Environ. Sci. Technol. 1997, 31, 2626-2633
Ex-Situ Process for Treating PAH-Contaminated Soil with Phanerochaete chrysosporium R O B E R T M A Y , †,‡ P E T E R S C H R O ¨ DER,† AND H E I N R I C H S A N D E R M A N N , J R . * ,† GSF Forschungszentrum fu ¨ r Umwelt und Gesundheit GmbH, Institut fu ¨ r Biochemische Pflanzenpathologie, Ingolsta¨dter Landstrasse 1, D-85764 Oberschleissheim, Germany, and ghb Gebru ¨ der Huber Bodenrecycling GmbH, Tannenwaldstrasse 2, D-81375 Munich, Germany
Based on the known ability of the white rot fungus Phanerochaete chrysosporium to metabolize PAHs, a fungal reactor system with separate soil extraction and fungal incubation units was constructed. The design of the system allowed samples to be easily removed at strategic positions and to ascertain mineralization. The highly contaminated soil (1-2 mm particle diameter), with a total EPA Method 610 concentration of 41 g of PAHs kg-1, was spiked with [7,10-14C]benzo[a]pyrene in order to follow the fate of this tracer by HPLC and high-performance gel permeation chromatography. While mineralization amounted to only 2.5%, it was observed that the fungus reduced the total soil PAH concentration by 45% through polymerization processes. For [7,10-14C]benzo[a]pyrene, a value of 4.9 mg kg-1 day-1 or overall 5.5% was obtained. The polymers remained associated with soil, and no monomeric PAHs were detected in the medium. In parallel experiments without soil, high molecular weight polymers could be found in the medium. Sterile soil and medium controls revealed no polymerization. The results were consistent with literature reports that P. chrysosporium converts PAHs primarily to quinones, which have a strong tendency to polymerize. On the basis of the success of this system, scaling up appears to be justifiable.
Introduction The white rot fungus Phanerochaete chrysosporium (Burdsall and Eslyn) is best known for its superior role in nature as a lignin degrader (1, 2). The extracellular lignin and manganese peroxidases coupled with an H2O2-producing system generate Fenton-type radicals that can oxidatively attack, degrade, and mineralize a number of aromatic substrates (3-10). The degrading enzymes of the organism also include cellulases, proteases, quinone reductases, and ring fission enzymes (1113). During idiophasic growth, the secondary metabolite veratryl alcohol acts as a redox mediator, allowing compounds to be oxidized that are not normally substrates for the peroxidases (14-16). Veratryl alcohol is usually added to the nutrient solution (14-17). In addition, the nonionic detergent Tween 80 may be included in order to increase the solubility of hydrophobic xenobiotics (18). The number of discovered or registered sites that seriously require bioremediation continues to grow. In 1988, approximately 50 000 sites in Germany were suspected to be * Corresponding author e-mail address:
[email protected]; telephone: +49 89 3187 2285; fax: +49 89 3187 3383. † GSF Forschungszentrum fu ¨ r Umwelt und Gesundheit GmbH. ‡ ghb Gebru ¨ der Huber Bodenrecycling GmbH.
