Peroxidase-Mediated Removal of a Polychlorinated Biphenyl Using

(Morris Plains, NJ), Aldrich Chemical Co. ... CA) and an electron capture detector was used to measure concentrations of PCB-4 ...... Sutton, Rebecca;...
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Environ. Sci. Technol. 2007, 41, 891-896

Peroxidase-Mediated Removal of a Polychlorinated Biphenyl Using Natural Organic Matter as the Sole Cosubstrate LISA M. COLOSI,† DANIEL J. BURLINGAME,‡ QINGGUO HUANG,‡ AND W A L T E R J . W E B E R . , J R . * ,‡ Departments of Civil and Environmental Engineering and of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109

Natural organic matter (NOM) of hydroxylated aromatic character can undergo catalyst-mediated self-coupling reactions to form larger molecular aggregates. Indeed, such reactions are central to natural humification processes. Nonhydroxylated persistent aromatic contaminants such as polychlorinated biphenyls (PCBs) are, conversely, inert with respect to such reactions. It is here demonstrated however that significant coincidental coupling and removal of a representative aqueous-phase PCB occurs during horseradish peroxidase (HRP)-catalyzed oxidative coupling reactions of a representative aquatic NOM. Experiments with Suwannee River fulvic acid as a reactive cosubstrate indicate that 2,2′-dichlorobiphenyl (PCB-4) is covalently incorporated into aggregating NOM, likely through fortuitous cross-coupling reactions. To develop a better understanding of potential mechanisms by which the observed phenomenon occurs, two hydroxylated monomeric cosubstrates of known molecular structure, phenol and 4-methoxyphenol, were investigated as alternative cosubstrates. PCB-4 removal appears from these experiments to relate to certain molecular characteristics of the native cosubstrate molecule (reactivity with HRP, favorability for radical attack, and hydrophobicity) and its associated phenoxy radical (stability). The findings reveal potential pathways by which PCBs, and perhaps other polyaromatic contaminants, may be naturally transformed and detoxified in nature. The results further provide a foundation for development of enhanced-humification strategies for remediation of PCBcontaminated environmental systems.

Introduction Biogeochemical degradation and humification comprise two classes of complex counterdirectional processes governing the ongoing environmental transformation and turnover of natural organic matter (NOM) (1). Degradation processes effect reductions in the sizes of macromolecules to smaller labile molecules for eventual mineralization, while humification involves processes in which small organic molecules are aggregated into macromolecular/supramolecular struc* Corresponding author phone: (734) 763-1464; fax: (734) 936 4391; e-mail: [email protected]. † Department of Civil and Environmental Engineering. ‡ Department of Chemical Engineering. 10.1021/es061616c CCC: $37.00 Published on Web 12/23/2006

