Environ. Sci. Technol. 2009, 43, 7068–7073
Removal of Acetaminophen Using Enzyme-Mediated Oxidative Coupling Processes: II. Cross-Coupling with Natural Organic Matter JUNHE LU AND QINGGUO HUANG* Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223, USA
Received January 14, 2009. Revised manuscript received July 31, 2009. Accepted July 31, 2009.
The influence of natural organic matter (NOM) on the transformation of acetaminophen in laccase-mediated oxidative coupling systems was investigated in this study. It was found that the removal of acetaminophen was enhanced while the self-coupling of acetaminophen was suppressed in thepresenceofdissolvedNOM,likelyresultingfromcross-coupling betweendissolvedNOMandacetaminophen.Inadditiontocrosscoupling with acetaminophen, NOM moieties could couple to each other upon reaction with laccase. This was evidenced by the development of a characteristic absorbance band centered at 472 nm. According to the rate of the absorbance change at 472 nm, the NOM coupling reactions in four different NOM solutions were evaluated. Apparently, the tendency of NOM coupling reactions correlates with the tendency of acetaminophen cross coupling with NOM in these solutions. Possible reaction pathways of cross-coupling were explored using guaiacol as a model NOM proxy, and the products were extracted and analyzed with mass spectrometry (MS). The results suggested that acetaminophen and guaiacol molecules were cross-coupled via the formation of CsOsC bonds.
Introduction Trace levels of human-derived pharmaceuticals and endocrine disrupting chemicals (EDCs) in water represent an emerging concern to the public and research communities. Most of these micropollutants are relatively hydrophilic which enables them to escape the conventional water treatment processes (1-5). As a consequence, they are released to the environment in a continuous mode and pose a potential risk to the aquatic ecosystem and eventually to humans (3, 6). The water/wastewater treatment industry faces a potential challenge to develop effective and practicable methods to address these contaminants in order to control and reduce their environmental exposure. Chemical oxidation and UVenhanced oxidation have been found effective to remove these micropollutants (1, 4, 7-9). However, the efficiency of these methods is often limited by the extremely low concentration of the contaminants (ppt or lower). In addition, large-scale application of these strategies can be costly due to the requirement of high energy and reagent input. As an alternative to oxidative degradation, enzymemediated oxidative coupling processes have been proposed to be a potentially cost-effective strategy to remove certain * Corresponding author phone: (770) 229-3302; fax: (770) 4124734; e-mail:
[email protected]. 7068
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micropollutants. Auriol et al. tested the feasibility of using this process to remove estrogen hormones, and demonstrated that both the hormone compounds and the estrogen activity at environmentally relevant levels could be effectively removed using horseradish peroxidases (HRP) or laccase to catalyze oxidative coupling reactions under mild conditions (10-12). Performance of the enzyme was not significantly affected by municipal wastewater matrices in the laccasecatalyzed systems (11). In our previous work (13), acetaminophen was found to be able to actively participate in laccasecatalyzed oxidative coupling reactions and couple to each other to form products with varying degrees of polymerization via a radical reaction mechanism. Our result suggests that laccase mediates a single-electron oxidation of the substrates yielding free radicals, and the resultant radicals can then couple with each other by CsC bonds formation resulting in polymeric products. The products that are soluble can continue to serve as substrates for further oxidative coupling and form larger polymers that can be more readily removed from water. Laccase-catalyzed oxidative coupling processes require little energy and minimal reagent input, and can be easily integrated into conventional water treatment systems without significant capital