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Journal of Environmental Quality 32:63-69 (2003)
© 2003 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Bioremediation and Biodegradation

Enzymatic Oxidative Transformation of Chlorophenol Mixtures

J.-M. Bollag*,a, Horng-Lun Chua, M. A. Raob and L. Gianfredab

a Laboratory of Soil Biochemistry, Center for Bioremediation and Detoxification, 129 Land and Water Research Building, The Pennsylvania State University, University Park, PA 16802
b Dipartimento di Scienze del Suolo, della Pianta e dell'Ambiente, Università di Napoli Federico II Via Università 100, 80055 Portici, Italy

* Corresponding author (jmbollag{at}psu.edu)

Received for publication March 4, 2002.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorinated phenols are major industrial and agricultural xenobiotics that pollute soil and ground water. It has been shown that laccases catalyze the oxidative coupling of phenolic compounds. Therefore, the transformation of one or a mixture of several chlorinated phenols by a laccase from the fungus Trametes villosa was studied. Generally, if more than one phenol was added, the transformation of chlorinated phenols decreased, and if the concentration of the laccase was increased, the transformation of the phenols was enhanced. There were exceptions to these observations: for instance, the transformation of 0.1 mM 4-chlorophenol incubated with 1 mM 2,4-dichlorophenol in buffered salt solutions was not enhanced if the concentration of the laccase was increased from 2 to 20 DMP units/mL. The reason for the reduced transformation of chlorinated phenols in the presence of additional phenols is still unknown. However, in spite of some limitations, the application of laccase to decontaminate wastewater polluted with chlorinated phenols appears feasible.

Abbreviations: 4-CP, 4-chlorophenol • 2,4-DCP, 2,4-dichlorophenol • 2,4,6-TCP, 2,4,6-trichlorophenol


   INTRODUCTION
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 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HALOGENATED ORGANIC chemicals, hazardous waste products, are often discharged into the environment through wastewater. They may reach concentrations of up to hundreds of parts per million in industrial wastewater, or in urban and agricultural runoff (Annachhatre and Gheewala, 1996). Chlorinated phenols are a major group of halogenated organic waste products that pollute soil and ground water (Mills et al., 1985). The most important sources of these compounds can be traced either to paper manufacturers or fungicide and herbicide producers (Annachhatre and Gheewala, 1996). Additionally, wastewater from industries such as polymeric resin production, oil refining, and coking plants also contain chlorophenols (Beltrame et al., 1980).

Because of the toxicity of chlorophenols and their persistence in the environment, techniques for their removal are urgently needed. Currently, many physical and chemical methods are available for decontaminating chlorinated phenols. But, considering the high costs of these procedures, attention has been focused on oxidative coupling of chlorophenols. The oxidative process gives rise to less soluble, higher molecular weight polymers; these can consequently be removed by sedimentation or filtration.

Extracellular oxidoreductases (i.e., peroxidases, laccases, and tyrosinases), mostly of microbial origin, are involved in oxidative coupling processes of chlorophenols (Bollag et al., 1995; Gianfreda and Bollag, 2002). These enzymes have been shown to remove a variety of organic chemicals, including chlorophenols, from aqueous solutions and wastewater (Klibanov et al., 1983; Davis and Burns, 1990; Dec and Bollag, 1990; Tatsumi et al., 1996; Sun and Payne, 1996). The effect of laccases from different sources, both in their free and immobilized forms, on the transformation of chlorophenols, has been extensively studied (Gianfreda et al., 1999 and references therein). It has been demonstrated that dechlorination usually occurs as a secondary reaction of chlorophenol laccase-mediated oxidation (Dec and Bollag, 1990; Roy-Arcand and Archibald, 1991; Gianfreda and Bollag, 2002).

Although much work has been done on the effect of laccases on the transformation of individual phenolic compounds, few studies have been performed to elucidate the transformation of mixtures of chlorophenols by laccases. Wastewater usually contains many different pollutants rather than one single chemical. Indeed, a better understanding of possible enhancing or inhibiting effects because of the simultaneous presence of more than one phenolic compound on the efficiency of an enzymatic catalytic process may be of great environmental significance.