 2007 American Chemical Society

tures. These structures may be classified as humic substances. Both classes of transformations can be exploited to achieve engineered remediation of soil and sediment systems that have been contaminated by organic xenobiotics; however, the humification pathway has generally received much less attention in this regard than has the degradation/mineralization pathway (2). Oxidative coupling processes constitute a class of reactions critically involved in NOM humification processes. These reactions, generally catalyzed by metal oxides or oxidative enzymes, have been shown to facilitate incorporation of both natural and anthropogenic phenols and anilines into actively aggregating or “polymerizing” NOM matrixes (2-5). In this sense, humification becomes an especially attractive remediation strategy because the coupling of xenobiotic monomers can mediate irreversible immobilization and ultimate detoxification (6-12). It has also been demonstrated that the removal of substrate compounds may be enhanced by the addition of a cosubstrate that is comparatively more reactive toward the selected catalyst than the target xenobiotic itself (7, 13-15). This enhancement has been attributed to an increased quantity of nonspecific free radicals generated during reactions between the catalyst and the cosubstrate and to subsequent participation of these radicals in crosscoupling reactions between substrates (6, 7). Finally, it has been suggested that phenolic cosubstrates undergoing oxidative coupling may, perhaps by the same mechanism, result in concomitant transformation of inert contaminants. That is, the presence of an active substrate has been shown to engender polymerization of compounds (e.g., polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs)) that, lacking a phenolic or anilinic subunit, may not themselves serve as active donors for the catalyst (2, 16). It has become increasingly evident, as our understanding of humification processes has evolved, that the traditional characterization of humic substances as macromolecular polymers may require reconsideration. It has indeed been suggested that these materials may be better classified as supramolecular associations of relatively small heterogeneous molecules (17, 18). This view is at least more consistent with observed direct polymerizations of humic substances in the presence of suitable catalysts. In these reactions activated aromatic constituents of the loosely bound humic structures presumably undergo the same radical-mediated polymerizations as do phenolic or anilinic monomers (17, 19-21). That humic constituents may influence the transformation of xenobiotics during oxidative coupling by participating in cross-coupling with the latter has in fact been documented (3, 5, 6, 22). Such observations support a hypothesis that certain contaminants, even though themselves inert with respect to a catalyst, may be transformed and incorporated into humic substances, presumably through free radical reactions mediating the oxidative cross-coupling reactions of cosubstrates. Motivated by the hypothesis articulated above, we in this paper describe experiments conducted to evaluate that hypothesis. Specifically, an inert xenobiotic, a PCB, is shown to be transformed effectively during the oxidative coupling reactions between a humic substance serving as the sole cosubstrate and horseradish peroxidase (HRP) as a model catalyst. In search of an understanding of associated mechanisms, we also investigated two specific monomeric test cosubstrates of well-known molecule structure, phenol and 4-methoxyphenol, and evaluated the manner in which structural features of these cosubstrates relate to the facilitation of PCB coupling and removal. Because no attempt was VOL. 41, NO. 3, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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made to measure radical species, it remains impossible to ascertain that the PCBs react directly with cosubstrate radicals; however, the cosubstrate molecular data are presented as indirect support for their presumed involvement. After decades of discharge into the natural environment, PCBs have become widely distributed in aquatic sediments, threatening human and environmental health as they make their way into both aquatic and terrestrial food chains (23). The findings presented here reveal a potential means by which these contaminants can be transformed and detoxified in the natural environment. They thus lay the groundwork and foundation for development of enhanced-humification strategies for engineered remediation of PCB-contaminated environments.

