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Use of Cyclic Voltammetry as an Effective Tool for Selecting Efficient Enhancers for Oxidative Bioprocesses: Importance of pH Elisabetta Aracri,*,† Tzanko Tzanov,‡ and Teresa Vidal† †

Department of Textile and Paper Engineering and ‡Group of Molecular and Industrial Biotechnology, Department of Chemical Engineering, Universitat Politècnica de CatalunyaBarcelonaTech, E-08222 Terrassa, Spain S Supporting Information *

ABSTRACT: Seven natural phenols and two synthetic compounds were evaluated by means of cyclic voltammetry as enhancers for the oxidation of the lignin model compound veratryl alcohol (VAl) and a sulfonated lignin (SL). Their electrochemical behaviors and catalytic efficiencies (CEs) against both substrates were assessed as a function of pH. A general increase in CE of the phenols was for the first time observed in the oxidation of VAl at pH 7 and 8. Methyl syringate (MS), syringic acid (SRC), and syringaldehyde (SRD) exhibited the highest CEs against VAl among the studied phenolic compounds despite the reduced stabilities of their phenoxy radicals. This was a result of favorable stability−reactivity balances, which were apparently influenced by both the chemical structures of the enhancers and the experimental conditions. Violuric acid (VAc) proved the most efficient compound in oxidizing lignin, followed by SRD and MS, which showed regeneration in the interval of pHs studied. soils,15 and waste pulping liquors16 makes them economically attractive in relation to synthetic mediators. According to the literature,17−21 the mechanism by which the phenolic mediators oxidize non-phenolic substrates should be similar to that of >N−OH compounds, based on a hydrogen atom transfer (HAT mechanism), where phenoxy radicals (PhO•) or aminoxyl radicals (>N−O•) are the reactive species that abstract one hydrogen atom from the substrate. Such a radical mechanism circumvents the low tendency of non-phenolic lignin moieties to take part in electron-transfer reactions with laccase, thereby facilitating their oxidation.22 However, phenoxy radicals resulting from the oxidation of phenolic compounds tend to couple or fragment, which prevents reduction to their original forms by the non-phenolic substrate they are intended to oxidize.22,23 The formation of long-living radicals has been suggested as a determining factor for the efficiency of phenolic compounds as laccase mediators.19 Such efficiency is increased by resonance stabilization and the presence of electrondonating groups in ortho position preventing 5−5′ coupling reactions.13 Laccase activity on phenols is also enhanced by the presence of electron-donating groups at the benzene ring that decrease their electrochemical potentials, thus making them more easily oxidizable. Another factor playing a significant role in the enzymatic catalysis is the pH of the reaction medium, which affects not only the catalytic activity of laccase, but also the redox potentials of its substrates.24 Thus, the phenoxy anion is a better one-electron reductant than the protonated phenol; therefore, increasing the solution pH should increase the fraction of phenol in its phenolate form, which is easier to oxidize to phenoxy radical by electron transfer.19 The effect of pH on the mediating efficiencies of laccase enhancers has been

1. INTRODUCTION Laccases (EC 1.10.3.2) are multicopper oxidases occurring in higher plants, fungi, bacteria, and insects that play a variety of roles in nature including their participation in lignin biosynthesis and biodegradation.1 Intensive research has been carried out in the past few decades in the pulp and paper industry for pulp delignification and bleaching with laccases.2−4 Lignin is a natural polymer consisting of a network of phenolic and nonphenolic compounds. Due to the rather low redox potential of laccases (0.5−0.8 V),5 these enzymes are able to oxidize directly only the less abundant (15%)6 phenolic moieties in lignin. However, the substrate spectrum of laccases can be extended to the non-phenolic moieties in lignin by using these enzymes in combination with a mediator or enhancer.7 The ideal mediator should be a good laccase substrate, able to generate stable radicals upon oxidation that do not inactivate the enzyme, and should not be consumed during the redox transformation. Most reported candidates fail to meet the requirements to be considered as mediators owing to the low stability of their oxidized intermediates, which tend to undergo undesired chemical transformations. These compounds, which are capable of mediating the catalytic action of laccases over a limited number of redox cycles, should be more properly designated enhancers.7 Several synthetic compounds belonging to the hydroxylamine (>N−OH) class have been studied with a view to improving the efficiency of laccases in various bioprocesses. 1Hydroxybenzotriazole and violuric acid have proved highly efficient in delignifying pulp.8−11 However, the high cost and toxicity of synthetic mediators hinder their use for industrial applications.12 The search for natural compounds as a costeffective, environmentally friendly alternative to synthetic mediators has focused on phenolic species present in the natural environment of laccase (viz. fungal metabolites, or fragments derived from the biodegradation of lignin).13 The widespread availability of natural mediators in plants,14 forest © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1455