and operational costs. In real conditions, however, a number of factors may affect the performance of the enzyme and the efficiency of the transformation, which have to be fully understood prior to a rational design. How the presence of NOM could impact such coupling reactions of micropollutants represents an important factor to be understood in light of the facts that (i) NOM is ubiquitous in water and often a critical factor influencing various physicochemical treatment processes (14); and (ii) micropollutants are usually present in waters at concentrations orders of magnitude lower than NOM. This study was conducted with the attempt of addressing how the presence of NOM may impact on laccase-mediated oxidative coupling reactions of acetaminophen. Water-soluble NOM is primarily comprised of organic acids that are often referred to as humic substances, which can be operationally categorized into a relatively smaller and more soluble fulvic acid fraction, and a larger and less soluble humic acid fraction (14, 15). NOM, depending on origin, contains varying amounts of aromatic moieties with various functional substituents, including carboxyl, hydroxyl, methoxyl, carbonyl, and amino groups (16). It has been suggested that NOM is present in water as supramolecular assemblies, collections of relatively small molecules that are selfassociated (17-21). Because NOM almost invariably contains phenolic functionalities, it can potentially serve as active substrates to laccase and other oxidative coupling catalysts. Experimental evidence indicates that NOM molecules can under appropriate conditions be effectively reconfigured at both molecular and supramolecular levels by oxidative coupling, yielding products with larger molecular sizes (21-26). Molecular characterization suggested that oxidative coupling resulted in a decrease of aromatic hydroxyl (phenolic) groups and an increase of aromatic ether groups, indicating the cross-linking of NOM moieties via CsOsC bonds (22, 24). Cross-linking leads to a significant reduction of the potential of the NOM to form disinfection byproducts (DBPs) upon chlorination, since phenolic moieties in NOM molecules are major precursors that cause DBP formation (24). Because both NOM and acetaminophen can serve as active substrates of laccase, we hypothesize that crosscoupling can occur in laccase-mediated reaction systems. This possibility was systematically investigated in the present 10.1021/es9001295 CCC: $40.75
2009 American Chemical Society
Published on Web 08/13/2009
study, with a particular aim of examining how such crosscoupling may be dependent on NOM characteristics and how it may impact acetaminophen removal.
Experimental Section Reagents. All reagents were ACS grade or better. Acetaminophen (98%) and guaiacol (99%) was obtained from SigmaAldrich Co (St. Louis, MO). A stock solution of each at the concentration of 1 mM was prepared and stored at 4 °C. Laccase was also purchased from Sigma-Aldrich, and its activity was determined immediately prior to each experiment, using a photometric method as described in the companion paper (13). NOM. Fulvic and humic materials were kindly provided by the Department of Chemical Engineering at the University of Michigan which were extracted from Canadian peat and Chelsea soil using the standard International Humic Substances Society (IHSS) protocol up to the steps before desalting (27). Both fulvate and humate fractions extracted from each soil were used in the experiments as model NOM materials, which were respectively designated as Canadian peat fulvates (CPFV), Canadian peat humates (CPHM), Chelsea soil fulvates (CSFV), and Chelsea soil humates (CSHM). Each model NOM was reconstituted in 0.01 M phosphate buffer (pH 7.0) and filtered through 0.45 µM membrane to obtain NOM solutions that were used throughout the study. The total dissolved organic carbon (DOC) content of each NOM solution was determined using a Shimadzu 5050A TOC analyzer (Shimadzu, Columbia, MD) and the UV absorbance measured with a Beckman DU640B spectrophotometer (Beckman, Fullerton, CA). The data are given in Supporting Information (SI) Table S1 for each NOM material. It should be