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contaminated (19). In North America, a similar situation prevails. For example, the Environmental Protection Agency (EPA) listed, at the end of 1994, 40 000 uncontrolled hazardous waste sites and 1300 National Priorities List (Superfund) sites (20). Over 300 000 landfills receive both hazardous and nonhazardous waste (21). Indeed, all major world countries are focusing more and more on the waste site problems (22). Based on these facts, many companies (including Firma Gebru ¨ der Huber Bodenrecycling, Munich, Germany) have developed soil purification plants. The Gebru ¨ der Huber system was constructed based on the decision to wash and bioremediate soil instead of depositing at a landfill site (23, 24). This system efficiently combines mechanical washing with biological degradation. Furthermore, efforts are made either to re-deposit the decontaminated soil at the original site or to use it as building material. In a continuing effort to expand and improve the bioremediation capacity of the Gebru ¨ der Huber system, we examined the degradation rates of a highly PAH-contaminated carbonate soil originating from the grounds of a former gas work in Munich, Germany. These ubiquitous xenobiotics are among the most recalcitrant, toxic, and/or cancerogenic of all xenobiotics (25) and are formed through both natural and anthropogenic routes whenever organic matter is not fully oxidized (for example, see ref 26). Due to their poor water solubility, they are almost always associated with particulate matter but have been detected in soils up to 4 m deep and in groundwater (27). The degradation of benzo[a]pyrene and other PAHs by P. chrysosporium has already been thoroughly studied (8-10). A main pathway leads to primary quinoid metabolites that are in small yield mineralized to carbon dioxide and in higher yield transformed to polymeric metabolites (8-10, 28). The latter are thought to be non-bioavailable and have lost the toxicological hazard of the parent PAHs (29). Nontoxic polymeric metabolites formed by P. chrysosporium are expected to eventually become part of the soil humus pool, as recently described for pentachlorophenol and its quinoid pathway metabolites (30). The present paper was intended to document the decontamination strategy of converting PAHs to polymeric metabolites. The decision to apply ex-situ strategies over their in-situ counterparts has many supporting arguments, especially when using white rot fungi. For an excellent treatise about cleanup strategies, the reader is referred to the book from Kamely et al. (31). They describe different reactor systems efficiently combining soil extraction with physically separate biological treatment, similar to the Gebru ¨ der Huber system, thereby allowing scaleup and increased fungal bioavailability of the bound PAHs. Furthermore, the system is closed and fitted with gas scrubbers, thereby preventing toxic metabolite escape and stripping, while permitting the simultaneous determination of mineralization rates and mass recovery.
Experimental Section Chemicals. All chemicals were of the best available purity and, unless otherwise mentioned, were used without prior purification. Veratryl alcohol (Aldrich, Steinheim, Germany) was purified by the method of Kirtikara and co-workers (32) or by vacuum distillation. Hydrogen peroxide (Merck, Darmstadt, Germany) was freshly prepared every week. Its concentration was determined at 240 nm (33). Radiochemicals. [7,10-14C]Benzo[a]pyrene was obtained from Amersham-Buchler, Braunschweig, Germany (specific activity 58 mCi mmol-1). Radiopurity was controlled by HPLC and found to be g98%. [UL-ring-14C]toluene (AmershamBuchler, specific activity 0.337 µCi mL-1) was used for internal
S0013-936X(97)00041-2 CCC: $14.00
1997 American Chemical Society
TABLE 1. Overview of Reactor Operation with Some Interim Results time (days)
event
comment/result
1 2 6
