Journal of Environmental Quality 31:1509-1515 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORTS
Bioremediation and Biodegradation
Treatment of 2,4-Dichlorophenol Polluted Soil with Free and Immobilized Laccase
Mi-Youn Ahna,
Jerzy Deca,
Jang-Eok Kimb and
Jean-Marc Bollag*,a
a Laboratory of Soil Biochemistry, The Pennsylvania State University, 129 Land and Water, University Park, PA 16802
b Dep. of Agricultural Chemistry, Kyungpook National University, Taegu, 702-701, Korea
* Corresponding author (jmbollag{at}psu.edu)
Received for publication October 8, 2001.
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ABSTRACT
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Enzyme treatment is currently considered for remediation of terrestrial systems polluted with organic compounds. In this study, two soils from Pennsylvania with 2.8 or 7.4% organic matter contents (Soils 1 and 2, respectively) were amended with 14C-labeled 2,4-dichlorophenol (2,4-DCP) and incubated with a laccase from Trametes villosa (free or immobilized on montmorillonite). 2,4-DCP was either transformed to methanol-soluble polymeric products (11–32%) or covalently bound to soil organic matter (53–85%); unaltered 2,4-DCP could be recovered from soil by methanol extraction (0–38%) at the completion of a 14-d incubation period. In Soil 1, both free and immobilized laccase removed 100% of 2,4-DCP without regard for moisture conditions. In Soil 2, immobilized laccase removed more 2,4-DCP (about 95%, regardless of moisture conditions) than free enzyme (55, 75, and 90% at 30, 55, and 100% of maximum water-holding capacity, respectively). Binding of 2,4-DCP in the humin fraction was nearly the same for free and immobilized laccase. More 2,4-DCP, however, was bound to humic and fulvic acids in the presence of immobilized laccase than in the presence of free laccase. In general, immobilized laccase performed better than free laccase. However, for practical applications, the higher activity of immobilized laccase is offset by a 23% loss in enzyme activity during immobilization, which approximates the 30% increase in free laccase needed to achieve the same level of remediation. Furthermore, immobilized laccase is more costly than free T. villosa laccase.
Abbreviations: 2,4-DCP, 2,4-dichlorophenol • HPLC, high-performance liquid chromatography • WHC, water-holding capacity
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INTRODUCTION
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ENZYMATIC TREATMENT is currently considered an alternative method for the removal of toxic xenobiotics from the environment (Dick and Tabatabai, 1993; Gianfreda et al., 1999; Karam and Nicell, 1997). Laboratory experiments have demonstrated that phenols and aromatic amines may be removed from water by the application of various phenoloxidases, such as peroxidase, laccase, or tyrosinase (Bollag, 1992; Fillazzola et al., 1999; Dec and Bollag, 2000). The underlying mechanism of the removal involves enzymatic oxidation of the pollutants to free radicals or quinones that subsequently undergo polymerization and partial precipitation (Dec and Bollag, 1994).
Similar enzyme-mediated transformation reactions may also occur in soil, where the oxidation products bind covalently to humus (Bollag, 1992). For instance, Flanders et al. (1999) found that up to 92% of 2,4-dichlorophenol (2,4-DCP) was irreversibly bound to soil constituents after application of minced horseradish roots containing large amounts of peroxidase. Hydrogen peroxide or calcium peroxide applied along with horseradish served as electron acceptors. Laccases from Trametes versicolor and Rhizoctonia praticola mediated extensive binding of 2,4-DCP (up to 65%) to dissolved humic materials (Sarkar et al., 1989). These enzymes required the presence of molecular oxygen as an electron acceptor. In soil contaminated with the pesticide 2,4-D (2,4-dichlorophenoxyacetic acid), transformation of phenolic derivatives occurred after applying cell-free enzymes (Shannon and Bartha, 1988).
Despite the abundance of promising experimental data, a number of limitations still restrict the use of enzymes to detoxify xenobiotics in the environment. First, many cell-free enzymes are short lived in soil environments. Enzymatic activity may be reduced or entirely eliminated through both nonbiological and biological deactivation factors, such as adsorption on soil colloids, extreme acidity or alkalinity, or biodegradation by proteases (Ruggiero et al., 1996). Additionally, soil organic matter may have an inhibitory effect on enzyme activity in terrestrial systems (Gianfreda and Bollag, 1994, 1996).
