Journal of Environmental Quality 31:1842-1847 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORTS
Bioremediation and Biodegradation
Polycyclic Aromatic Hydrocarbon Removal from Soil by Surfactant Solubilization and Phanerochaete chrysosporium Oxidation
Zhongming Zheng and
Jeffrey Philip Obbard*
Department of Chemical & Environmental Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore, 129791
* Corresponding author (chejpo{at}nus.edu.sg)
Received for publication November 22, 2001.
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ABSTRACT
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Surfactant soil washing can remove polycyclic aromatic hydrocarbons (PAHs) from contaminated soil, and the white rot fungus, Phanerochaete chrysosporium Burdsall in Burdsall & Eslyn, can oxidize PAHs. The objective of this study was to develop a novel bioremediation technology using a combination of abiological surfactant soil washing followed by PAH biological oxidation in soil washwater using P. chrysosporium in a rotating biological contactor (RBC) reactor. Soil used for experimentation was an 11-month aged contaminated soil spiked with a total of nine PAHs: acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, benzo(a)pyrene, dibenz(ah)anthracene, and benzo(ghi)perylene. After 11 months of aging, recovery percentages of high molecular weight PAHs [i.e., from chrysene to benzo(ghi)perylene] were greater than 86%, while those of low molecular weight PAHs (i.e., from acenaphthene to pyrene) were less than 19%. Total removal efficiency for any of the nine PAHs was greater than 90% using a combination of surfactant soil washing and P. chrysosporium oxidation of soil washwater in the RBC reactor when used in batch operation, and greater than 76% when used in continuous operation. The treatment of PAH-contaminated soil using a combination of surfactant soil washing and subsequent PAH removal from the resultant washwater in an RBC reactor, in the presence of immobilized P. chrysosporium, permits (i) a rapid abiological cleanup of soil for compliance with relevant soil quality standards and (ii) PAH biological removal in soil washwater for compliance with aqueous discharge standards.
Abbreviations: PAH, polycyclic aromatic hydrocarbon • RBC, rotating biological contactor
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INTRODUCTION
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POLYCYCLIC AROMATIC hydrocarbons (PAHs) are pollutants of major concern at many contaminated industrial sites throughout the world. Elimination of PAHs from soil is necessary for compliance with relevant "cleanup" standards based on individual PAH concentrations (e.g., United States Federal Register, 1993). The biodegradation of soil-bound PAHs is a two-step process that involves initial mobilization of PAHs from the solid to aqueous phase, followed by subsequent microbial catabolism. However, rapid PAH biodegradation is hindered by limited bioavailability, which is due to low PAH water dissolution rates, sorption to solid matrices, and incorporation into soil particle micropores (Cerniglia, 1992; Tiehm et al., 1997). Although laboratory studies have revealed that virtually all PAHs are biodegradable, it is generally accepted that sorbed hydrophobic pollutants are not readily available for microbial degradation (Guha and Jaffe, 1996). Consequently, it has been concluded that mass transfer processes associated with contaminant release into the aqueous phase limit the PAH attenuation rate, rather than explicit aqueous-phase biodegradation kinetics (Yeom and Ghosh, 1998).
Surfactants enhance the apparent PAH solubility in water as well as their dissolution rates from the solid phase. Surfactant molecules, above their critical micelle concentration (CMC), form aggregates in water, known as micelles. Hydrophobic compounds are readily solubilized into micelles, thereby enhancing bioavailability and subsequent biodegradation. In equilibrium, the concentration of solubilized PAH linearly depends on the surfactant concentration above the CMC (Edwards et al., 1991; Zheng and Obbard, 2002a). Solubilization, as well as decreases in surface and interface tension, are believed to be the main reasons for the facilitated transport of adsorbed pollutants to the aqueous phase (Tiehm et al., 1997). Soil washing with surfactant solutions has been shown to be an effective remediation technology for the rapid removal of hydrophobic compounds from soil, including PAHs (Edwards et al., 1994; Yeom et al., 1995). However, subsequent disposal of micelle-contaminated soil wash water can be a major environmental constraint. The acceptability of remediation technologies can be increased if the soil contaminants present in the washwater can be effectively oxidized in either aboveground reactors or in situ (Guha and Jaffe, 1996).