fungal inoculation into medium aeration 5 min; 50 mL of clean medium removed soil added at RT, clean aeration 60 min; 100 mL of clean medium removed; 100 mL fresh medium added; soil stir at 40 °C for 30 min; overnight sediment at 40 °C soil medium pumped to fungus for 30 min; aeration 5 min; 100 mL of clean medium removed; soil to RT soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; 50 mL of clean and 50 mL of soil medium removed; soil medium pumped to fungus for 30 min; aeration 60 min; 100 mL of fresh medium added soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; soil medium pumped to fungus for 30 min 100 mL of clean and 100 mL of soil medium removed; 100 mL of fresh medium added; added ca. 5 × 106 spores soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; soil medium pumped to fungus for 30 min aeration 60 min; soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; 50 mL of clean and 50 mL of soil medium removed; soil medium pumped to fungus for 60 min; 300 mL of fresh medium added aeration 60 min; soil stir at 40 °C for 30 min; pumped to fungus for 60 min; no sedimentation time allowed
spore concn ) 1.01 × 107 spores mL-1 pHclean ) 4.37; [O2] ) 40% pHclean ) 4.37; [O2]start ) 19%; [O2]end ) 62%
7 11 13 14 16 17 20 28 36
aeration 60 min; soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; soil medium pumped to fungus for 60 min aeration 60 min; soil stir at 40 °C for 30 min; 30 min sediment at 40 °C; soil medium pumped to fungus for 60 min experiment terminated
LSC standardization. [1-14C]Hexadecane (Amersham-Buchler, specific activity 3.94 × 10-4 µCi mL-1) was used to calibrate the biological oxidizer. NaH14CO3 (Sigma, Deisenhofen, Germany, specific activity 9.6 mCi mmol-1) was used to calibrate 14CO2 trapping. Fungal Growth and Incubation. The white rot fungus Phanerochaete chrysosporium (ATCC 24725) was a kind gift from Prof. I. Fiechter (ETH, Zurich). Basidiospores were stored at room temperature, on agar slants, and were transferred to fresh slants every 8 weeks (34). The concentration of spores was measured by optical density at 650 nm (17). The reactor system and incubation flasks were aerated for 30 min with moist, sterile (0.2 µm filter, SM 16534 Sartorius, Go¨ttingen, Germany) air at day 0 and thereafter with 100% oxygen every 3 days (for incubation flasks) and according to Table 1 for the reactor. After removing the growth medium, the reactor system tubing was blown empty and was aerated for a further 20 min. Incubations in the reactor were conducted at 39 °C for 36 days initially in 3500 mL of nitrogenlimited medium (17, 34), containing 0.1% (v/v) Tween 80. Parallel incubations with benzo[a]pyrene were conducted in 100 mL of Tween 80 (0.1%) containing nitrogen-limited medium at 39 °C without soil. In one benzo[a]pyrene series, the sodium salt of humic acid (Aldrich, H1, 675-2, technical grade) was added as a soil mimic at a concentration of 20 mg L-1. Aeration and sampling closely paralleled those used for the reactor. Quantification of Mineralization. The pressure exits of the fungal incubation flasks were serially connected to one impinger trap containing 20 mL of kerosene (Fluka, NeuUlm, Germany), followed by another trap containing 20 mL of 2-methoxyethanol/ethanolamine, 2:1 (v/v). Liquid Scintillation Counting (LSC). Aqueous solutions and fungal medium (maximum volume 2 mL) were measured in 10 mL of Hydroluma (J. T. Baker, Deventer, Germany). Organic solutions, kerosene, and CO2 trapping fluids (maximum volume 2 mL) were measured in 10 mL of counting fluid consisting of 5 g of PPO in 750 mL of toluene and 250 mL of methanol (Zinsser, Frankfurt, Germany). Counting was performed with a Wallac 1211 (LKB, Uppsala, Sweden). Sample Combustion. Freeze-dried samples were oxidized in a biological oxidizer (Zinsser) under the following condi-
pHclean ) 4.40; pHsoil ) 4.97; [O2]start ) 14%; [O2]end ) 45% pHclean ) 4.48; pHsoil ) 5.33; [O2]start ) 10%; [O2]end ) 80%