Immobilization on solid supports is a proven approach for increasing the stability of enzymes under unfavorable conditions (Gianfreda and Bollag, 1994). Enzymes have been successfully immobilized on porous glass beads (Weetall, 1969; Messing, 1970), various clay minerals (Shuttleworth and Bollag, 1986; Leonowicz et al., 1988; Naidja et al., 1997), and soil (Sarkar et al., 1989; Ruggiero et al., 1989). Immobilization has also been achieved by entrapment in a variety of matrices, including alginate beads or organic gels (Kennedy and Cabral, 1987; Davis and Burns, 1990; Crecchio et al., 1995). Use of natural supports, such as clay or soil, represents an attractive option for treatment of terrestrial systems (Gianfreda and Bollag, 1994).
The objective of this study was to determine the potential of the laccase from T. villosa to remediate soil polluted with 2,4-DCP. The experiments were performed with either free laccase or laccase immobilized on montmorillonite. The specific objectives of the study were to (i) determine the effect of different factors (incubation time, laccase concentration, soil moisture, aging of laccase in soil) on the extent of 2,4-DCP binding and polymerization; (ii) determine the distribution of 2,4-DCP transformation products between different soil fractions; and (iii) evaluate the performance of the immobilized laccase in soil as compared with that of free laccase.
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MATERIALS AND METHODS
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Chemicals
The chemicals 2,4-dichlorophenol (2,4-DCP) and 2,6-dimethoxyphenol were purchased from Aldrich Chemical Co. (Milwaukee, WI). Uniformly ring-14C-labeled 2,4-DCP with a specific activity of 7.47 x 108 Bq mM-1 was bought from Sigma Chemical Co. (St. Louis, MO). The chemicals for immobilization of laccase (glutaraldehyde and 3-aminopropyltriethoxylane) were obtained from Acros Organics (Pittsburgh, PA). Extracellular laccase (T. villosa, EC 1.10.3.2) was obtained from Novo Nordisk (Danbury, CT). Montmorillonite (Wyoming bentonites; surface area = 31.82 m2 g-1; cation exchange capacity = 76.4 cmol kg-1) and kaolinite (Georgia kaolin; surface area = 10.05 m2 g-1; cation exchange capacity = 2.0 cmol kg-1) were purchased from Ward's Natural Science Establishment (Rochester, NY).
Soils
Two soils used in the experiments were collected (top 10 cm) from two sites in Centre County, Pennsylvania. They differed considerably in organic matter contents (2.8 and 7.4% organic matter in Soils 1 and 2, respectively); their names and physical and chemical properties are summarized in Table 1. Prior to testing, the soils were air-dried, passed through a 2-mm sieve, and stored at 4°C.
Laccase Immobilization
Laccase was immobilized on montmorillonite and kaolinite as described by Ruggiero et al. (1989) and Gianfreda and Bollag (1994). Briefly, the minerals were prepared for immobilization by consecutive shaking with 0.5 M HNO3 and a 0.09 M solution of 3-aminopropyltriethoxylane in acetone. The activated mineral was treated with a 0.5 M solution of glutaraldehyde in a phosphate buffer (pH 7.0) and stirred for 24 h with specific amounts of the laccase dissolved in the buffer. The immobilized enzyme was recovered by centrifugation, washed with buffer until no laccase activity was detected in the washings, resuspended in the buffer, and stored at 4°C until used.
Measurement of Laccase Activity
The activity of free and immobilized laccase was determined with a biological oxygen monitor (Model 5300; Yellow Spring Instruments Co., Yellow Springs, OH) equipped with a Clark oxygen electrode (5357 Micro Oxygen Probe; Yellow Springs Instruments Co.). A specific amount of free enzyme in solution or a suspension of immobilized enzyme in 0.1 M citrate–phosphate buffer (2.7 mL, pH 3.8) was incubated at 25°C in the sample chamber under agitation with a Teflon-coated magnetic bar. The enzymatic reaction was initiated by the addition of a 10 mM 2,6-dimethoxyphenol solution in the same buffer (0.1 mL stored previously at 25°C) through a side port next to the electrode. One unit of enzymatic activity was defined as the amount of free or immobilized laccase required to consume 1 mol min-1 of O2.