October 11, 2012 November 28, 2012 January 4, 2013 January 4, 2013 dx.doi.org/10.1021/ie3027586 | Ind. Eng. Chem. Res. 2013, 52, 1455−1463

Industrial & Engineering Chemistry Research

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scarcely investigated to date19,25 due to the restricted to acid pH stability of most fungal laccases. Cyclic voltammetry is a powerful technique for studying the electrochemical oxidation of lignin or a lignin model compound by mediators/enhancers. Some authors26,27 have recently assessed the efficiencies of a number of natural phenols as mediators or enhancers for the oxidation of lignin or a lignin model compound (veratryl alcohol) at a single, acid pH value. To our knowledge, no study of the electrochemical behaviors of laccase enhancers as a function of pH has so far been reported. Therefore, this work aimed at studying the effect of pH 3−8 on the electrochemical behaviors and catalytic efficiencies of seven natural phenols and two synthetic compounds for oxidizing veratryl alcohol and a sulfonated lignin. The results obtained would reveal the potential of using natural enhancers to improve the oxidative efficiencies of a broad range of laccases acting in acidic, neutral, and alkaline conditions.

(0.125 mg/mL) at pH 4, 6, and 8, at a scan rate of 5 mV/s. The potential was scanned from −50 to 1200 mV against the reference electrode after holding the electrochemical system at the initial potential for 10 s. All tests were performed at room temperature at least twice, and all measurements found to be highly reproducible.

3. RESULTS AND DISCUSSION 3.1. Cyclic Voltammetry of Natural and Synthetic Compounds in Tartrate Buffer at pH 4: Catalytic Efficiency in Oxidizing Veratryl Alcohol. In the first part of this study, five natural phenols (FRC, CLD, SNC, SLD, and MS) and two synthetic compounds (HBT and VAc) were assayed by cyclic voltammetry at a concentration of 0.2 mM in 50 mM tartrate buffer, pH 4, which had previously been used in laccase-based biobleaching processes.28,29 Table 1 shows the structures of the seven compounds studied. >N−OH compounds were included in the study on the grounds of the similarity of their oxidation pathway (based on radical Habstraction) to that of phenolic compounds. Although a cyclic voltammetry study under similar conditions was previously reported for most of the compounds examined here,26 our tests were intended to facilitate comparison of the electrochemical properties of the selected compounds at different pH values and substrate-to-enhancer ratios. The first and second scans of the cyclic voltammograms (CVs) obtained for the phenolic and >N−OH compounds at low and high scan rates, 5 and 200 mV/s respectively, are shown in Figure S1 (see the Supporting Information). All compounds except violuric acid (VAc) underwent irreversible oxidation. Interestingly, the CVs for the p-hydroxycinnamic compounds with identical degrees of substitution on the benzene ring (viz. 4-hydroxy-3methoxycinnamic for CLD and FRC, and 4-hydroxy-3,5dimethoxycinnamic for SLD and SNC) were quite similar. Moreover, the decrease of the anodic current in the second scan (due to the formation of a passivating layer on the electrode surface) was more pronounced in the case of CLD and FRC than in the case of SLD and SNC, suggesting a clear tendency of the former to polymerize. On the other hand, the electrode responses obtained at high and low scan rates differed markedly, especially among the phenolic compounds. Thus, at low scan rate, no cathodic signal was detected for CLD, and a weak cathodic peak well apart from the oxidation peak potential was exhibited by the other phenolic compounds, suggesting the formation of oxidized intermediates that are rapidly removed by a chemical reaction such as phenoxy radical coupling. By increasing the scan rate to 200 mV/s, all phenolic compounds exhibited a more reversible process, which evidenced the instability of their oxidized intermediates. The CVs obtained in this work for the phenolic compounds differ from those shown ́ by Diaz-Gonzá lez et al.26 in having reduced cathodic peak currents, which can be due to the use of a higher phenol concentration resulting in increased probability to undergo coupling reactions. The above-described compounds were assayed in the presence of VAl in order to determine their catalytic efficiencies in oxidizing the lignin model compound. Cyclic voltammograms (Figure 1) were recorded at low scan rate (5 mV/s), using a substrate-to-enhancer ratio of 10:1. As can be seen from Figure 1, HBT was the only compound showing an increased oxidation peak in the presence of VAl. Since the alcohol is electroinactive over the potential range scanned, the increase in anodic current can only have resulted from regeneration of