Experimental Section Materials. Extracellular horseradish peroxidase (type I, RZ ) 1.3), hydrogen peroxide (29.9%, ACS reagent grade), 2,2′azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (98% in diammonium salt form), and radiolabeled phenol (U-14Cphenol in toluene solution) were purchased from Sigma Chemical Co. (St. Louis, MO). Cosubstrates including phenol (99%), 4-methoxyphenol (99%), and Suwannee River fulvic acid (SRFA) were obtained from Acros Chemical Co. (Morris Plains, NJ), Aldrich Chemical Co. (Milwaukee, WI), and the International Humic Substances Society (IHSS), respectively. 2,2′-Dichlorobiphenyl (PCB-4) (99.35%) was obtained from Neosyn Laboratories Inc. (New Milford, CT). Quantifying Apparent Removal of PCB-4 at Selected Cosubstrate/PCB Ratios Over Consecutive Reaction Periods. Reactions mediated by HRP/H2O2 were carried out at room temperature in 5 mL of a 2.0 µM stock solution of PCB-4 prepared in a phosphate buffer solution (PBS; 10 mM, pH 7.0). Agitated Teflon-capped 11 mL glass test tubes were used as completely mixed batch reactors (CMBRs). In initial experiments designed to determine optimal cosubstrate ratios, different volumes of cosubstrate solution and complementary PBS were added, such that the total volume in each reactor was always 6 mL. In this way, an array of reactors containing varying ratios of cosubstrate to PCB was created in quadruplicate, and each CMBR contained the same final PCB concentration (1.8 µM). A 100 mg/L total SRFA stock solution was prepared and then filtered using 0.45 µm glass fiber filters to yield a soluble total organic carbon (TOC) concentration of 52.44 mg/L. Because the molecular weight of SRFA is not well characterized, the SRFA/PCB-4 ratios were then computed on a TOC basis. Concentrated stock solutions of two test cosubstrates (phenol and 4-methoxyphenol) were prepared and delivered in methanol to achieve high cosubstrate/PCB-4 ratios without significant dilution of the PCB concentration. To ensure uniform HRP activity at different ratios, each CMBR received the same total volume of methanol in the form of a stock cosubstrate solution, dilution methanol, or some combination of the two to yield a final 5.5% methanol (v/v) content. HRP activities for the phenol, 4-methoxyphenol, and SRFA ratio experiments were 0.01, 0.01, and 2 U/mL, respectively, where 1 unit (U) of HRP activity is defined as the amount catalyzing the oxidation of 1 µmol of ABTS/min. The same excess concentration of hydrogen peroxide (150 µM) was used for the reactions with phenol and 4-methoxyphenol, but a more aggressive condition (2 mM) was used with SRFA to compensate for its decreased aromaticity compared to that of phenol and 4-methoxyphenol and to ensure saturation of the increased HRP dose required in the SRFA experiments. Following addition of the hydrogen peroxide, each CMBR was agitated gently on a shake table for 30 min prior to the addition of 96 µL of 1.0 N HCl to terminate the reaction. Following termination, samples were immediately centrifuged at 2205g for 25 min to remove polymerized reaction 892

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byproducts. Equal volumes of the supernatant and isooctane were then added to a fresh tube and vortexed vigorously for 30 s. Upon separation of the phases, analytical samples were taken from the isooctane. Extraction recovery for the liquidliquid extraction was between 89% and 97%. Once the optimal ratio of cosubstrate to PCB was determined for the SRFA and the monomeric cosubstrates, experiments were conducted using a time-sequenced multiple addition reaction scheme and an initial PCB-4 concentration of 1.8 µM. In each experiment, an appropriate volume of phenol, 4-methoxyphenol, or SRFA was added to achieve the predetermined optimal cosubstrate/PCB-4 ratios. Specifically, 24 identical CMBRs were prepared to test each cosubstrate. Four blank CMBRs each received a volume of PBS equal to the sum of the HRP + H2O2 doses and served as the sample for time t ) 0. The remaining reactors were dosed with the enzyme and catalyst and set on the shaker table to react. At preselected 30 min time intervals, sets of four test tubes were “sacrificed” by additions of the acid to terminate the reactions. The remaining test tubes were then redosed, set back on the shake table, and allowed to react for additional 30 min intervals ranging from 60 min to 3 h. A control reaction was performed using this same protocol (HRP + H2O2 + PCB-4) in the absence of any cosubstrate. It should be noted that HRP dosages were selected to ensure saturation of the enzyme with respect to the cosubstrate at time zero for a subsequent 30 min reaction period, i.e., 2 U/mL for SRFA, 0.75 U/mL for phenol, and 0.007 U/mL for 4-methoxyphenol. Although an excess concentration of hydrogen peroxide (150 µM) was used for both monomeric cosubstrates, an even more aggressive condition (2 mM) was used with SRFA. A Hewlett-Packard 5890 series II gas chromatograph equipped with a Zebron ZB-1 column (30 m × 0.25 mm × 0.25 µm; Phenomenex, Torrance, CA) and an electron capture detector was used to measure concentrations of PCB-4 in the isooctane samples. Nitrogen was used as the carrier gas at a flow rate of 6.4 mL/min. Injected sample volumes were 4 µL, and initial temperatures for the injector, oven, and detector were 280, 350, and 325 °C, respectively. The retention time for PCB-4 was 10.3 min using the following heating protocol: from 110 to 200 °C in 8 min and held until 15 min, from 200 to 250 °C in 2 min and held until 20 min. Following GC analysis, the difference between the average control (prereaction) and each experimental (postreaction) concentration was computed to determine apparent PCB removal in each reacted sample. Characterizing Adsorptive versus Reactive Removal of PCB-4. PCB removal via adsorption to precipitated products of phenol coupling was also evaluated using a method previously described by Weber and Huang (2). Six triplicate sets of test tube reactors received 4 mL each of 1.8 µM PCB-4 stock solution and varying dosages of dry preformed polymer solids (adsorbent). The contents of these reactors were mixed on a shaker table for 2.5 h and the reactors then centrifuged to separate solids from the aqueous phase. Aliquots of supernatant were then transferred to fresh test tubes, in which aqueous-phase PCBs were extracted into equal volumes of isooctane for subsequent GC analysis. The doses of adsorbent were selected to simulate the amount of precipitated polymers formed as a function of time during reaction of phenol with HRP: 0 mg/L (0 min), 50 mg/L (∼60 min), 100 mg/L (∼90 min), 300 mg/L (∼120 min), and 600 mg/L (∼150 min). The doses were selected on the basis of a time-course evaluation of the reaction of radiolabeled phenol (14C-phenol) with HRP. Briefly, 2 L of 1500 µM 14C-phenol stock solution was prepared in PBS, stirred continuously, and dosed at 30 min intervals with 0.5 U/mL HRP and 150 µM H2O2. Prior to each dose, 2 mL samples were collected and centrifuged to remove solids. The supernatant was then analyzed in a