Lignin-degrading white rot fungi can rapidly oxidize PAHs into metabolites, which are then more readily mineralized by bacteria and fungi (Barr and Aust, 1994; Bezalel et al., 1996; Bogan and Lamar, 1996; Kotterman et al., 1998). It has been previously reported that Phanerochaete chrysosporium can degrade PAHs present in anthracene oil, from which at least 22 major hydrophobic organic compounds were degraded by 70 to 100% after 27 d of incubation (Bumpus, 1989). Nonionic surfactants are known to stimulate PAH biodegradation through enhanced PAH bioavailability (Zheng and Obbard, 2001). For example, the surfactant Tween 80 (Sigma–Aldrich, St. Louis, MO) and polyoxyethylene 10 lauryl ether (PLE) are known to increase anthracene, pyrene, and benzo(a)pyrene oxidation rates by two- to fivefold (Kotterman et al., 1997). Recently, research on white rot fungi in soil showed limited PAH oxidation, in contrast to liquid fungal cultures, where PAHs were degraded due to enhanced bioavailability (Boyle et al., 1998).
Bench scale studies using white rot fungi in rotating biological contactor (RBC) reactors have successfully demonstrated the biodegradation of organic munitions wastes (Sublette et al., 1992), as well as the decolorization of bleach plant effluent and paper mill wastewater (Yin et al., 1989). It has also been reported that pentachlorophenol is efficiently degraded by fixed films of white rot fungi in rotating tube bioreactors (Alleman et al., 1995). Our recent study showed that an RBC reactor with immobilized P. chrysosporium could efficiently remove more than 90% of phenanthrene, pyrene, and benzo(a)pyrene from a surfactant solution with PAHs, by both biological oxidation and abiological adsorption (Zheng and Obbard, 2002b).
Although research has been undertaken on both soil PAH washing with surfactants and PAH oxidation by white rot fungi, there are no studies that have addressed soil washing and subsequent PAH removal in soil washwater in an RBC reactor using immobilized white rot fungi. Such treatment is required to avoid a secondary pollution problem caused by the generation of PAH-contaminated washwater (Zheng and Obbard, 2000).
The objective of this study was to investigate a novel bioremediation technology for the treatment of PAH-contaminated soil based on a combination of initial surfactant soil washing and subsequent white rot fungal oxidation of PAHs in soil washwater in an RBC reactor. This objective was to achieve a rapid dissolution and abiological PAH removal from the soil solid phase, followed by PAH removal in the contaminated washwater using immobilized P. chrysosporium in an RBC reactor.
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MATERIALS AND METHODS
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Preparation of Polycyclic Aromatic Hydrocarbon–Contaminated Soil
The soil used for experimentation was an uncontaminated topsoil of medium sandy texture with a sieved particle size of 75 to 600 µm. Two grams of a total of nine PAHs [i.e., acenaphthene (300 mg), fluorene (300 mg), phenanthrene (300 mg), fluoranthene (300 mg), pyrene (300 mg), chrysene (300 mg), benzo(a)pyrene (50 mg), dibenz(ah)anthracene (75 mg), and benzo(ghi)perylene (75 mg)] were dissolved in 0.2 L of acetone and methylene chloride (1:1 ratio), prior to spiking 3.5 kg of soil (i.e., soil moisture content = 2.6%). Subsequently, the soil was left in a fume cupboard for two days with occasional mixing to evaporate the organic solvents. The soil was then homogenized in an end-over-end tumbler (Reax20; Heidolph Elektro, Kelheim, Germany) in darkness at 23°C for 72 h. Subsequently, soil was kept in darkness at room temperature and aged over an 11-mo period with regular measurement of residual PAH concentrations (see below).
Soil Washing with Nonionic Surfactant Tween 80 Solution
The PAH concentration remaining in the soil, Csoil,n, on the nth soil washing is defined by:
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where Csoil,n and Csoil,ini are PAH concentration remaining in the soil on the nth soil washing (mg kg-1) and initial soil PAH concentration (mg kg-1), respectively; Kd is the soil–water PAH partition coefficient (L kg-1); Vw is volume of bulk solution (L); and Wsoil is weight of soil (kg).