2. EXPERIMENTAL SECTION 2.1. Apparatus and Electrodes. Voltammetric tests were performed using a μAutolab Type III potentiostat/galvanostat from EcoChemie (Utrecht, The Netherlands) controlled by Autolab GPES software, version 4.9. All tests were carried out in a thermostatic 40 mL, three-electrode configuration cell from Metrohm (Utrecht, The Netherlands). The working electrode was a glassy carbon electrode (GCE) with a surface diameter of 3 mm from Metrohm. The counter and reference electrodes were platinum and Ag/AgCl, respectively, both from Metrohm. The glassy carbon surface was renewed by polishing with 1.0 and 0.3 μm α-alumina (Micropolish, Buehler, Germany) on a microcloth polishing pad (also from Micropolish), followed by a thorough washing with ultrapure water obtained with a MilliQ plus 185 (Millipore Ibérica S.A., Madrid, Spain). 2.2. Reagents and Solutions. 3,4-Dimethoxybenzyl alcohol (veratryl alcohol, VAl), 1-hydroxybenzotriazole (HBT), 2,4,5,6(1H, 3H)-pyrimidinetetrone 5-oxime (violuric acid, VAc), and the natural phenolic compounds 3,5dimethoxy-4-hydroxybenzoic acid (syringic acid, SRC), 3,5dimethoxy-4-hydroxycinnamic acid (sinapic acid, SNC), 4hydroxy-3-methoxycinnamic acid (ferulic acid, FRC), 3,5dimethoxy-4-hydroxybenzaldehyde (syringaldehyde, SRD), 3,5-dimethoxy-4-hydroxycinnamaldehyde (sinapyl aldehyde, SLD), and 4-hydroxy-3-methoxycinnamaldehyde (coniferyl aldehyde, CLD) were purchased from Sigma (Madrid, Spain). Methyl syringate (MS) was provided by Novozymes (Bagsvaerd, Denmark). Sulfonated lignin (SL) was supplied by Borregaard (Sarpsborg, Norway). Boric acid, acetic acid, phosphoric acid, tartaric acid, and sodium hydroxide were purchased from Sigma. Solutions of the different enhancers, with and without VAl or SL, were prepared in either 50 mM tartrate buffer, pH 4, or Britton−Robinson (B−R) buffer (40 mM boric acid, 40 mM acetic acid, 40 mM phosphoric acid plus 0.5 mM NaOH to the desired pH) of different pHs immediately before use. All solutions were prepared using ultrapure water. 2.3. Measurement Procedures. The voltammetric responses of each enhancer were first recorded at a 0.2 mM concentration in tartrate buffer, pH 4, using scan rates of 5 and 200 mV/s, and in the presence of 2 mM VAl at 5 mV/s. A second test set was performed on 1 mM enhancer solutions in the presence and absence of 0.75 mM VAl in B−R buffers at pH 3−8, using a scan rate of 5 mV/s. Enhancer solutions (1 mM) in B−R buffer were also assayed in the presence of SL 1456