scintillation counter to determine the concentration of 14Cphenol in the aqueous phase. This quantity was then subtracted from the starting concentration to determine how much phenol had been converted to precipitated solids as a function of time. An identical reaction, using phenol rather than 14C-phenol, was performed to generate the required quantities of preformed adsorbent. Following termination of this reaction, the entire contents were centrifuged at 5000 rpm for 20 min. The pellets were then combined into a single batch and then evaporated to dryness under nitrogen gas. The masses required for the doses specified previously were weighed out individually. Evaluating the Effects of PCB-4 on the Rates of HRP Reactions with Test Cosubstrates. Following determination of optimal cosubstrate/PCB-4 ratios for both test cosubstrates, the effects of the PCB on the initial rate of reaction between HRP and each test cosubstrate were evaluated. To this end, an approach similar to that described in a previous paper by Colosi et al. was taken (24). Stock solutions of phenol (1600 µM) and 4-methoxyphenol (500 µM) in PBS were prepared with and without the addition of PCB-4 (1.8 µM) to achieve the optimal cosubstrate/PCB-4 ratios in those CMBRs containing both chemicals. Reactors containing 5 mL of stock solution were run in triplicate, both with and without PCBs, and then dosed with sufficiently low HRP to ensure saturation of the enzyme with respect to the cosubstrate, i.e., 0.75 and 0.01 U/mL for phenol and 4-methoxyphenol, respectively. Reactions were initiated upon addition of 150 µM H2O2 and the CMBRs shaken by hand for 20 s prior to addition of 100 µL of 1.0 N HCl to terminate the reaction. Blank CMBR tests were run in triplicate with and without PCB-4 and dosed with equivalent volumes of PBS in place of H2O2. Immediately following acid addition, all CMBRs were centrifuged at 2205g for 25 min, and the supernatants from each reactor were transferred to amber HPLC vials. An Agilent 1100 series high-performance liquid chromatograph equipped with a Phenomenex C18 column (250 × 2.0 mm, 5 µm particle size) was used to detect pre- and postreaction concentrations of phenol (mobile phase 40% reagent-grade acetonitrile (ACN), 60% distilled deionized water (DDI), flow rate 0.3 mL/min, tR ) 4.7 min) and 4-methoxyphenol (mobile phase 30% ACN, 70% DDI, flow rate 0.4 mL/min, tR ) 3.9 min). For both chemicals, concentrations were measured using UV absorbance with external five-point calibration. Following measurement of the cosubstrate concentration in the blank (S0) and reaction CMBRs (S20), initial reaction rates (r0) were defined for the first reaction interval according to r0 ) (S0 S20)/∆t. Determinations of Pertinent Molecular Descriptors of Cosubstrate Molecules. Correlations between molecular descriptors and PCB-4 removal efficiency were attempted for both monomeric cosubstrates evaluated. Literature values for octanol-water partitioning coefficients (log P) were employed to assess their relative hydrophobicity (25). Electronic parameters utilized in a comparison of molecular reactivity for the selected cosubstrates were computed using the HyperChem Molecular Modeling System, professional version 7.1 (Hypercube, Inc., Gainesville, Florida) (26). Cosubstrate structures were optimized using the OPLS molecular mechanics force field and the Polak-Ribiere optimization algorithm set to a convergence gradient criterion of less than 0.1 kcal/(Å mol). Subsequent quantum optimization was achieved using the ZINDO/1 semiempirical method with the same convergence algorithm and criterion. After optimization, EHOMO (energy of the highest occupied molecular orbital), ELUMO (energy of the lowest unoccupied molecular orbital), and the HOMO-LUMO gap (H-L gap ) ELUMO - EHOMO) were computed for each cosubstrate molecule. Polarizability, an indicator of reactivity that parametrizes