The PAH fraction in bulk solution after N washings can then be given by:
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Therefore, a smaller Kd value, a larger water volume to soil mass ratio, or an increased number of soil washings will facilitate PAH removal from the soil solid phase to surfactant solution. Aliquots (0.2 kg) of the 6- or 11-mo aged PAH-contaminated soil and 2 L of 5 g L-1 Tween 80 were magnetically stirred in a 5-L conical flask for 24 h. The soil and surfactant solution was then transferred to 0.2-L tubes and centrifuged at 8000 rpm for 20 min. The supernatant was then collected and the residual soil was washed another four times in the flask with an identical procedure. The 6-mo soil was used to study PAH removal in each of the five successive washings, while the 11-mo soil was washed with five successive washings, and all supernatant was amalgamated for PAH treatment in the RBC reactor.
Polycyclic Aromatic Hydrocarbon Removal with the Rotating Biological Contactor Reactor with Immobilized White Rot Fungi
A schematic diagram of the RBC reactor system is shown in Fig. 1 , and its physical specifications are listed in Table 1. Discs were made of polyurethane foam and were 50% immersed in solution. Total disc surface area (A) was 0.138 m2. Total liquid volume in the RBC reactor (VL) was 2.5 L. Disc surface area per unit liquid volume (A/VL) was 55.4 m-1. Five discs were mounted on a horizontal steel shaft and were rotated with a varying speed from 5 to 30 rpm with an electric voltage drive motor. Controlled gas-phase aeration was provided via an air pump through a 0.45-µm filter.
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Fig. 1. A schematic diagram of rotating biological contactors (RBC). 1, Polycyclic aromatic hydrocarbon (PAH) contained wastewater tank; 2, effluent wastewater tank; 3, air condenser; 4, RBC reactor; 5, electrical motor; 6, air filter; 7, airflow meter; 8, air pump; 9, recycling water bath.
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Experiments were initiated with the RBC in batch mode. Basal III medium (2.5 L) (Tien and Kirk, 1988) was autoclaved and placed in the RBC reactor, inoculated with the white rot fungus P. chrysosporium spores (i.e., 2 x 108 spores L-1), and then operated in batch mode for five days to immobilize the fungal biomass on the rotating discs. After immobilization, the RBC reactor was ready for either continuous or batch operation. Process variables for continuous operation (i.e., PAH hydraulic loading rate and PAH concentration in influent solution) and batch operation (i.e., initial PAH concentration, airflow rate, disc rotating speed, and PAH removal kinetics) have been previously investigated and reported (Zheng and Obbard, 2002b). In this study, the RBC reactor airflow rate was 1.0 L min-1, disc-rotation speed was 15 rpm, and temperature was kept at 37°C. A hydraulic parameter A/Q of 1442 h m-1 (i.e., a flow rate of 1.6 mL min-1 and a retention time of 26 h) was adopted in continuous operation (i.e., 10 d), while a 5-d retention time was used in batch operation.
Polycyclic Aromatic Hydrocarbon Extraction
Polycyclic aromatic hydrocarbons in soil were extracted using Soxhlet extraction (Falc, Lurano, Italy). One gram of sample was transferred to a cellulose extraction thimble (25 x 80 mm), and subjected to extraction with 0.2 L of dichloromethane for 8 to 10 h at a rate of 5 to 6 min per cycle. Solvent extract was then concentrated in a rotary evaporator at 45°C to near dryness, and the residue was then dissolved in 3 mL of dichloromethane prior to gas chromatography–mass spectrometry (GC–MS) analysis. Five milliliters of PAH surfactant solution was extracted with 3 mL dichloromethane three times, after adding 2 drops of 12 mol L-1 HCl. The extracts were then passed through a column (100 x 10 mm) filled with 2 g of sodium sulfate and collected. The column was then rinsed with another 5 mL dichloromethane. The extracts were pooled and evaporated under argon gas to dryness, and then redissolved in 1 mL dichloromethane prior to GC–MS analysis.
Gas Chromatography–Mass Spectrometry Protocol for Polycyclic Aromatic Hydrocarbon Analysis
A GC–MS (Hewlett-Packard [Palo Alto, CA] 6890 with auto injection G1512A; MS Hewlett Parkard 5972) was used for PAH analysis, and a capillary column (HP-5, 5% phenyl methyl siloxane, 30.0 m x 320 µm x 0.25 µm nominal) was used for PAH separation. The oven temperature was programmed as follows: hold 60°C, 3 min; ramp rate 8°C min-1 to 150°C, hold 3 min; ramp rate 5°C min-1 to 280°C, hold 10 min; The injection volume was 1 µL and injection was via a split-less injection port maintained at 310°C. Helium was used as a carrier gas at a flow rate of 1.2 mL min-1.