Experimental Procedures
To determine enzyme stability, free or immobilized laccase was aged in 2-g soil samples (3800 unit g-1 soil; triplicates) for 0, 1, 7, or 14 d at 25°C and 55% of the maximum water-holding capacity (WHC, which was 354 g kg-1 for Soil 1 and 679 g kg-1 for Soil 2). During the incubation period, 0.1-g subsamples were withdrawn and analyzed for enzyme activity in the biological oxygen monitor, as described above.
To evaluate the feasibility of enzymatic treatment, the soils were mixed with uniformly ring-labeled 14C-2,4-DCP (8.3 x 105 Bq kg-1) to result in an initial concentration of 1000 mg kg-1, distributed into 15-mL test tubes (1 g; dry weight) and incubated in triplicate with varying amounts of free or immobilized laccase (950, 1900, 3800, or 7600 unit g-1) for 0, 1, 7, and 14 d. In a separate experiment, free or immobilized laccase was incubated with soil for 0 to 14 d prior to the addition of 14C-2,4-DCP and evaluated after a 24-h incubation. The experiments were performed under different moisture conditions: 30, 55, or 100% of the maximum WHC. After incubation, soil samples were extracted two times with 5.0 mL of methanol to remove free residues of 2,4-DCP and/or its metabolites (on Day 0, the samples were extracted 1 min after the incubation started). Each of the combined methanol extracts was analyzed for the remaining 2,4-DCP by high-performance liquid chromatography (HPLC) and for radioactivity by liquid scintillation counting. The methanol-extracted soil (0.1 g) was combusted to 14CO2 in an OX-600 biological oxidizer (R.J. Harvey Instrument Corporation, Hillsdale, NJ) and analyzed for radioactivity with a liquid scintillation counter. The combustion temperatures were 900°C (combustion chamber) and 680°C (catalyst zone).
Soil Fractionation
Free or immobilized laccase (3800 unit g-1) was aged in 5-g soil samples amended with 2,4-DCP (1000 mg kg-1 soil) and 14C-labeled 2,4-DCP (8.3 x 105 Bq kg-1 soil) for 0, 1, 7, or 14 d at 25°C and 55% of the maximum WHC. After incubation, the samples were extracted two times with methanol (by stirring at room temperature for 1 h with a magnetic bar) to remove free 2,4-DCP and/or methanol-soluble transformation products and centrifuged at 10 000 x g. The solids were air-dried overnight and extracted for 24 h by shaking with 30 mL of 0.5 M NaOH under nitrogen. The NaOH extract was separated by centrifugation, and the soil pellet was washed three times with 0.1 M NaOH. The combined NaOH extract and washings were acidified (pH < 1) with 5 M HCl and stored overnight at 4°C to facilitate humic acid precipitation. Humic acid was separated from the fulvic acid fraction by centrifugation. The fulvic acid–containing supernatant was extracted two times with 70 mL of methylene chloride. The combined methylene chloride extracts were evaporated and redissolved in 2 mL of methanol. The humic acid pellet was dissolved in 1 mL of 0.5 M NaOH after washing with acidified water. The NaOH-extracted soil (designated as humin) was dehydrated by freeze-drying. All three fractions were then analyzed by radiocounting.
Analytical Procedures
Before HPLC analysis, the methanol extracts were passed through a 0.45-µm nylon membrane filter (Millipore Corporation, Milford, MA). The analysis was conducted on a Waters (Milford, MA) HPLC system consisting of a 2690 Alliance separation module, a Waters 2487 dual 
absorbance detector operating at 280 nm, and a Supelcosil 15-cm x 4.6-mm LC-18 DB column of 5-µm particle size with a LC-18 DB guard column (Supelco, Bellefonte, PA). The mobile phase, delivered at a flow rate of 1.0 mL min-1, was composed of an aqueous component A (0.33 M acetic acid, 0.018 M ammonium acetate) and an organic component B (methanol, 0.33 M acetic acid, 0.018 M ammonium acetate) mixed at a 35:65 A to B ratio. Radioactivity measurements were conducted on a Beta Trac 6895 liquid scintillation counter (Tracor Analytic, Elk Grove Village, IL). Aliquots of methanol extracts (1 mL) and fulvic acid fraction (1 mL) were analyzed in 10 mL of Ecoscint (Manville, NJ). The methanol-extracted soil (0.1 g), the humin fraction (0.1 g), and the humic acid fraction (0.1 mL) were combusted in an OX 600 biological oxidizer to 14CO2, which was trapped in Harvey 14C cocktail for radiocounting.