FIGURE 1. Apparent PCB-4 removal following reaction of HRP with the cosubstrate at an array of selected cosubstrate/PCB ratios. Enzyme concentrations used in conjunction with SRFA (top), phenol (middle), and 4-methoxyphenol (bottom) as the cosubstrate are 2, 0.01, and 0.01 U/mL, respectively. Error bars represent 95% confidence intervals for the mean value (n ) 4). the flexibility of a molecule’s electron cloud, was also calculated. Finally, quantum-optimized structures of each cosubstrate’s respective radical (phenoxy and 4-methoxy) were computed using the ZINDO/1 algorithm in conjunction with unrestricted Hartree-Fock (UHF) calculations and the same convergence criterion. In each case, the total charge was set to zero with doublet multiplicity. The quantumoptimized structures were used to compute the total energy (Etotal) for each radical.

Results and Discussion Quantifying Apparent PCB-4 Removal at Selected Cosubstrate/PCB Ratios. Figure 1 depicts apparent removal of PCB-4 following 30 min reactions of HRP with SRFA, phenol, and 4-methoxyphenol at an array of selected cosubstrate/ PCB ratios. Despite dramatic differences in the cosubstrate molecules investigated, their impacts on PCB-4 removal share a common general trend, as shown in Figure 1. In each instance, PCB-4 removal increases with increasing cosubstrate concentration from zero to a maximum and then begins to decrease with a further increase in cosubstrate concentration. This is consistent with the hypothesis that HRP reactions generate free radicals in their reactions with cosubstrates that can then nonselectively attack PCB molecules, leading to their incorporation in precipitable products of the oxidative coupling reactions. The PCB molecule, because it lacks a hydroxylated aromatic moiety, is not itself a direct HRP substrate, such that some cosubstrate must be reacted with the enzyme to generate nonspecific phenoxy radicals. At low concentrations of cosubstrate (corresponding to the lower ratios), rates of PCB-4 removal appear to be limited by suboptimal rates of radical formation. In contrast, when cosubstrate concentrations are higher than optimal (corresponding to higher ratios), interactions between radicals and PCB molecules become increasingly less probable; i.e., the preponderance of cosubstrate molecules effectively outcompetes the PCB. Thus, most efficient PCB removal will occur at the optimal ratio of cosubstrate to PCB. As indicated in Figure 1, this ratio is 10× for SRFA, 900× for phenol, and 200× for 4-methoxyphenol. It is noteworthy that while each of the plots in Figure 1 shares the same general trend, each reaches a different maximum value at dramatically different optimal ratios. Characterizing Apparent PCB-4 Removal over Consecutive Reaction Periods. Figure 2 depicts the results of VOL. 41, NO. 3, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. PCB-4 concentrations over the course of consecutive 30 min reaction periods after multiple reagent additions for three cosubstrates and a cosubstrate blank (control). Enzyme concentrations used in conjunction with SRFA, phenol, and 4-methoxyphenol as the cosubstrate are 2, 0.75, and 0.007 U/mL, respectively. Error bars represent 95% confidence intervals for the mean (n ) 4).