The major goals of this study were to (i) investigate the transformation of chlorinated phenols, when present as a mixture, by a fungal laccase of T. villosa, and (ii) evaluate the counteracting effects of the phenolic compounds on their enzymatic transformation. The chlorophenols used in this study were 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP). Two of them, 2,4-DCP, and 2,4,6-TCP, are USEPA priority pollutants (Mills et al., 1985).


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Chlorophenols were purchased from Aldrich Chemical Co. (Milwaukee, WI). Methanol (high-performance liquid chromatography [HPLC] grade) was obtained from Fisher Scientific (Pittsburgh, PA).

Enzymes
An extracellular laccase of T. villosa was obtained from Novo Nordisk Biotech (Davis, CA). One unit of laccase activity (DMP unit) is defined as the amount of enzyme that causes a change in absorbance at 468 nm of 1.0/min at pH 4 when 2,6-dimethoxyphenol (3.24 µmol) is used as the substrate in 3.5 mL of 1 mM citrate–phosphate buffer (Dec and Bollag, 1990; Roper et al., 1995). Absorbance was measured with a Bausch and Lomb (Rochester, NY) Model 2000 spectrophotometer.

Enzyme Assays
Preliminary experiments with citrate (0.1 M)–phosphate (0.1 M) buffer (pH 3.0 to 6.0) and 0.2 M phosphate buffer (pH 7.0 to 8.0) were performed to select the most appropriate incubation pH. Substrates (0.5 mM) were incubated in triplicate at 25°C for 24 h with specified amounts of enzyme (2 or 4 DMP) in 10 mL of 0.2 mM phosphate buffer at pH 7.0. Incubations without enzymes served as controls.

Experiments with different initial concentrations of substrates (0.1 to 1 mM) in binary and ternary mixtures and for different incubation times were also performed. Specific reaction conditions adopted in each experiment are detailed in the legends of the figures and tables.

High-Performance Liquid Chromatography Analysis
At specified times, the reaction assays were stopped with 0.5 mL of 5 M phosphoric acid. The acidified samples and controls were stored at 4°C and filtered before HPLC analysis. Aliquots of 20 µL were then analyzed by HPLC. Analysis was performed with a Waters Associates (Milford, MA) system consisting of two Model 510 delivery systems, a Waters Model 717 plus autosampler, a Bischoff Chromatography Nucleosil 120-5-C18 (250 x 4 mm) column, a Supelco (Bellefonte, PA) LC18-DB guard column, a Waters–Millipore Lambda-Max Model 480 LC spectrophotometer operated at 280 nm, a Waters–Millipore automated gradient controller, and a Hewlett-Packard (Palo Alto, CA) 3392A integrator. The mobile phase consisted of A (99% water + 1% acetic acid) and B (100% methanol) delivered at a flow rate of 0.8 mL/min in various ratios of A to B according to the substrate analyzed (30:70 for 2,4,6-TCP and 40:60 for 4-CP and 2,4-DCP). Retention times for the various substrates were between 4 and 15 min.


   RESULTS
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transformation of Chlorinated Phenols by a Laccase of Trametes villosa
Preliminary tests were performed to select the experimental conditions (e.g., pH, amount of the laccase of T. villosa, incubation time) to be used in this study. Assays were performed by incubating 0.5 mM chlorophenols at 25°C and for 24 h in a pH range between 3 and 8. Results demonstrated that the enzyme was active over a broad pH range for all chlorophenols. Consistent transformation of 4-CP (approximately 45%), 2,4-DCP (approximately 100%), and 2,4,6-TCP (approximately 100%) was measured at pH values between 4.0 and 7.0, 5.0 and 7.0, and 4.0 and 7.0, respectively. At pH 8.0 a significant reduction of activity was observed for all chlorophenols with a complete loss of activity for 4-CP. For further studies, pH 7.0 was chosen for maximizing the activity of the laccase with all three substrates. At this pH, the transformation efficiency of the three chlorophenols was 2,4-DCP > 2,4,6-TCP > 4-CP.