dx.doi.org/10.1021/ie3027586 | Ind. Eng. Chem. Res. 2013, 52, 1455−1463


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regenerate their reduced forms. However, an increase in oxidation current was detected at potentials around 1000 mV; this indicates that the oxidation of VAl occurs at potentials below that for the alcohol alone (Ep,a ∼ 1.2 V). These results show that the studied phenolic compounds and VAc act as effective catalysts in the oxidation of the lignin model compound.26 The electrochemical behavior of VAc in the presence of VAl differed significantly from that recently reported by González-Arzola et al.,27 who found VAc in the presence of a large excess of VAl (a substrate-to-enhancer ratio of 200:1) in McIlvaine buffer at pH 6 to be efficiently regenerated. The catalytic efficiency (CE) of the enhancers was calculated as ΔI/IE (Table 2). For phenolic compounds and VAc, ΔI was the increase in anodic current at 1020 mV and IE was the oxidation current of the enhancer at the same potential; for HBT, ΔI was the increase in anodic current corresponding to its peak potential and IE was the oxidation current of the enhancer at the same potential. The results showed the effect exerted on the redox potentials of phenolic compounds by the substituents on the aromatic ring. Thus, the presence of two electron-releasing groups (OMe) in SNC and SLD decreased the redox potentials in relation to FRC and CLD, which bore a single substituent; also, the presence of an alkenyl chain between the aromatic rings and the electron-withdrawing carbonyl groups in the p-hydroxycinnamic compounds resulted in a decreased redox potential relative to MS. Therefore, the compounds with reduced redox potentials are more easily oxidized by virtue of the increased stabilities of their phenoxy radicals. As expected for an H-abstraction oxidation mechanism, the CEs of the phenolic compounds on VAl did not increase with the redox potential, but rather with the presence of stabilizing functional groups. 3.2. Effect of pH on the Redox Potentials and Catalytic Efficiencies of Enhancers in Oxidizing VAl and Sulfonated Lignin. In the second part of this study, the natural phenols SRC and SRD were assayed in addition to the above-described compounds in order to more accurately examine the relationship between the electrochemical behaviors and chemical structures of the phenolic compounds. Since the oxidation of the phenolic compounds involved proton exchange, the solution pH should play a central role in their redox chemistry. Although the redox potential is known to be pH dependent, no study has to our knowledge ever examined the effect of pH on the electrochemical behaviors and catalytic efficiencies of laccase phenolic enhancers for oxidizing lignin or a lignin model compound. In this work, we used solutions of the different enhancers in the presence or absence of VAl prepared in B−R buffers spanning the range pH 3−8 that were prepared immediately before use. Figure 2 illustrates the influence of pH on the redox potentials of the studied compounds. As can be seen, the potentials of the phenolic compounds and VAc decreased linearly with increasing pH, whereas that of HBT varied little after an initial decrease from pH 3 to 5. The phenolic compounds studied exhibit acid−base transitions in the neutral-to-alkaline pH range, with pKa values from 7.3 to 9.5. Therefore, the decrease in their redox potentials can be ascribed to an increase in the proportion of their phenolate forms, which are more easily oxidized than the phenol. Interestingly, a clear relationship was found between the substitution pattern of the phenols and their potential profiles: the presence of two OMe groups and a CHCHCOR group in the p-hydroxycinnamic compounds SNC and SLD resulted in their having the lowest potentials, and similar for the

Table 1. Names, Abbreviations, and Chemical Structures of the Natural Phenols and Synthetic Compounds Used in This Study

HBT at the electrode surface after abstraction of a benzyl hydrogen atom from the substrate (a homogeneous chemical reaction).17 Despite the low stability of the HBT radical, the observed regeneration indicates that the radical is long-living enough to efficiently oxidize the lignin model compound. The voltammograms for the other compounds in the presence of VAl exhibited no increase in anodic current in proportion to their oxidation potentials, which suggests that they failed to 1457



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Figure 1. Cyclic voltammograms of 0.2 mM solutions of phenolic and synthetic compounds (gray lines), 2 mM solution of VAl (dashed lines), and compounds plus VAl (black lines). 50 mM tartrate buffer pH 4. Scan rate: 5 mV/s.