FIGURE 3. PCB-4 removal during adsorption to preformed polymers of phenol coupling (]) and PCB-4 removal during reaction as mediated by HRP with phenol as the cosubstrate (9). Error bars represent 95% confidence intervals for the mean (n ) 3). sequential 30 min reaction experiments with three different cosubstrates and in the absence of a cosubstrate (control). In each experiment, the optimal ratio of cosubstrate to PCB depicted in Figure 1 was used. As expected, results from the control reaction confirm that no significant removal of PCB-4 can be achieved in the absence of a cosubstrate. In contrast, appreciable removal of PCB-4 is achieved in the presence of each cosubstrate after each cumulative sequential reaction period over the course of 3 h: 77% removal with SRFA, 97% removal with phenol, and 87% removal with 4-methoxyphenol. Although the nature of the experiments in which the data presented in Figure 2 were collected precludes direct mechanistic interpretation of reaction rates, it is nonetheless evident that the cumulative transformation and removal process for each cosubstrate appears to be pseudo-first-order with respect to PCB-4 concentration. The PCB-4 disappearance rate constants (k1), obtained using a first-order reaction rate to simulate the data in Figure 2, are 0.007 min-1 (R2 ) 0.99), 0.025 min-1 (R2 ) 0.85), and 0.014 min-1 (R2 ) 0.99) for reactions with SRFA, phenol, and 4-methoxyphenol, respectively. Such apparent PCB removal rates provide a relative comparison of the effectiveness of PCB removal among systems in the presence of different cosubstrates. Characterizing Apparent Removal: Differentiating between Adsorption and Chemical Reaction. Following characterization of reactive PCB removal over time, the strictly adsorptive removal was evaluated using preformed polymer products as the adsorbent. Figure 3 depicts the fraction of PCB-4 remaining as a function of time, where removal is thought to occur solely by reversible adsorption (]). To reiterate, abscissa values for the adsorption system were not measured directly in time units. Instead, these were achieved using the 14C-phenol reaction to track precipitate formation as a function time as described in the Experimental Section 894