Figure 1 shows the time course for transforming 0.5 mM of various chlorinated phenols by T. villosa laccase (2 DMP units/mL) in phosphate buffer at pH 7.0 and at 25°C. 2,4-DCP was quickly and completely transformed by the laccase after a 24-h incubation; 4-CP was least transformed and 2,4,6-TCP showed an intermediate behavior. Most of the transformation of all three compounds occurred within the first 6 h of the 24-h incubation period (44, 66, and 47% transformation of 4-CP, 2,4-DCP, and 2,4,6-TCP, respectively). After that, an additional 20% of 4-CP, 30% of 2,4-DCP, and 30% of 2,4,6-TCP were transformed (Fig. 1). The time necessary to achieve 50% of transformation of the initial substrate concentration was 42.3 x 103, 14.7 x 103, and 26.4 x 103 s for 4-CP, 2,4-DCP, and 2,4,6-TCP, respectively, thus confirming a compound oxidation rate in the order 2,4-DCP > 2,4,6-TCP > 4-CP.



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  		Fig. 1. Time course for transformation of 0.5 mM chlorinated phenols by 2 DMP units/mL laccase of Trametes villosa. Incubations were performed in phosphate buffer at pH 7 and 25°C (standard deviation was within 5%).

 
The time course for the transformation of 4-CP in buffered salt solutions by different concentrations of the laccase is shown in Fig. 2 . As previously described, when incubated with 2 DMP units/mL laccase, most of 4-CP transformation occurred within the first 6 h of incubation. After 48 h of incubation, 31% of 4-CP was still present in the reaction mixture. When 4-CP was incubated with double the amount of enzyme (4 DMP units/mL laccase), the reaction rate increased correspondingly. After a 6-h incubation 69% and after 48 h 100% of the substrate were transformed. In contrast with this result, a lower laccase efficiency was observed when the 4 DMP units/mL laccase were added stepwise. A first dose of 2 DMP/mL laccase was added in the beginning, followed by a second dose of 2 DMP/mL laccase after 6 h of incubation. After 24 h of incubation 4-CP transformation was increased by 20%, while 6% of untransformed 4-CP remained in the solution 48 h after the first addition of the enzyme (Fig. 2).



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  		Fig. 2. Time course for transformation of 4-chlorophenol (0.5 mM) by various concentrations of a laccase of Trametes villosa. Incubations were performed in phosphate buffer at pH 7 and 25°C (standard deviation was within 5%).

 
Transformation of a Mixture of Two Chlorinated Phenols by the Laccase of Trametes villosa
Table 1 shows the transformation of 0.5 mM chlorophenols when they were incubated for 24 h at 25°C and pH 7.0 alone and as binary and ternary mixtures. The simultaneous presence of 4-CP and 2,4-DCP in the reaction mixture negatively affected their transformation. Compared with the transformation as single substrates, a decrease of about 15% of both 4-CP and 2,4-DCP transformation was measured. However, while a marked reduction (approximately 18%) was observed for 2,4,6-TCP incubated in the presence of 4-CP, no significant decrease was measured for 4-CP transformation. Similarly, when 2,4-DCP and 2,4,6-TCP were incubated together, the transformation of 2,4-DCP was reduced by 12% while that of 2,4,6-TCP remained the same.