pHclean ) 4.51; pHsoil ) 5.58; [O2]start ) 30%; [O2]end ) 78% pHclean ) 4.57; pHsoil ) 5.51; [O2]start ) 16%; [O2]end ) 65%; pHclean ) 4.59; pHsoil ) 5.52; [O2]start ) 13%; [O2]end ) 72%; sand enters fungal reactor
tions: 350 mL min-1 O2 flow rate, 350 mL min-1 N2 flow rate, 4 min burn program (900 °C chamber temperature, 680 °C catalyst temperature), using Oxysolve C-400 scintillation fluid (Zinsser). Soil samples were oxidized with ca. 50 mg of powdered cellulose mixed into the soil matrix. Combustion efficiency and linearity were standardized with [1-14C]hexadecane (Amersham). Determination of Ligninase Activity. Ligninase activity determination was conducted by the veratryl alcohol oxidation assay (35) and was recorded at 30 °C in plastic cuvettes at 310 nm shortly after an aeration.The assay contained 1 M sodium phosphate buffer, pH 3.2 (500 µL), 100 mM veratryl alcohol (20 µL), medium (100 µL), and water (860 µL) and was initiated by the addition of freshly prepared 27 mM H2O2 (20 µL). After 30 s preincubation, samples were centrifuged at 13000g for 3 min at room temperature prior to the test. Determination of Carbohydrate Concentration in Solutions. The concentration of total carbohydrate in the reactor medium (Figure 1, K1) was determined spectrophotometrically by the dinitrosalicylic acid method basically according to Miller (36) and Ghose (37). Samples (typically 20 µL) and standards were mixed with 3 mL of dye solution in a small test tube and boiled at 100 °C for 5 min. This was followed by cooling under flowing tap water. Since some confusion exists in the literature as to the optimal wavelength and choice of blank (36, 37), four calibration curves at 540 and 575 nm using both water and heated reagent as blanks were performed. The best correlation coefficient was obtained at 575 nm with water as a blank. The reagent was prepared as follows: To 250 mL of H2O were added in this order: dinitrosalicylic acid (Fluka, 1.87 g, 8.19 mmol), NaOH (3.5 g, 87.5 mmol), potassium sodium tartrate tetrahydrate (72.66 g, 257.36 mmol), melted phenol (1.34 mL, 15.2 mmol), and sodium metabisulfite (1.47 g, 7.73 mmol). A standard solution of D(+)-glucose (J. T. Baker) was used for calibration. Soil Characterization. The carbonate-type soil originated from the grounds of a former gas work in Munich, Germany. The soil was highly basic in nature (pH ) 8.2) and was wet sieved into several fractions under a gentle stream of tap water using an EML 200 (Haver and Boecker, Oelde, Germany) apparatus. The 1-2 mm diameter fraction was chosen for these experiments. The soil pH was measured according to
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FIGURE 1. Overview and construction of the reactor system. McLean (38) as follows. To air-dried soil (6.85 g) was added CaCl2 (6.85 mL, 0.01 M), and the mixture was vortexed for 10 s at room temperature. After a 10-min settling period, the pH was measured with an electrode calibrated from pH 7-10. Sterilization was conducted directly in the soil reactor for 1 h at 121 °C at 1410 kPa. After drying under a clean bench for 48 h, the benzo[a]pyrene stock solution, dissolved in toluene, was distributed throughout the soil under sterile conditions via a syringe. Determination of Humic and Fulvic Acids. The humic and fulvic acid contents of soil were determined by the modification of a published procedure (39). Soil (10 g; pH 8.2) was allowed to stand in dilute HCl (50 mL of 0.01 M) for 10 min until the bubbling of CO2 had ceased. The acid was removed, and the soil was rinsed with water (5 × 10 mL). The soil (pH ) 5.7) was spread onto tared aluminum foil and was allowed to air-dry for 3 days at room temperature. The soil was placed in a 500-mL polyethylene flask and extracted with NaOH (100 mL, 0.5 M). The headspace of the flask was purged with nitrogen, and the flask was then sealed and shaken for 24 h at room temperature in the dark. After centrifugation (10000g, 20 min), the soil was washed with water (50 mL) and again