two, followed by the p-hydroxycinnamic compounds FRC and CLD, which bear a single OMe group. Also, FRC and CLD exhibited potential profiles similar to that of SRC, which bears two OMe groups and a COOH group instead of a CH CHCOR group. Consistent with the electron-withdrawing effect of the α-carbonyl group in para position, the potential profiles of SRC, MS, and SRD decreased in the following sequence: Ep,a,SRC < Ep,a,MS < Ep,a,SRD. The synthetic enhancers had the highest potentials, with Ep,a,HBT > E°VAc, and a less pronounced decrease with increasing pH. The influence of pH on the CEs of the enhancers in VAl oxidation was examined at a substrate-to-enhancer ratio of 0.75:1 in order to reproduce

the conditions of biobleaching processes previously applied to low-lignin pulp.28,29 Figure 3 shows the variation of CE for the phenolic compounds and VAc in oxidizing VAl over the range pH 3−8; CE for HBT was expressed in a different form and is not included in the graph. Interestingly, all phenolic compounds succeeded in catalyzing VAl oxidation with variable efficiency in tartrate buffer at pH 4, but exhibited little or no efficiency at the same pH and a substrate-to-enhancer ratio of 0.75:1. Similarly, the efficiency of HBT dropped from 10.14 to 1.46 when measured in substrate deficiency (results not shown). On the other hand, VAc catalyzed VAl oxidation nearly 2 times more efficiently than in the previous tests. These 1458



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toward the substrate. SRD and MS are known to be effective laccase mediators for pulp bleaching and oxidation of nonphenolic lignin model compounds;11,30−32 by contrast, phydroxycinnamic compounds are scarcely effective in enhancing pulp delignification.28,33 As can also be seen from Figure 3 in relation to the phenol-catalyzed oxidation of VAl, CE increased markedly at pH 7−8 with all enhancers except CLD. The general increase in CE versus VAl of the phenolic compounds most probably reflected a better balance between reactivity and stability in the ensuing radicals. These results may boost the search for new laccases in, for example, bacterial sources possessing pH stability in the neutral-to-alkaline range for applications where phenolic compounds are typically used as laccase enhancers.25 The catalytic efficiency of VAc peaked over the range pH 3−5, decreased markedly at pH 6−7, and then increased to a level similar to the initial value at pH 8. Similarly to its redox potential, the efficiency of HBT changed little over the pH studied (results not shown). Since the ability of an enhancer to oxidize a non-phenolic lignin model compound does not suffice to oxidize lignin itself, additional experiments were performed in the presence of lignin to assess the actual potential of the studied compounds for pulp delignification. To this end, solutions of the different enhancers, with or without sulfonated lignin, were prepared in B−R buffers at pH 4, 6, and 8 immediately before use. CE in lignin oxidation was determined as ΔI/IE, where ΔI is the increase in anodic current corresponding to the enhancer peak potential and IE the oxidation current of the enhancer at the same potential. As can be seen from Figure 4, the ability of the enhancers to regenerate their reduced forms by oxidizing ligninvia homogeneous chemical reactionsvaried markedly with pH and from compound to compound. Thus, VAc exhibited the best catalytic performance and greatest increase in anodic peak current at pH 6 among all enhancers. Similarly to the results of González-Arzola et al.,27 HBT was much less efficient than VAc in regenerating its reduced form in the presence of lignin despite its increased CE relative to VAl. MS and SRD were the only phenolic compounds capable of regenerating their initial forms by lignin oxidation at any pH; this result makes them especially attractive for industrial use (e.g., in pulp biobleaching) in small amounts and hence with

Table 2. Redox Potential and Catalytic Efficiency against VAl of Phenolic and Synthetic Compounds (Substrate-toEnhancer Molar Ratio 10:1)a compd

Ep,a (V)

CE

MS FRC CLD SNC SLD VAc HBT

0.69 0.58 0.58 0.47 0.44 0.72b 0.87

0.02 0.28 0.26 0.33 0.31 0.76 9.14c

a

With 50 mM tartrate buffer, pH 4. Scan rate: 5 mV/s. bE° = (Ep,a + Ep,c)/2. cΔI taken in correspondence of its peak potential.