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  		Table 1. Transformation of 0.5 mM chlorophenols alone and as binary and ternary mixtures by a laccase of Trametes villosa.{dagger}

 
Experiments were performed to assess the influence of the initial substrate concentration on the transformation of chlorophenol mixtures. Figure 3 presents the transformation of 4-CP and 2,4-DCP when incubated together at different initial concentrations. With increasing 2,4-DCP concentrations, the transformation of 4-CP was reduced. For example, in the absence of 2,4-DCP, 67% of 0.1 mM 4-CP was transformed, while in the presence of 1.0 mM 2,4-DCP, only 36% of 0.1 mM 4-CP was oxidized. However, the higher the original concentration of 4-CP, the smaller was the inhibitory influence of 2,4-DCP on 4-CP transformation. For instance, the addition of 1.0 mM 2,4-DCP decreased the transformation of 1.0, 0.5, and 0.1 mM 4-CP by 20, 28, and 31%, respectively.



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  		Fig. 3. Transformation by 2 DMP units/mL laccase of Trametes villosa of 4-chlorophenol in the absence and presence of various concentrations of 2,4-dichlorophenol, and 2,4-dichlorophenol in the absence and presence of various concentrations of 4-chlorophenol. Incubations were performed at 25°C in phosphate buffer at pH 7 for 24 h (standard deviation was within 5%).

 
Similarly, 4-CP inhibited the transformation of 2,4-DCP at all concentrations investigated (Fig. 3). For example, the transformation of 1.0 mM 2,4-DCP in the presence of 0.0, 0.1, 0.5, and 1.0 mM 4-CP was 88, 71, 60, and 44%, respectively. In contrast to 4-CP transformation in the presence of 2,4-DCP, the higher the original concentration of 2,4-DCP, the larger was the inhibitory effect of 4-CP on 2,4-DCP transformation. In the presence of 1.0 mM 4-CP, the transformation of 1.0, 0.5, and 0.1 mM 2,4-DCP was reduced by 44, 35, and 34%, respectively.

Table 2 presents the transformation of 0.1 mM 4-CP and 2,4-DCP by a laccase of T. villosa in the absence and presence of 1 mM 2,4-DCP and 4-CP, respectively, as a function of enzyme activity. By increasing the concentration of the enzyme, the transformation of 4-CP was enhanced whereas 2,4-DCP was completely transformed regardless of the laccase concentration. In the binary mixture, the transformation of 2,4-DCP at the lowest enzyme concentration was appreciably decreased by the presence of 4-CP. When the enzyme concentration was increased from 2 to 20 DMP units/mL, however, no inhibition was evident and 2,4-DCP was completely transformed. By contrast, the transformation of 0.1 mM 4-CP in the presence of 1 mM 2,4-DCP was not appreciably enhanced even when the enzyme concentration was increased from 2 to 20 units/mL.


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  		Table 2. Transformation of 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP) at different concentrations of a laccase from Trametes villosa as influenced by the addition of a 10-fold concentration of the two phenols.{dagger}

 
Transformation of a Mixture of Three Chlorinated Phenols by a Laccase of Trametes villosa
When 0.5 mM 4-CP, 2,4-DCP, and 2,4,6-TCP were incubated as a ternary mixture, combined effects were observed (Table 1). For instance, 2,4-DCP was transformed less than if alone or in a binary mixture. By contrast, 4-CP transformation was less than that measured when the phenol was incubated alone or in the presence of 2,4,6-TCP but higher than in the binary mixture with 2,4-DCP. In regard to 2,4,6-TCP transformation, it was practically the same if 2,4,6-TCP was part of a ternary mixture or a binary mixture together with 4-CP, indicating a prevalent inhibitory effect by the presence of 4-CP.

Figures 4 through 6 show the transformation of the three chlorinated phenols when incubated in a ternary mixture at different initial concentrations. For comparison purposes, the transformation of each phenol in binary mixtures is also depicted. In the presence of 2,4,6-TCP at all the initial concentrations of 4-CP, 4-CP was transformed at about the same level or slightly higher than it was in the absence of 2,4,6-TCP. When the three chlorinated phenols were incubated together, the transformation of 4-CP was about 5% less than its transformation in the absence of the other two phenols, regardless of the concentration of 4-CP.



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  		Fig. 4. Transformation of various amounts of 4-chlorophenol by 2 DMP units/mL laccase of Trametes villosa in the absence and presence of 2,4-dichlorophenol and 2,4,6-trichlorophenol. Incubations were performed at 25°C in phosphate buffer at pH 7 for 24 h (standard deviation was within 5%).