Ligninolytic Enzyme Activity
Culture supernatant lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were assayed colormetrically on an PerkinElmer (Wellesley, MA) Lambda 20 UV/Vis spectrophotometer using the extracellular fluid of the culture solutions. Lignin peroxidase was determined at 310 nm based on the maximum rate of oxidation of veratryl alcohol to veratraldehyde (Tien and Kirk, 1988). Enzyme activity was expressed in enzyme units, U, with 1 enzyme unit defined as 1 µmol veratraldehyde produced per min. The MnP was assayed at 610 nm with N,N,N,N-tetramethyl-1,4-phenylenediamine(-2HCl) (TMPD) as the substrate with an extinction coefficient of 1160 mol-1 m2. One unit (U) of MnP activity is defined as that required to oxidize 1 µmol of TMPD per min (Paszczynski et al., 1988).
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RESULTS AND DISCUSSION
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Polycyclic Aromatic Hydrocarbon Concentrations in Eleven-Month Aged Soil
The characteristics of the soil used for experimentation are shown in Table 2. The PAH concentrations in soil at different ages are shown in Table 3. The lower recovery percentages of acenaphthene and fluorene after one month of aging are most likely due to volatilization during the preparation of PAH-contaminated soil. After 6 mo of aging, recovery percentages of four-ring and above PAHs were greater than 84% [i.e., from pyrene to benzo(ghi)perylene], while those of three-ring PAHs were less than 9.3% recovered (i.e., acenaphthene, fluorene, and phenanthrene). After 11 mo of aging, recovery percentages of high molecular weight PAHs [i.e., chrysene, benzo(a)pyrene, dibenz(ah)anthracene, and benzo(ghi)perylene] were still greater than 86%, while those of low molecular weight PAHs (i.e., acenaphthene, fluorine, phenanthrene, and pyrene) were less than 19.4%. It is well known that low molecular weight PAHs are semivolatile and easily exist in the gas phase (Cerniglia, 1992), therefore, the low recovery of low molecular weight PAHs from the soil during the aging test is most likely to have been due to volatilization from the spiked soil.
Successive Soil Washing with Nonionic Surfactant
Figure 2
shows accumulative recovery percentage of pyrene, a representative example, from 6-mo aged contaminated soil in five successive soil washings compared with predicted values with a Tween 80 concentration of 5 g L-1 and a soil to aqueous ratio of 1:10. Results show that after five successive surfactant soil washings, approximately 90% of pyrene was removed from the soil solid phase to surfactant solution with a low surfactant concentration of 5 g L-1 Tween 80, and a soil to aqueous ratio of 1:10. Table 4 shows the results of PAH removal from the 11-mo aged contaminated soil after five successive washings. Results clearly show that a significant PAH amount (i.e., >94%) was removed from the soil solid phase to surfactant solution. It can be calculated that approximately 0.8 mg of Tween 80 was needed per milligram of total PAH (i.e., the sum of nine PAHs) removal from the soil solid phase with five successive washings.
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Fig. 2. Accumulative recovery percentage (%) of pyrene in five successive soil washings ( ) compared with predicted values (solid line) with a Tween 80 concentration of 5 g L-1 and a soil to aqueous ratio of 1:10.
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Table 4. Polycyclic aromatic hydrocarbon (PAH) removal from 11-month aged contaminated soil after five successive washings with 5 g L-1 Tween 80 solution at a water volume to soil mass ratio of 10.