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(2). Additionally, data from the time course reaction experiment at the optimal phenol/PCB-4 ratio (repeated from Figure 2) is presented as a reference (9). At every measurement time other than the initial, the reaction + adsorption system contains less remaining PCB than the strictly adsorptive system. Predictably, the extent of PCB-4 removed in a strictly adsorptive system increases with the dosage of preformed polymer, ultimately achieving in excess of 60% removal over the course of what would be equivalent to a reaction of approximately 150 min (600 mg/L adsorbent present). In comparison, the simultaneously reactive and adsorptive system (9) accomplishes a more significant reduction in PCB4, on the order of 98%, in the same period of time. Using the difference between the two curves at each measurement as a crude indicator, Figure 3 seems to suggest that at least 40-50% of the removal exhibited during simultaneous reaction + adsorption may be attributed to a mechanism other than reversible adsorption. Because the precipitated reaction polymers tend to dissolve in the solvents most efficiently capable of desorbing the PCBs, no desorption data were collected. This makes it impossible to definitively apportion the remainder of the removal between irreversible covalent binding and semi-irreversible sorption at inaccessible locations within the precipitated products. The occurrence of both mechanisms has been previously demonstrated by Weber and Huang (2). Importantly, either results in more permanent sequestration and detoxification than might be achieved by removal solely via reversible adsorption. It is also noteworthy that SRFA does not form any precipitate during HRP-mediated oxidative coupling reactions at conditions tested in this study. The high levels of PCB removal observed in the presence of SRFA can thus be attributed principally to PCB association with humic substances in forms resistant to isooctane extraction, likely covalent bonds formed through fortuitous oxidative coupling reactions. Rationalizing Differential PCB-4 Removal for the Selected Cosubstrates. As a first step toward understanding reasons for the observed differences in reactivity among the three cosubstrates tested, we investigated the potential effects of the PCB-4 on initial rates of cosubstrate coupling and removal. One-parameter t tests indicate that the presence of PCB-4 has no significant effect on the initial rate of reaction at the optimal ratio of cosubstrate to PCB for either of the monomeric cosubstrates. P values are 0.15 for phenol and 0.41 for 4-methoxyphenol. As we have seen, the selection of cosubstrate has a dramatic effect on the rate of apparent PCB removal despite the fact that the presence of PCB seems to have no effect on the rate of cosubstrate removal. It is possible that the dramatic differences in apparent PCB removal exhibited in the presence of each cosubstrate are directly related to the rate at which the cosubstrate itself reacts with HRP. In previous investigations of HRP-mediated degradation of phenolic compounds we measured Michaelis-Menten (M-M) parameters rmax (maximum reaction rate) and KM (halfsaturation concentration) for an array of chemicals including both phenol and 4-methoxyphenol (24). According to the classical M-M model, rmax is achieved only when the substrate concentration (S0) is large enough to saturate the enzyme according to the relationship rmax ) kcatE0, where E0 is the initial enzyme concentration. Rearrangement of this formulation yields an expression for the enzyme-normalized maximum reaction rate (kcat) according to kcat ) rmax/E0. HRP dosages for investigating HRP-mediated PCB removal were selected to ensure that the enzyme was saturated with respect to the cosubstrate at time zero. As such, the rate constant for the removal of PCB-4 in the presence of each test cosubstrate obtained through fitting to the data presented in Figure 2

TABLE 1. Observed Constants for Apparent PCB-4 and Cosubstrate Removal as Well as Selected Molecular Characteristics for the Monomeric Cosubstrates Evaluated Cosubstrate

ln(kPCB) ln(kcat) EHOMO ELUMO H-L gap polariz- log P (s-1) (s-1) (eV) (eV) (eV) ability (A3) (25)