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RESULTS
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Enzyme activity measurements indicated that montmorillonite had a greater capacity for laccase immobilization than kaolinite. Maximum immobilization on montmorillonite was 77% of the added enzyme, whereas only 6% was immobilized on kaolinite (data not shown). The difference probably resulted from the fact that the surface area and cation exchange capacity of montmorillonite (31.82 m2 g-1 and 76.4 cmol kg-1, respectively) were greater than those of kaolinite (10.05 m2 g-1 and 2.0 cmol kg-1, respectively). After a 14-d exposure to soil, free laccase lost 80% (Soil 1) and 100% (Soil 2) of the initial activity, whereas the activity of immobilized laccase was reduced by 17 and 33%, respectively (Fig. 1)
. The loss of enzyme activity in aqueous solution was negligible (<5%).
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Fig. 1. Activity (% of initially added; 3800 unit g-1) of free or immobilized laccase of Trametes villosa in soils under 55% of maximum water-holding capacity (WHC) moisture condition, as determined by a biological oxygen monitor with 2,6-dimethoxyphenol as substrate. The standard error (SE) for activity (%) ranged between 0.1 and 2.3.
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Incubation of Soil 1 both with free and immobilized laccase led to more than 88% disappearance of 2,4-DCP after 1 d, and the chemical completely disappeared after 14 d, regardless of moisture conditions (Fig. 2)
. After a 1-d incubation in Soil 2, the removal of 2,4-DCP in the presence of free laccase was 49, 23, and 30% at 30, 55, and 100% WHC, respectively, and it increased to 62, 76, and 92%, respectively, after 14 d. Immobilized laccase induced more than 95% removal of 2,4-DCP after 14 d as compared with 74, 60, and 53% removed on Day 1 at 30, 55, and 100% WHC, respectively (Fig. 2).
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Fig. 2. Disappearance of 2,4-DCP (measured by high-performance liquid chromatography, HPLC) following the treatment of 14C-2,4-DCP-polluted Soils 1 and 2 with free or immobilized laccase of Trametes villosa (3800 unit g-1) under different moisture conditions (30, 55, and 100% of maximum water-holding capacity, WHC). The standard error for 2,4-DCP (%) ranged between 0.1 and 3.5.
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The disappearance of 2,4-DCP was largely due to its binding to soil. During a 14-d incubation under different moisture conditions, binding of 2,4-DCP to Soil 1 in the presence of free or immobilized laccase ranged from 53 to 76% or from 57 to 80%, respectively, while the control samples (without enzyme addition) showed a binding of only 3 to 17% and 5 to 13%, respectively, depending on moisture conditions. In Soil 2, the enzyme-mediated binding was 23 to 75% and 62 to 85%, respectively, as compared with 4 to 22% and 3 to 20% binding for controls (Fig. 3)
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Fig. 3. Binding of radioactivity following the treatment of 14C-2,4-DCP-polluted Soils 1 and 2 with free or immobilized laccase of Trametes villosa (3800 unit g-1) under different moisture conditions (30, 55, or 100% of maximum water-holding capacity, WHC). The SE for radioactivity (%) ranged between 0.1 and 4.0. A * indicates controls obtained by treating the 14C-2,4-DCP-polluted soils with Milli-Q water or montmorillonite without the enzyme.
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Fractionation of soil incubated with free laccase (Table 2) revealed that in Soil 1, the greatest amounts of bound radioactivity were present in the humin fraction (24–27%), whereas in Soil 2, most binding occurred in humic acid (20–33%). For immobilized laccase (Table 3), the greatest amounts of bound radioactivity were found in the humic acid fraction of Soil 1 (23–36%) and Soil 2 (32–49%).
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Table 2. Distribution of radioactivity in different fractions of soils during incubation with free laccase of Trametes villosa (3800 unit g-1) for 0, 1, 7, and 14 d under 55% water-holding capacity (WHC) moisture condition.