noted that the model NOM materials are of terrestrial origin and may differ from those typically present in water or wastewater in terms of certain property and reactivity. Coupling Reaction. Acetaminophen coupling reactions mediated by laccase in NOM and NOM-free control solutions were conducted in 50 mL flasks at room temperature. Each reactor contained 20 mL of a model NOM solution (DOC concentrations given in SI Table S1) containing acetaminophen (initial concentration 20 µM). The initial dosage of enzyme was 1.0 unit/mL. After 1 h of reaction, 0.5 mL solution was withdrawn from the reactor and immediately mixed with 0.5 mL methanol which was found to be sufficient to completely denature the enzyme and stop the reaction. Blank control samples were also prepared that contain acetaminophen in NOM solutions in the absence of laccase. No acetaminophen loss was observed in these control samples after 1 h of incubation. All samples were analyzed using a Shimadzu LC 20AT HPLC equipped with a Shimadzu 20A photo diode array (PDA) detector (Shimadzu, Columbia, MD) as described in the companion paper (13). Acetaminophen was quantified by the chromatogram peak areas measured by absorbance at 260 nm with an external calibration. The relative yields of three major self-coupling products, including a dimer, a trimer, and a tetramer were also determined based on their respective peak areas at 260 nm normalized to the peak area of acetaminophen before reaction. These selected products are those shown in Figure 6 of the companion paper (13). The Impact of Oxidative Coupling Reactions on NOM. The oxidative coupling reactions of NOM were induced by dosing 1.0 unit/mL laccase to 20 mL of each model NOM solution without acetaminophen added.The reaction mixture was then transferred to a 1 cm quartz cuvette. At each 10 min interval over a one-hour period, the UV absorbance spectrum of the solution in the cuvette was scanned using a Beckman DU640B spectrophotometer. Characterization of Cross-Coupling Products. Oxidative coupling reactions were induced by adding 1.0 unit/mL
FIGURE 1. Yields of coupling products in laccase catalyzed systems when acetaminophen was incubated with NOM as cosubstrates or without NOM. Concentrations of NOM as DOC were given in SI Table S1. Acetaminophen was initially at 50 µM, laccase was dosed at 1.0 unit/mL, pH 7.0, and reaction time 1 h. (a) Relative concentrations of residual acetaminophen remaining after the reaction and the relative yields of the three major coupling products; (b) Yields of the coupling products in relative to the total removal of acetaminophen. Error bars represent the standard deviation of three replicates. laccase to a 50 mL phosphate buffer solution (pH 7.0) containing 50 µM acetaminophen and 10 µM guaiacol as a model NOM compound. After 60 min, a few drops of concentrated H2SO4 were added to adjust the pH to below 2. The solution was then loaded onto a C18 solid-phase extraction (SPE) cartridge (Restek, 6 mL, 500 mg) that had been preconditioned with 5 mL methanol at a flow rate of 10 mL/min. After loading, the SPE cartridge was eluted with 5 mL methanol and the elution was collected, dried by a gentle nitrogen gas flow, and then reconstituted to 3 mL with methanol. A reaction system that contained only guaiacol but not acetaminophen was also tested following the same procedure described above. A Waters Micromass Quattra LC tandem mass spectrometer (MS/MS) (Waters, Milford, MA) was used to characterize the coupling products extracted by SPE. The MS was operated in positive electronspray ionization mode (ESI+), with the capillary potential set at 3.5 kV. Nitrogen (Airgas, >99.999% purity) was used as nebulizer and drying gas and maintained at flow rates of 45 and 300 L/h, respectively. Desolvation temperature was at 350 °C and source block temperature at 100 °C. Cone voltage was set to 58 V and the extractor 4 V. The mass analyzer was first run at scanning mode, and secondary MS spectra were then generated for selected ions using collision induced dissociation. The collision gas was Ar (Airgas, >99.999% purity). Fragmentor voltages and collision energy (CE) were experimentally optimized.