 


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  		Fig. 6. Transformation of various amounts of 2,4,6-trichlorophenol by 2 DMP units/mL laccase of Trametes villosa in the absence and presence of 4-chlorophenol and 2,4-dichlorophenol. Incubations were performed at 25°C and pH 7 for 24 h (standard deviation was within 5%).

 


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  		Fig. 5. Transformation of various amounts of 2,4-dichlorophenol by 2 DMP units/mL laccase of Trametes villosa in the absence and presence of 4-chlorophenol and 2,4,6-trichlorophenol. Incubations were performed at 25°C and pH 7 for 24 h (standard deviation was within 5%).

 
The transformation of 2,4-DCP at the 0.1 mM concentration was not influenced by the presence of one or two of the other phenols (Fig. 5). The presence of 4-CP reduced the transformation of 2,4-DCP, when its initial concentration was increased. The transformation of 0.1 mM 2,4-DCP was also reduced by the addition of 2,4,6-TCP, but to a lesser degree. When the three chlorinated phenols were incubated together, 2,4-DCP was transformed less than it was when incubated alone, especially at 0.5 and 1 mM concentrations.

When comparing the transformation of 0.1 to 1.0 mM 2,4,6-TCP alone or with equal amounts of 4-CP or 2,4-DCP, the addition of 4-CP caused various degrees of decreases in transformation, while the addition of 2,4-DCP had barely any effect (Fig. 6). At 0.1 mM 2,4,6-TCP the addition of an equal amount of 4-CP decreased the transformation of 2,4,6-TCP, while the addition of equal amounts of 2,4-DCP or 4-CP plus 2,4-DCP had no effect on 2,4,6-TCP transformation. While at 0.25 mM 2,4,6-TCP the addition of an equal amount of 4-CP had no effect on 2,4,6-TCP transformation, the addition of an equal amount of 4-CP at 0.5 and 1.0 mM caused significant decreases in the transformation of 2,4,6-TCP. At 0.25 to 1.0 mM 2,4,6-TCP the greatest decreases in transformation were seen when equal amounts of both 4-CP and 2,4-DCP were added.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Laccase from T. villosa was able to transform chlorinated phenols whether they were present alone or in combinations of two or three. According to the results obtained by Dec and Bollag (1990) and Shuttleworth and Bollag (1986) with a laccase from Trametes versicolor and Rhizoctonia praticola, 2,4-DCP was transformed more easily than 2,4,6-TCP and 4-CP, in the order listed (Fig. 1). The greater suitability of 2,4-DCP as a substrate for T. villosa laccase activity is also evident from the results of kinetic experiments (Fig. 1) and values for the time necessary to achieve 50% transformation of the initial substrate concentration. When 2,4-DCP was the substrate, the reaction proceeded more rapidly and 50% transformation of the initial phenol concentration was achieved in a shorter time period.

A different response to laccase catalysis by the three chlorophenols was also evident in the results reported in Fig. 3 through 6. The higher the initial concentration of 4-CP, the lower was its enzymatic transformation. No similar effects were evident with 2,4-DCP and 2,4,6-TCP.

When investigating the effect of incubation time on the transformation of 4-CP by various amounts of enzyme, the results indicate that the enzyme loses much of its activity after 6 h of incubation (Fig. 2). When a second dose of 2 DMP units/mL laccase was added 6 h after incubating 4-CP with 2 DMP unit/mL laccase, it caused almost complete transformation of the remaining 4-CP. However, even with this addition, transformation did not reach the same level (100%) as using 4 DMP units/mL laccase from the beginning. These findings suggest that a step-wise process for transforming 4-CP is not necessary, but that an initial concentration of 4 DMP units/mL laccase is the most efficient method.