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Table 3. Distribution of radioactivity in different fractions of soils during incubation with immobilized laccase of Trametes villosa (3800 unit g-1) for 0, 1, 7, and 14 d under 55% water-holding capacity (WHC) moisture condition.
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The rate of 2,4-DCP binding to soil increased with enzyme concentration (Fig. 4)
. For instance, from 10 to 80% of 2,4-DCP was immobilized in Soil 1 during 14 d of incubation as the concentration of free laccase increased from 950 to 7600 unit g-1. With immobilized laccase, 2,4-DCP binding to Soil 1 increased from 48 to 75% with increasing enzyme concentration. In Soil 2, the increase in 2,4-DCP binding was from 19 to 80% for free laccase and from 75 to 80% for immobilized laccase.
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Fig. 4. Binding of radioactivity following the treatment of 14C-2,4-DCP-polluted Soils 1 and 2 with different amounts of free or immobilized laccase of Trametes villosa under 55% of maximum water-holding capacity (WHC) moisture condition. The standard error for radioactivity (%) ranged between 0.1 and 4.0. A * indicates original enzyme activity applied for immobilization.
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When free or immobilized laccase was aged in Soil 1 for 0, 1, 7, and 14 d prior to the addition of 14C-2,4-DCP, the percentage of 2,4-DCP binding was 63 to 69% after a 24-h incubation, regardless of the aging period (data not shown).
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DISCUSSION
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The concentration of 2,4-DCP (1000 mg kg-1) in this study was in the range of soil cleanup criteria established by the New Jersey Department of Environmental Protection (Frasco, 1999) for residential and nonresidential areas (170 and to 3100 mg kg-1, respectively). The 2,4-DCP was not aged or equilibrated in soil before enzyme treatment to model an emergency scenario or a post-spill situation. However, the results are also relevant to 2,4-DCP, which following aging or equilibration, is present in soil solution or can be readily desorbed from soil under wet conditions.
The ability of phenoloxidases to polymerize phenols and mediate covalent binding of these chemicals to humic substances is well documented (Bollag, 1992). Immobilization of phenoloxidases on solid supports proved to enhance the enzymatic transformation of phenols in aqueous solutions (Gianfreda et al., 1999); little was known, however, about the performance of immobilized phenoloxidases in soil. This study demonstrated that phenols can be efficiently transformed in soil by both free and immobilized laccase from T. villosa.
The activity of free laccase quickly decreased during a 14-d incubation with soil (from 100% to 20 and 0% in Soils 1 and 2, respectively), whereas in the absence of soil, only a 5% decrease was observed (Fig. 1). Due to immobilization on montmorillonite, laccase activity in both soils remained practically unaffected for 7 d, and it dropped to only 80 and 70% in Soils 1 and 2, respectively, by Day 14 (Fig. 1). These results are consistent with previous studies (Sarkar et al., 1989) in which immobilized laccases showed increased stability in the presence of soil as compared with free enzymes. The decrease in enzyme activity (for both free and immobilized laccase) was faster in Soil 2 than in Soil 1, which explains the reduced rate of 2,4-DCP disappearance in Soil 2 compared with Soil 1 (Fig. 2).
In Soil 1, there were no significant differences between the disappearance of 2,4-DCP in the presence of free or immobilized laccase. In Soil 2, however, the disappearance of 2,4-DCP in the presence of immobilized laccase was faster than with free laccase (Fig. 2). The difference was possibly due to the enhanced resistance of the immobilized enzyme to the inhibitory action of soil organic matter, which was about three times greater for Soil 2 (7.4%) than for Soil 1 (2.8%). Enzyme inhibition by soil organic matter has been demonstrated for an oxidoreductase (Pflug, 1980; Sarkar and Bollag, 1987), a protease (Ladd and Butler, 1969), an invertase, and a phosphatase (Malcolm and Vaughan, 1979).