Results and Discussion Oxidative Coupling Reactions of Acetaminophen in the Presence of NOM. Figure 1a displays the fraction of acetaminophen remaining (A) after one hour reaction mediated by laccase as well as the relative yields (Pi) of three major self-coupling products in reaction systems that contained different NOM and a reaction system that did not contain NOM. It is evident that the removal of acetaminophen was VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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enhanced in the reaction systems with NOM as a cosubstrate. Interestingly, the relative yields of the three major selfcoupling products were all suppressed in the presence of NOM, although the total acetaminophen conversion was increased. Based on the results, we hypothesize that NOM can participate in the reactions. As potential substrates toward laccase and other oxidative coupling catalysts, NOM molecules bearing phenolic functionalities can be oxidized to form free radicals. These radicals can further react with acetaminophen through hydrogen exchange mechanism to convert them to free radicals, resulting in enhanced removal of acetaminophen. In addition, NOM radicals can crosscouple with acetaminophen radicals. Hence relatively less self-coupling products of acetaminophen were generated despite more acetaminophen was transformed in systems containing NOM. To quantitatively compare this effect in systems with different NOM, the yield of each product in each reaction system was further normalized against the removal ratio of acetaminophen using eq 1. Yi )
Pi 1-A
(1)
It is evident in Figure 1b that the yields of self-coupling products relative to total transformed acetaminophen (Yi) were significantly reduced in the presence of NOM. Among the different reaction systems, such an effect of reduction in self-coupling products formation seems to be the least strong in CSHM solution while those in the other three are similar to each other. The difference in this effect in different NOM solutions may reflect the tendency of NOM to participate in cross-coupling reactions dependent on the NOM concentration and reactivity. In this regard, CSHM solution appears to be least reactive among the four tested. Differential UV Absorbance Analysis. In addition to crosscoupling with other contaminants, NOM molecules are presumed to be able to couple with each other during oxidative coupling processes (21, 22). As described in the Experimental Section, we conducted experiments in which each NOM solution in the absence of acetaminophen was incubated with laccase and the UV absorbance spectrum was scanned every ten min to probe the reaction. Differential UV absorbance spectra, a measure that has often been used in NOM studies, were calculated using eq 2 to quantify the NOM spectral changes. ∆Aλ ) Aλ - Ainit λ
(2)
In eq 2, Ainit λ and Aλ are the absorbance of light at wavelength λ prior to and after reaction, respectively, and ∆Aλ is the differential UV absorbance at that wavelength (28-32). The differential UV spectra for each of the NOM solutions show a similar pattern when incubated with laccase, as illustrated in Figure 2 and SI Figure S1. Two prominent features stand out in these Figures. First, no time-dependent changes of absorbance occurred at wavelength below 320 nm, where structural features related to aromaticity is located. The ∆Aλ at this range is primarily the contribution of enzyme which is not included in Ainit λ . Second, a chromophore at 472 nm was generated regardless of the origin and types of NOM, and ∆A472 nm increased linearly with the incubation time (Figure 3). This wavelength is within the visible range and corresponds to long-range conjugation of electrons. The pattern of the observed spectral changes appears to be indicative of the coupling between NOM molecules. As noted earlier, oxidative coupling facilitates polymerization reactions by forming CsOsC, CsNsC, and/or CsC bonds (22, 33-36). This process bridges aromatic rings directly or indirectly, thus making the conjugation of electrons between two or more aromatic rings possible. Because this process did not 7070
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FIGURE 2. Time based differential UV absorbance of CSFV solution treated by laccase. Laccase dosage was 1.0 unit/mL, pH 7.0.