centrifuged. The soil was discarded, and the combined supernatant was acidified to pH 2.0 with 2 M HCl and kept in the dark for 24 h. The supernatant was again centrifuged at 10000g for 20 min. The resulting precipitate and supernatant fractions were freeze-dried for 3 days. The mass of humic acid was determined by weighing the dried precipitate. The freeze-dried supernatant was redissolved in H2O (3 mL) and desalted on a commercially available PD-10 column (Pharmacia, Freiburg, Germany) using water as the eluent. The eluate was freeze-dried, and the mass of fulvic acid was determined. On average, 1 g of soil dry weight led to 1.6 mg of fulvic acid and 5.6 mg of humic acid. PAH Extraction. Air-dried soil samples (10 g) were vigorously shaken in Teflon-sealed glass flasks with 10 g of Na2SO4 and 50 mL of 3:1 (v/v) acetone/CH2Cl2 for 15 min. This was followed by 15 min of ultrasonication (Bandelin, Berlin, Germany). After a 15-min settling time, a 1.2-mL aliquot was removed and centrifuged in a 2-mL Eppendorf
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tube at 13000g for 10 min. From the supernatant, 1 mL was added to a separatory funnel containing 50 mL of CH2Cl2, 100 mL of H2O, and 5 mL of 125 g Na2SO3 L-1 solution. The aqueous phase was extracted two more times with 50 mL of CH2Cl2. The combined organic phases were collected in an Erlenmeyer flask and dried over 10 g of Na2SO4, and 250 µL of HOAc was added. After 20-min drying time, the solution was filtered through Schleicher & Schuell No. 604 filter paper into a round bottom flask, and the glassware was rinsed with 3 × 10 mL portions of CH2Cl2. Solvent was evaporated on a rotary evaporator at 50 °C, with no applied vacuum, to an oil. The oil was then dissolved in 1 mL of CH3CN/CH2Cl2 1:1 (v/v) and quantitively transferred into a 10-mL volumetric flask using 1-mL portions of this solvent mixture. Further dilutions were conducted with pure CH3CN as needed. HPLC Conditions. Sample extracts were analyzed on a Waters-Millipore (Eschborn, Germany) HPLC system comprised of two 510 pumps, a 996 PDA detector, a 470 fluorescence detector, a Ramona (Raytest, Straubenhardt, Germany) radiodetector, and a 717 Plus autosampler. The PDA detector was set at a wavelength region of 200-550 nm at 1.2 nm resolution, and 0.1 spectra s-1 was collected. For peak matching against spectral libraries, search depth 3 with retention time presearch of 20% was chosen. The analytical column was a VYDAC 210 TP 54 (Sep/a/ra/tions Group, Hesperia, USA) operated under the following conditions. Eluent A, acetonitrile; eluent B, water acidified with 1 mL (0.17%, w/w) of ortho phosphoric acid/L-1. Gradient: 0-5 min, 50% A; 5-25 min, linear to 100% A, at a flow rate of 1.5 mL min-1. For the optimal detection of the PAHs, a fluorescence time program was applied (40). All HPLC samples were filtered through a 1-mL polyethylene syringe with a rubber-tipped plunger (Becton Dickinson) fitted with a 0.45 µM filter (Millipore, SJHV LO4NS) prior to analysis. HPLC solvents were filtered and degassed under vacuum while stirring, and during column equilibration, solvents were purged with helium gas (28). Incubation System Workup. At the end of the incubation and final aeration, the contents of the incubation flasks were vacuum filtered through tared Schleicher & Schuell No. 604
FIGURE 2. Molecular weight calibration of the GPC system as a function of the retention time. DMF at 50 °C at 1 mL min-1 was the mobile phase. The chromatogram in the upper right corner represents a typical resolution for a mixture of 10 polystyrene standards. filter paper into a tared vacuum flask. Typically, the incubation flask was washed with 2 × 50 mL H2O followed by 2 × 5 mL acetone/dichloromethane 3:1 (v/v). Extraction of Medium Samples. Medium samples after separation from the biomass and insoluble substrates by vacuum filtration or by centrifugation were either directly analyzed by HPLC or extracted as follows. The media (ca. 90 mL from flasks or 500 mL each from soil and fungal reactors) was extracted with 3 × 50 mL (200 mL reactor) CH2Cl2. The aqueous phase was acidified to pH 3.0 with 1 M HCl and was extracted with another 3 × 50 mL CH2Cl2 (200 mL reactor). The aqueous phase was then adjusted to pH 10, with 1 M NaOH, and extracted with a further 3 × 50 mL CH2Cl2 (200mL reactor). The combined organic extracts were pooled and dried for 30 min with Na2SO4 (ca. 10 g L-1 CH2Cl2), filtered through Schleicher & Schuell No. 604 filter paper, and concentrated to an oil at 35 °C on a rotary evaporator. Biomass Treatment. Weighed, lyophilized, or air-dried fungal biomass was typically treated by swirling three times each with 1 mL acetone/CH2Cl2 3:1 (v/v) in a closed vessel for 5 min. The solution was carefully decanted after each washing. The combined organic extracts were pooled and concentrated under a gentle stream of nitrogen gas. After redissolving to a known volume with CH3CN/CH2Cl2 1:1 (v/ v), the extract was analyzed by HPLC. Gel Permeation Chromatography (GPC). For the determination of molecular weight profiles, samples were analyzed by GPC. The Nucleogel highly cross-linked polystyrene-divinylbenzene matrix column system (Macherey Nagel, Du ¨ ren, Germany) consisted of a precolumn, GPC-5P; 5 µm spheres, column 1 (GPC 105-5, 4000 × 103 Da cutoff, 5 µm spheres, 105 Å pore size), and column 2 (GPC 103-5, 60 × 103 Da cutoff, 5 µm spheres, 103 Å pore size). The complete
system was driven with HPLC-grade degassed (30 min 20 Torr under stirring) N,N-dimethylformamide (DMF, Aldrich) warmed to 50 °C with a flow rate of 1 mL min-1. Commercially available polystyrenes (Aldrich) with molecular masses of ca. 1.47 × 106 down to 687 Da served as calibration standards and were detected at 270 nm. The PDA detector was set to a wavelength region of 250-400 nm at 1.2 nm resolution. Each standard was dissolved in DMF (ca. 2 mg mL-1), allowed to stand for at least 24 h, and injected three times. The resolution of a mixture of standards as well as the associated calibration curve for all the individual standards are depicted in Figure 2. Gpc Sample Preparation. Aqueous samples were frozen in liquid nitrogen and, depending on volume, were lyophilized in either a Speed Vac (Uniequip, Martinsried, Germany) or a freeze dryer (Leybold-Heraeus, Hanau, Germany). After incubation with a minimum volume of DMF/LiCl (5% w/v, Aldrich) with a spatula tip of anhydrous Na2SO4 (Fluka) for at least 24 h, the samples were centrifuged for 10 min at 13 000 rpm in a bench-top centrifuge (Hettich, Tuttlingen, Germany) and injected. A minimum time of 20 min was allowed between runs to ensure complete equilibration and washing of the columns. Samples in organic solvents and soil samples were treated similarly, but were not lyophilized. Construction and Operation of the Reactor System. The current need for efficient soil decontamination demands that new technologies be developed to optimally exploit the capabilities of general microbial degradation. The model reactor was constructed to treat soil physically separate from the fungus (Figure 1). The only contact between soil and the fungal biomass was through the nutrient medium containing Tween 80 (0.1% v/v). The latter served to extract the PAHs out of the soil. The system was a closed, batch feed design.
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TABLE 2. Concentration of PAHs from Starting Soil and from Various Samples after Incubationa EPA Method 610 PAH
soil start (mg kg-1)
soil end (mg kg-1)
% PAH deg in soil
fungal med (mg L-1)
soil med (mg L-1)
biomass (mg)
acenaphthene acenaphthylene anthracene benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene benzo[g,h,i]perylene benzo[k]fluoranthene chrysene dibenz[a,h]anthracene fluoranthene fluorene indeno[1,2,3-c,d]pyrene naphthalene phenanthrene pyrene sum
4 700 4 962 278 4 062 3 234 2 938 2 454 2 388 810 2 789 2 647 544 2 587 1 294 1 148 4 361 41 196