As a result of enzymatic treatment, 2,4-DCP was either transformed to methanol-soluble polymeric products or covalently bound to soil organic matter (Tables 2 and 3). Both polymerization and binding to soil are believed to have a detoxification effect (Sjoblad and Bollag, 1981; Berry and Boyd, 1985; Shannon and Bartha, 1988). Svenson et al. (1989) and Oberg et al. (1990) reported the formation of dioxins and dibenzofurans during these reactions, which raised some environmental concerns. However, the yields of such products were extremely low (0.0001% of the transformed substrate) and probably resulted from a 10-fold excess of the added H2O2. It is also important that phenoloxidase-mediated reactions lead to the dehalogenation of chlorinated phenols (Dec and Bollag, 1994). Dehalogenated products are usually less toxic and more susceptible to biodegradation than the halogenated parent compounds.
Increasing soil moisture was expected to (i) enhance 2,4-DCP transformation by facilitating enzyme contact with the substrate and (ii) reduce the inhibitory effect of soil organic matter following its dilution with water. However, increasing water content in Soil 1 did not have any effect on the disappearance of 2,4-DCP in the presence of either free or immobilized laccase (Fig. 2). In Soil 2, the disappearance of 2,4-DCP was somewhat enhanced as moisture increased in the presence of free laccase. Nevertheless, during incubation with immobilized laccase, soil moisture had little effect on 2,4-DCP disappearance. A similar pattern was observed for 2,4-DCP binding: little effect of soil moisture in Soil 1 and some increase in 2,4-DCP binding in Soil 2, especially for free laccase (Fig. 3).
The transformation of 2,4-DCP in the presence of either free or immobilized laccase was practically over after 1 d of incubation in Soil 1 and after 7 d of incubation in Soil 2 (Fig. 2). The time courses for 2,4-DCP binding show similar patterns (Fig. 3), which indicates that 2,4-DCP was simultaneously transformed to methanol-soluble products and bound to soil organic matter and that the soluble products hardly participated in binding. In view of this outcome, the rapid decrease in the activity of free laccase (Fig. 1) is of little consequence. By Day 7, when free laccase was still active (40 and 20% of the initial activity in Soils 1 and 2, respectively; Fig. 1), the process of binding of 2,4-DCP to soil was almost completed (Fig. 3).
Methanol extracts from soil amended with 14C-2,4-DCP and then incubated with the T. villosa laccase contained up to 59% of the initial radioactivity (Tables 2 and 3). At the completion of incubation, the extractable radioactive material mainly consisted of methanol-soluble products of 2,4-DCP transformation; virtually no unaltered 2,4-DCP was present in the extracts from Soil 1, as determined by HPLC, and only 5 to 38% of unaltered 2,4-DCP was found in the methanol extracts from Soil 2 (Fig. 2).
In general, immobilized laccase performed better than free laccase, particularly in Soil 2 (Fig. 3). However, for practical soil remediation applications, immobilized T. villosa laccase may have a limited advantage over free laccase. The greater longevity of immobilized laccase compared with free laccase (Fig. 1) is not critical during a 14-d incubation (Fig. 3). This was shown when soil was incubated with free or immobilized laccase for 14 d followed by 2,4-DCP addition, which resulted in no significant differences in 2,4-DCP binding after 24 h (data not shown). Furthermore, immobilized laccase had no advantage over free laccase for binding within soil fractionation. The most stable binding of xenobiotics is expected to occur in humin (Barriuso and Koskinen, 1996); however, 2,4-DCP binding in humin in the presence of immobilized laccase was comparable with that determined for free laccase (Tables 2 and 3).
The rates of 2,4-DCP binding for immobilized laccase were greater than those for free laccase (Fig. 4), but only at low laccase concentrations (950 and 1900 unit g-1). At higher laccase concentrations (3800 and 7600 unit g-1), the rates of 2,4-DCP binding for free and immobilized laccase were comparable. For future soil remediation applications, the loss of enzyme activity during immobilization must be considered. For each unit of immobilized laccase, there was a 23% loss in activity from the immobilization (Fig. 4). Consequently, the advantage of immobilized laccase is minimal because the amount of activity lost with immobilization is roughly similar to the added amount of free laccase needed to maintain the same level of remediation. Then, factoring in the high cost of immobilization, using free T. villosa laccase for soil remediation becomes a more practical option.
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ACKNOWLEDGMENTS
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Funding for this research was provided by the USEPA Office of Research and Development (Grant no. R-823847). M.-Y. Ahn was partially supported by the Penn State Biogeochemical Research Initiative for Education (BRIE) sponsored by NSF (IGERT) Grant DGE-9972759.
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