xia, Y., et al., 2012. Optimization of diesel oil biodegradation in seawater using statistical experimental methodology. Water Sci. Technol. 66 (6), 1301–1309.
Please cite this article in press as: Xia, W.-x., et al. Studies on crude oil removal from pebbles by the application of biodiesel. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.11.026