phenol -8.74 4-methoxy- -5.09 phenol

7 10

-8.18 7.85 -7.09 7.62

16.03 14.71

11.07 13.54

1.46 1.58

can be further normalized using the appropriate enzyme concentration. Table 1 presents the normalized apparent rate of PCB-4 degradation, kcat values measured for degradation of the cosubstrate in the absence of PCB-4, and selected molecular descriptors of the evaluated cosubstrates. The cosubstrate 4-methoxyphenol, which reacts more quickly with HRP than phenol, also mediates more effective removal of PCB-4. The data presented in Table 1 are thus in accord with our conjecture that the apparent rate of PCB removal is dependent on the rate of reaction between HRP and the cosubstrate. Consistent with the hypothesized mechanism by which HRP reactions enable cross-coupling and ultimately mediate removal of PCB-4, it seems likely that those cosubstrates degraded most efficiently by the enzyme generate a larger quantity of radicals per unit of time, enabling faster polymerization of the PCB, which itself has little effect on the reaction rate. While each cosubstrate’s ability to generate radicals appears to be associated with its optimal rate of PCB removal, the ratio at which the PCB removal exhibits optimal rates seems to depend on the electronic character of a cosubstrate’s molecular structure. It seems reasonable that characteristics facilitating radical attack might improve the probability that the cosubstrate is preferentially polymerized over the PCB, effectively reducing the rate at which the PCB will be removed from solution. Of the molecular descriptors in Table 1, the H-L gap and polarizability are related to the overall favorability of radical attack toward a molecule of a given cosubstrate. As indicated, phenol exhibits a higher H-L gap than 4-methoxyphenol. This suggests that phenol molecules are more stable and less excitable than molecules of 4-methoxyphenol (14, 27). Similarly, the difference in polarizability between the two cosubstrates indicates that the electron cloud surrounding phenol is less flexible than that of 4-methoxyphenol. On the basis of these criterion comparisons, 4-methoxyphenol is in both instances a better target for radical attack, consistent with Figure 1 in which the optimal ratio for phenol (900×) is significantly higher than the optimal ratio for 4-methoxyphenol (200×). At ratios higher than that shown to be optimal for PCB removal in Figure 1 (bottom), the inherent favorability of 4-methoxyphenol radical attack makes it such that PCB polymerization becomes increasingly improbable. In contrast, a much higher concentration of the phenol molecules may be present during the reaction because they are unable to compete as effectively with the PCB. Extrapolated, this trend can be used to infer the relative favorability of radical attack toward the PCB in the presence of the SRFA as the cosubstrate. As indicated in Figure 1, the observed optimal ratio of SRFA to PCB-4 (10×) is an order of magnitude smaller than even that of 4-methoxyphenol. This suggests that SRFA is even more optimally suited for attack by radical species, scavenging as it does radicals that might otherwise mediate the incorporation of a PCB molecule. This is consistent with the fact that it mediates slower PCB-4 removal than phenol and 4-methoxyphenol, as well as with previous investigations in which NOM was found to be a good radical scavenger (3, 29, 30). More subtle than the inference regarding differences in optimal ratios across cosubstrates, Figure 1 also indicates that the optimal extent of reactive removal could be related

to the stability of each cosubstrate’s associated radical. All things being equal, a stable radical species will be longerlived, increasing its opportunity to interact with a molecule of PCB. The 4-methoxy radical (ZINDO-computed Etotal ) -52703 kcal/mol) is more stable and ostensibly more likely to promote PCB polymerization than the phenoxy radical (ZINDO-computed Etotal ) -37083 kcal/mol). This is consistent with a decreased extent of PCB removal for phenol (30%, middle of Figure 1) relative to 4-methoxyphenol (37%, bottom of Figure 1) at their respective optimal ratios. Again, this trend may be extrapolated to make inferences about the structure of radicals formed during reaction of HRP with SRFA as the cosubstrate. The extent of PCB removal for SRFA (29%, top of Figure 1) is somewhat smaller but not significantly different from that for phenol. This perhaps indicates that electron-donating aliphatic substituents of SRFA radicals make them structurally more similar to phenoxy than 4-methoxy radicals, but less stable than either. Finally, it seems possible that the differences in hydrophobicity between the test cosubstrates may affect the relative effectiveness of adsorptive removal as the values of their octanol-water partitioning coefficients (P) are slightly different. As indicated in Table 1, 4-methoxyphenol (log P ) 1.58) is slightly more hydrophobic than phenol (log P ) 1.46). By extension, polymers formed from the oxidative coupling of 4-methoxyphenol might more readily adsorb the highly hydrophobic PCB-4 molecules, contributing to the greater apparent removal as indicated by the enzyme-normalized rate constants presented in Table 1. Significantly, as noted earlier, no precipitation of polymers was observed during reactions with the SRFA. This implies that PCB removal in the case of this natural cosubstrate resulted principally from its covalent incorporation into the SRFA matrix.

Acknowledgments We thank Jennifer Gehle for her diligent laboratory assistance and Tom Yavaraski for important guidance with respect to the analytical procedures and methodologies. This research was financed in large measure by Research Grant P42ES0491114 from the National Institute of Environmental Health Sciences. L.M.C. appreciates the support provided by a National Science Foundation Graduate Research Fellowship.

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Received for review July 7, 2006. Revised manuscript received October 19, 2006. Accepted November 2, 2006. ES061616C