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FIGURE 3. Formation of ∆A472 nm against reaction time due to the coupling of NOM molecules during laccase-catalyzed oxidative coupling process. DOC of each of the NOM solution is given in SI Table S1, and laccase dosage was 1.0 unit/mL, pH 7.0. The inset plot indicates the rate of change of ∆A472 nm for each NOM solution that is calculated by linear regression. Error bars represents 95% confidence intervals. break down aromatic moieties per se, it did not cause timedependent changes in absorbance below 320 nm. The differential spectra exhibit greater noise around 370 nm, which may arise primarily from the fact that the light sources switch (UV-visible) within this range. Nonetheless, noise in this range does not obscure the two spectral features discussed above, which occur respectively below 320 nm and above 420 nm. Based on this analysis, the increase in absorbance at 472 nm likely resulted from NOM coupling. As such, the rate of such absorbance change ∆A472 nm/∆t as shown in Figure 3 may be indicative of the relative tendency of coupling reactions between NOM molecules in each model NOM solution. The ∆A472 nm/∆t values related to each of the NOM solutions, obtained by linear regression analysis of the ∆A472 nm data against time, are also presented in Figure 3. Because the reactivity of NOM toward laccase is determined primarily by the abundance of phenolic functionalities, which does not necessarily correlate directly to organic carbon content, we did not further normalize ∆A472 nm/∆t to DOC. The ∆A472 nm/∆t data in Figure 3 in fact reflect the reactivity of each NOM solution resulting from the collective effect of NOM intrinsic reactivity and its quantity in the solution. The data reveal that CSHM solution is less reactive than the other three NOM solutions, which have similar extent of reactions. Apparently, less NOM radicals were available in the reaction system containing CSHM and the extent of cross-coupling
FIGURE 4. MS spectra of the products resulting from laccase-catalyzed reaction systems, laccase dosage 1.0 unit/mL, pH 7.0. (a) Guaiacol (10 µM) along as a control; (b). Acetaminophen (50 µM) incubated with guaiacol (10 µM) as NOM model compound; (c) MS/MS spectrum of the cross-coupling products between acetaminophen and guaiacol. with other compound would be less. This is consistent with the data presented in Figure 1 in which the removal of acetaminophen was less (Figure 1a) but the yields of acetaminophen self-coupling products relative to the total acetaminophen removal (Figure 1b) were greater in CSHM solution than those in the other three NOM solutions. Reaction Pathways. It is generally believed that phenolic/ anilinic moieties are the active electron donors enabling a substrate active toward laccase (21, 24). It may thus be presumed that the relative reactivity of NOM with respect to laccase catalysis is correlated to such functionalities present in NOM molecules. Because of the complexity and uncertainty of NOM molecular structures, model compounds having well-defined structures representing the reactive moieties in NOM molecules have been widely employed in NOM studies (37-42). We used guaiacol in this study to mimic the phenolic moieties in NOM molecules to elucidate the possible cross-coupling reaction pathways. Guaiacol was incubated in reaction systems mediated by laccase in the presence and absence of acetaminophen. The product solutions after one hour of reactions were extracted and analyzed using positive ESI-MS, and the MS spectra are
presented in Figure 4. As shown in Figure 4a, a series of products (m/z ) 489, 611, 733, 855) with a periodical increment of mass (122) were prominent when guaiacol alone was incubated with laccase. Such a pattern of mass distribution likely resulted from the self-coupling of guaiacol (M ) 124) with 4-8 guaiacol monomer units. However, the relative yields of self-coupling products with a lower degree of polymerization were not significant in comparison to the higher degree coupling products. When acetaminophen was present in the system (Figure 4b), the self-coupling of guaiacol was largely suppressed with the relative intensities of the mass peaks corresponding to self-coupling products having more than three guaiacol units significantly reduced in comparison to a peak corresponding to dimer (m/z 247). This suggests that the self-coupling led to primarily dimer products with a lesser extent to form higher degree polymers, likely resulting from cross-coupling interferences. A peak (m/z ) 274) is pronounced in Figure 4b for the system with acetaminophen, but is absent in Figure 4a for the system without acetaminophen. This peak likely corresponds to a product that resulted from cross-coupling between guaiacol and acetaminophen. Unlike other products, the compound VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Fragmentation pathways of the cross-coupling products between acetaminophen and guaiacol during MS/MS analysis. corresponding to this peak has an odd number of molecular weight (M ) 273), indicating that it contains an odd number of nitrogen atoms based on nitrogen rule. The nitrogen was apparently from an acetaminophen molecule (M ) 151) which, if coupled with a guaiacol molecule (M ) 124) via radical-radical mechanism, would form a product with a molecule weight of 273. Upon oxidation by the laccase, a substrate loses an electron and subsequently a proton from the hydroxyl oxygen or acetamide nitrogen to form a radical. The unpaired electron in the nitrogen/oxygen atom tends to relocate to the conjugated sites on the benzene ring through resonance. In theory, a total