results testify to the prominent effect of the substrate-toenhancer ratio on the CEs of the enhancers. The studied phenolic compounds and VAc exhibited no regeneration in the pH range examined here; by contrast, González-Arzola et al.27 found several phenolic compounds and VAc to be regenerated in the presence of VAl at pH 6a likely result of their using a much higher substrate-to-enhancer ratio. As can be seen from Figure 3, SLD was the phydroxycinnamic derivative exhibiting the highest CE over the range pH 5−7 and higher values than FRC and CLD at pH 8; also, SNC was more efficient than FRC and CLD at pH 7 and the most efficient at pH 8. The increased efficiency of the syringyl-type compounds was probably a result of the increased stability of their oxidized forms. Overall, MS, SRC, and SRD proved more efficient than the p-hydroxycinnamic compounds at any pH despite the lower stability of their phenoxy radicals. Thus, SRC exhibited the highest CE over the range pH 3−5 and was more efficient than SRD at pH 6−8; likewise, MS was less efficient than SRD at pH 3−5 but more efficient at pH 6− 8. The greater efficiency of MS, SRC, and SRD in catalyzing VAl oxidation may reside in a more favorable balance between the reactivity and stability of their oxidized forms. The presence of a more extended conjugated double bond system in the phydroxycinnamic compounds must have facilitated electron delocalization after the oxidation step, thus resulting in greater stability but in also lower reactivity of the phenoxy radicals

Figure 2. Dependence of redox potentials of phenolic and synthetic compounds on pH. 1459



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Figure 3. Variation of catalytic efficiency against VAl of natural compounds and VAc (substrate-to-enhancer molar ratio 0.75:1) over the range pH 3−8. 40 mM Britton−Robinson buffer. Scan rate: 5 mV/s.

Figure 4. Variation of catalytic efficiency against SL (0.125 mg/mL) of natural compounds and synthetic compounds (1 mM) at pH 4, 6, and 8. 40 mM Britton−Robinson buffer. Scan rate: 5 mV/s.

of various natural and synthetic laccase enhancers for the oxidation of veratryl alcohol and sulfonated lignin. With tartrate buffer at pH 4 and a substrate-to-enhancer ratio of 10:1, all phenolic compounds succeeded in catalyzing VAl oxidation with an efficiency increasing with increase in the stability of their phenoxy radicals. VAc was much more efficient than the phenolic compounds. Increasing pH from 3 to 8 resulted in a linear decrease in redox potential of the enhancerparticularly in the natural compoundsthat was consistent with the Habstraction oxidation mechanism involved. Also, the resulting potential profiles for the phenolic compounds were consistent with the presence of groups stabilizing the phenoxy radicals formed. The redox potential of the phenolic compounds decreased in the following sequence: Ep,a,SRD > Ep,a,MS > Ep,a,SRC ∼ Ep,a,CLD ∼ Ep,a,FRC > Ep,a,SNC ∼ Ep,a,SLD. The influence of pH on the ability of the enhancers to oxidize VAl or lignin was

substantial cost savings. The decreased anodic peak currents exhibited by FRC and CLD at pH 6, and by SRC at pH 4 and 6, were probably due to adverse coupling reactions of the electrogenerated phenoxy radicals formed in the lignin structure. Although the enhancer was not regenerated, an increase in anodic charge was observed at high potentials in the presence of lignin in some cases (Figure 5); this is suggestive of oxidation of non-phenolic lignin components. Therefore, the ability of an enhancer to catalyze lignin oxidation should be assessed in terms of both its CEas calculated from the increase in anodic peak currentand the increase in oxidative charge at higher potentials.

4. CONCLUSIONS Cyclic voltammetry was used in this work to assess the effect of pH on the electrochemical behaviors and catalytic efficiencies 1460



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Figure 5. continued

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Figure 5. Cyclic voltammograms of 1 mM solutions of phenolic and synthetic compounds (gray lines), 0.125 mg/mL solution of SL (dashed lines), and compounds plus SL (black lines), at pH 4 (left), pH 6 (center), and pH 8 (right). 50 mM Britton−Robinson buffer. Scan rate: 5 mV/s.