The different response of the three chlorophenols to T. villosa laccase action could be ascribed to different mechanisms and, possibly different reaction products, occurring during the transformation process. First, interactions (adsorption and/or hydrogen binding) between phenoxy radicals, formed as intermediates during the oxidative reaction, and the enzyme's active site could take place, which would reduce the enzyme activity (Nakamoto and Machida, 1992). The presence of an additive, such as polyethylene glycol, reduced the adsorption of chemicals, and consequently removed enzyme inhibition (Nakamoto and Machida, 1992; Wu et al., 1993). Second, the laccase could be inhibited by the chloride ions released during the reaction. Chloride ions reportedly contribute to limited accessibility of the Type 2–Type 3 trinuclear copper cluster sites of the laccase (Xu, 1996).

Partial loss of enzymatic activity through either inhibition by reaction products or removal of active enzyme molecules from the reaction mixture also has to be considered. In experiments performed with different phenolic compounds, Gianfreda et al. (1998) and Filazzola et al. (1999) showed that a reduction of Cerrena unicolor laccase activity usually occurred. Further experiments, performed either after different incubation times or with different initial concentrations of 2,4-DCP and/or catechol, demonstrated that the residual enzyme activity decreased with increasing substrate concentration or incubation time. The authors explained their findings by assuming that laccase molecules may be incorporated into and/or adsorbed onto the newly formed polymeric products during the course of the reaction, thus partially losing their catalytic activity.

Previous research has shown that in the absence of cosubstrates the chlorophenols used in this study could be almost completely transformed by the addition of sufficient amounts of enzymes (Klibanov et al., 1983; Bollag et al., 1988; Dec and Bollag, 1990). Roper et al. (1995) further demonstrated that in the presence of guaiacol and 2,6-dimethoxyphenol, some chlorophenols, which without the cosubstrates produced only minor transformation, could be transformed up to 95% with even lower enzyme amounts.

In this study, when chlorophenols were incubated together, transformation of each chlorophenol was reduced, probably due to mutual competitive inhibition of enzyme activity. There were, however, exceptions to this finding. The transformation of 4-CP was not reduced when incubated with 2,4,6-TCP (Fig. 4). The transformation of neither 2,4-DCP nor 2,4,6-TCP was reduced when the two substrates were incubated together (Fig. 5 and 6). Generally, the influence of 4-CP prevailed over that of the other two phenols. For example, the inhibiting effect of 4-CP on 2,4,6-TCP transformation was observed when 2,4,6-TCP was incubated in the presence of 4-CP and 2,4-DCP (Fig. 6).

Various amounts of laccase were used to determine whether higher concentrations of the laccase could reverse the reduced transformation capacity when two chlorophenols were incubated together. The results showed that with an increase of 2 to 20 DMP laccase units/mL, the transformation of 0.1 mM 4-CP in the presence of 1 mM 2,4-DCP was not appreciably enhanced (Table 2). By contrast, 0.1 mM 2,4-DCP, in the presence of 1.0 mM 4-CP, was almost completely transformed when the enzyme concentration was increased from 2 to 20 DMP units/mL (Table 2).

Although not easily explainable, these results seem to support the hypothesis of a different mechanism involved in the enzymatic oxidation of the two chlorophenols. Furthermore, the assumption of an increased removal of laccase molecules from the reaction in the presence of high 2,4-DCP concentrations, not occurring at equal amounts of 4-CP, could partly justify the different behavior observed. Further studies, however, are needed to clarify these findings.

The results reported in this paper indicated that an oxidoreductive enzyme, such as a laccase, could oxidize mixtures of chlorinated phenols. Our experimental findings demonstrate that a laccase of T. villosa was efficient in the transformation of several chlorophenols, when incubated alone or in complex mixtures of two or three substrates. Its activity was, however, influenced by the simultaneous presence of more than one substrate in the reaction solution, generally by a reduction of its catalytic efficiency. In spite of this drawback the use of laccase for the bioremediation of complex wastewater appears feasible.


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 MATERIALS AND METHODS
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DiSSPA Contribution 0009.


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