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Removal of Polycyclic Aromatic Hydrocarbons in a Rotating Biological Contactor Reactor with Phanerochaete chrysosporium
The PAH removal rate and efficiency from soil washwater containing 5 g L-1 (w/v) Tween 80 at a hydraulic parameter A/Q of 1442 h m-1 (i.e., a retention time of 26 h) in the RBC reactor operated in continuous operation for 10 d with or without nutrient addition to soil washwater, are shown in Table 5. The removal efficiencies for all nine PAHs were greater than 60%, with removal of acenaphthene and fluorene reaching 100% without nutrient addition. Removal efficiencies for all nine PAHs were enhanced to in excess of 76% when carbon and nitrogen sources (i.e., 5 g L-1 glucose and 0.2 g L-1 ammonium tartrate) were added to the soil washwater. The thickness of the fungal mycelium on RBC discs increased, on average, from a mean of 0.01 m after 5 d of fungal biomass immobilization in RBC discs to 0.026 m at the end of the continuous flow experiment. Although concentrations of fluoranthene, pyrene, and benzo(a)pyrene in effluent were higher than those regulated as maximum concentrations for any monthly average discharge limit (USEPA, 1995), the concentrations of acenaphthene, fluorene, phenanthrene, and chrysene were lower than specified in the discharge limit (see Table 5). The performance of the RBC reactor in continuous operation can be enhanced to reduce concentrations of pyrene and benzo(a)pyrene to below their discharge limits by either increasing the residence time in the RBC reactor, or decreasing initial PAH concentration in soil washwater (Zheng and Obbard, 2002b).
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Table 5. Removal of polycyclic aromatic hydrocarbons (PAH) from soil washwater in the rotating biological contactor (RBC) reactor in continuous operation at A/Q = 1442 h m-1 (i.e., residence time of 26 h).
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The PAH removal from soil washwater containing 5 g L-1 (w/v) Tween 80 in the RBC in batch operation is shown in Table 6. The removal efficiency was more than 90% for all PAHs, and reached 100% for acenaphthene and fluorene after 5 d. The final PAH concentration in treated soil washwater was lower than relevant discharge limits, with the exception of chrysene, where its concentration was equivalent to the discharge limit (see Table 6). The batch operation was efficient for PAH removal from surfactant soil washwater, as there was a sufficient retention time for PAH oxidation.
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Table 6. Removal of polycyclic aromatic hydrocarbons (PAHs) from soil washwater in a rotating biological contactor (RBC) in batch operation.
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For both RBC continuous and batch operations, no lignin peroxidase activity was detected in solution medium, and only a low activity level of manganese peroxidase (i.e., 10–100 U L-1) was measured over the entire period of RBC operation. Although activity of ligninolytic enzymes detected in the RBC reactor was low, the PAH removal by the immobilized P. chrysosporium was high over both continuous and batch operation periods. Results support previous studies that show that the removal of pollutants is not always linked with lignin peroxidase activity (Dhawale et al., 1992; Alleman et al., 1995).
Overall Removal of Polycyclic Aromatic Hydrocarbons from Aged Contaminated Soil with Surfactant Washing and White Rot Fungal Oxidation
The removal efficiency of PAHs from aged contaminated soil by using a combination of surfactant soil washing and P. chrysosporium oxidation is given in Table 7. The overall removal efficiency (i.e., biological and abiological) for any PAH was more than 90% for combined surfactant soil washing and P. chrysosporium batch operation in the RBC reactor, while it was more than 76% for combined surfactant soil washing and P. chrysosporium continuous RBC operation. However, the removal rate in continuous RBC operation was greater as the residence time in continuous operation (i.e., 26 h) was approximately one-fifth of that in batch operation (i.e., 5 d).
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Table 7. Overall removal of polycyclic aromatic hydrocarbons (PAHs) from aged contaminated soil by using surfactant washing and white rot fungal oxidation.
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Our previous mass balance study on typical PAHs [i.e., phenanthrene, pyrene, and benzo(a)pyrene] in the RBC reactor indicated that biological oxidation was the main factor for removal of benzo(a)pyrene (i.e., 95.7%). However, for phenanthrene and pyrene, both biological oxidation (i.e., 49 and 56%, respectively) and RBC disc foam adsorption (i.e., 44 and 34%, respectively) made a significant contribution for the removal percentage (Zheng and Obbard, 2002b).
Tween 80 is a biodegradable polyxyethylene surfactant and has been used widely in the study of PAH-contaminated soil bioremediation (Yeom et al., 1995). Therefore, its fate and toxicity in the environment is not of scientific concern. Overall, the treatment of PAH-contaminated soil using a combination of surfactant soil washing and subsequent PAH removal of the resultant washwater in an RBC reactor in the presence of immobilized white rot fungus is an effective remediation technology. This has particular application for (i) rapid abiological cleanup of soil for compliance with relevant soil quality standards and (ii) the use of white rot fungi to oxidize PAHs in soil washwater for compliance with relevant aqueous discharge standards.
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ABSTRACT
INTRODUCTION