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ACKNOWLEDGMENTS This work was supported by Spain’s MICINN projects FUNCICELL (CTQ2009-12904), BIOFIBRECELL (CTQ2010-20238-CO3-01) and BIOSURFACEL (CTQ201234109).

assessed at a substrate-to-enhancer ratio of 0.75:1 in order to reproduce the conditions used in previous work to biobleach low-lignin nonwood pulp. All enhancers except VAc had a much lower CE for VAl oxidation at pH 4 than in the presence of a substrate excess; this indicates that the substrate-toenhancer ratio affects the efficiency of the enhancers. As revealed by our results for the phenol-catalyzed oxidation of VAl, CEs peaked at pH 7−8. MS, SRC, and SRD were more efficient than the p-hydroxycinnamic acids at any pH despite the lower stabilities of their radicals. This was most probably a result of a more favorable balance between the reactivities and stabilities of the ensuing radicals, which thus seemingly depend not only on the chemical structure of the enhancer but also on the experimental conditions used. Based on their CVs in the presence of sulfonated lignin, all enhancers except SRC were regenerated during lignin oxidation, to a variable extent depending on pH. Consistent with a number of pulp biobleaching studies, VAc was by far the most efficient enhancer, followed by SRD and MS. In the absence of regeneration, however, an increase in oxidative charge at high potentials suggestive of oxidation of non-phenolic structures in lignin was observed in most cases. As shown in this work, cyclic voltammetry provides a simple, yet powerful, tool for obtaining useful information with a view to predicting the efficiency of laccase−enhancer systems in oxidative processes under variable experimental conditions.

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(1) Mayer, A. M.; Staples, R. C. Laccase: new functions for an old enzyme. Phytochemistry 2002, 60, 551−565. (2) Bajpai, P. Biological bleaching of chemical pulps. Crit. Rev. Biotechnol. 2004, 24, 1−58. (3) Fillat, U.; Roncero, M. B. Optimization of laccase-mediator system in producing biobleached flax pulp. Bioresour. Technol. 2010, 101, 181−187. (4) Call, H. P.; Mücke, I. History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym-process). J. Biotechnol. 1997, 53, 163−202. (5) Baldrian, P. Fungal laccasesoccurrence and properties. FEMS Microbiol. Rev. 2006, 30, 215−242. (6) Adler, E. Lignin chemistry: past present and future. Wood Sci. Technol. 1977, 11, 169−218. (7) Morozova, O.; Shumakovich, G. P.; Shleev, S. V.; Yarapolov, Y. I. Laccase-mediator systems and their applications: A review. Appl. Biochem. Microbiol. 2007, 43, 523−535. (8) Fillat, A.; Roncero, M. B.; Vidal, T. Assessing the use of xylanase and laccases in biobleaching stages of a TCF sequence for flax pulp. J. Chem. Technol. Biotechnol. 2011, 86, 1501−1507. (9) Ibarra, D.; Camarero, S.; Romero, J.; Martínez, M. J.; Martínez, Á . T. Integrating laccase-mediator treatment into an industrial-type sequence for totally chlorine-free bleaching of eucalypt kraft pulp. J. Chem. Technol. Biotechnol. 2006, 81, 1159−1165. (10) Valls, C.; Vidal, T.; Roncero, M. B. Boosting the effect of a laccase-mediator system by using a xylanase stage in pulp bleaching. J. Hazard. Mater. 2010, 177, 586−592. (11) Moldes, D.; Díaz, M.; Tzanov, T.; Vidal, T. Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp. Bioresour. Technol. 2008, 99, 7959− 7965. (12) Xu, F. Applications of oxidoreductases: recent progress. Ind. Biotechnol. 2005, 1, 38−50. (13) Cañas, A. I.; Camarero, S. Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes. Biotechnol. Adv. 2010, 28, 694−705. (14) Lam, T. B. T.; Iiyama, K.; Stone, B. A. Determination of etherified hydroxycinnamic acids in cell walls of grasses. Phytochemistry 1994, 36, 773−775.

ASSOCIATED CONTENT

S Supporting Information *

Figure S1, cyclic voltammograms (first and seconds scans) of 0.2 mM solutions of phenolic and synthetic compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +34 937398190. Fax: +34 937398101. Notes

The authors declare no competing financial interest. 1462



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