of four resonance structures of acetaminophen radicals and three resonance structures of guaiacol radicals can be formed in this way as shown in SI Figure S2. Therefore, a total of 12 isomeric products could possibly be formed via cross-coupling between acetaminophen and guaiacol by CsC, CsOsC, CsNsC, NsO, or OsO bonds. However, given the energy status of different resonance structures as well as the steric hindrance and electrostatic interactions when two radicals approaching to each other, only some of them are favored energetically and/or kinetically. In order to further elucidate the structures of the crosscoupling products, the species corresponding to the peak of m/z 274 was analyzed by secondary MS, and the spectrum is given in Figure 4c. It is noted that the primary MS is not capable of differentiating isomers. The peak of m/z 274 in Figure 4b is in fact contributed by a mixture of isomers. Consequently, the MS/MS spectrum generated from m/z 274 (Figure 4c) represents a mixture of MS/MS spectra originating from different isomers. Because each isomer may have its characteristic secondary fragmentation pattern. MS/ MS spectrum in Figure 4c does provide hints as to how acetaminophen and guaiacol are cross-coupled. One of the principal fragments exhibits an m/z value of 200, which corresponds to the loss of a neutral methyl acetyl molecule (M ) 74) from the parent ion (m/z ) 274). This was most likely to occur by eliminating the methoxyl group from the guaiacol moiety and the acetate group from the acetaminophen moiety via formation of an intramolecular bond that bridges the two aromatic rings as shown in Figure 5a. This requires the two moieties in facile orientation that allows them to be able to approach to each other. Based on the above analysis, the parent ion of this fragment (m/z ) 200) was most likely originated from a cross-coupling product that was formed by covalently bonding between the hydroxyl oxygen in guaiacol and one of the ortho carbons of the acetamino group in the acetaminophen molecule. As shown 7072
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in Figure 5a, this structure allows the formation of a 6-member ring, which is a thermodynamically favorable configuration, through the elimination of a methyl acetyl molecule during MS fragmentation. None of the other possible configuration seems to be able to favor such a fragmentation pathway. Another fragment distinct in the MS/MS shown in Figure 4c has an m/z value of 242. This fragmentation corresponds to the loss of a neutral methanol molecule (M ) 32) from the parent ion (m/z ) 274). As indicated in Figure 5b, such a fragmentation pathway could happen only if the parent ion was originated from a cross-coupling product formed via bonding between the hydroxyl oxygen of guaiacol and one of the ortho carbons of the hydroxyl group in the acetaminophen molecule. A methanol molecule can be eliminated from such a configuration through the formation of a 6-member ring during the MS fragmentation (Figure 5b). In summary, the cross-coupling between acetaminophen and guaiacol is likely to proceed primarily via CsOsC bond formation. No direct evidence supporting the presence of other cross-coupling species is present in the MS/MS spectra. Nonetheless, the cross-coupling through CsC, CsNsC, or other bonds are also possible in real systems given the diversity of the functionalities and structural properties of NOM molecules (43-45). Both NOM and model compound studies suggest the possibility of formation of cross-coupling products between NOM and acetaminophen during laccase-mediated oxidative coupling processes. Such cross-coupling between NOM and pharmaceuticals could very likely play a more dominant role than self-coupling in micropollutant transformation because typical NOM concentrations (∼mg/L as TOC in water/ wastewater) are often 10 orders of magnitude higher than micropollutants (ppt or lower). Incorporation of the contaminants into NOM molecules via oxidative coupling will lead to deactivation of the biological effects of these contaminants, whereas the cross coupling between NOM molecules has been proven to enhance the removal of NOM from water (21, 22). This in fact provides a potential strategy to remove NOM and micropollutants simultaneously in water/wastewater treatment.
Acknowledgments The study was supported in part by U.S. EPA STAR grant G6M10518 and by HATCH funds. The content of the paper does not necessarily represent the views of the funding agencies. We thank Mr. Roger A. Pinto in the Department of Chemical Engineering at the University of Michigan for providing NOM samples.
Supporting Information Available (i) Time-dependent differential UV spectra of the model NOM upon oxidation by laccase; (ii) resonance structures of acetaminophen and guaiacol radicals; (iii) model NOM materials used in the experiment. This material is available free of charge via the Internet at http://pubs.acs.org.
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