Biocatalytic generation of Mn(III)-chelate as a chemical oxidant of different environmental contaminants

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Biocatalytic Generation of Mn(III)-Chelate as a Chemical Oxidant of Different Environmental Contaminants Roberto Taboada-Puig, Thelmo Lu´-Chau, Gemma Eibes, Marı´a Teresa Moreira, Gumersindo Feijoo, and Juan Manuel Lema Dept. of Chemical Engineering, E.T.S.E., Lope Go´mez de Marzoa, Santiago de Compostela 15782, Spain DOI 10.1002/btpr.585 Published online April 20, 2011 in Wiley Online Library (wileyonlinelibrary.com).

The objective of this study was to investigate the enzymatic generation of the Mn3þ-malonate complex and its application to the process of oxidizing several organic compounds. The experimental set-up consisted of an enzymatic reactor coupled to an ultrafiltration membrane, providing continuous generation of Mn3þ-malonate from a reaction medium containing versatile peroxidase (an enzyme produced by Bjerkandera adusta strain BOS55), H2O2, MnSO4, and malonate. The effluent of the enzymatic reactor was introduced into a batchstirred reactor to oxidize three different classes of compounds: an azo dye (Orange II), three natural and synthetic estrogens, and a polycyclic aromatic hydrocarbon (anthracene). The enzymatic reactor provided the Mn3þ complex under steady-state conditions, and this oxidative species was able to transform the three classes of xenobiotics considerably (90–99%) C with negligible loss of activity. V 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 668–676, 2011 Keywords: Mn(III)-malonate, enzymatic oxidation, versatile peroxidase, dye, anthracene, estrogen

Introduction Lignin is one of the most widespread substances in nature. One widely held principle concerning lignin degradation by fungi is the necessity of primary attack by extracellular oxidoreductases including lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase.1 In addition, some fungi contain another lignin-degrading enzyme: versatile peroxidase (VP). VP is also known as hybrid Mn-peroxidase and combines properties of LiP and MnP2 (i.e., it is able to oxidize Mn2þ and non-phenolic aromatic compounds). Nevertheless, the catalytic efficiency of VP in the presence of Mn2þ is considerably higher than its efficiency in the presence of other aromatic substrates.3 The catalytic cycle of VP includes successive conversions in which the enzyme is oxidized by H2O2 to a two-electronoxidized intermediate, Compound I. Compound I removes one electron from the substrate and passes the electron to a one-electron-oxidized species, Compound II. At this stage of the catalytic cycle, organic substances and Mn2þ can serve as electron donors. Compound II removes another electron from the substrate, and subsequently, the enzyme returns to its native form. The oxidation of Mn2þ generates Mn3þ, which is a strong oxidative species (1.54 V) and acts as a mediator in the degradation of organic compounds at locations remote from the enzyme active site.4 That is, target substrates are oxidized by Mn3þ and not directly by the enzyme. This ion is rather unstable and requires the presence of organic acids that

Correspondence concerning this article should be addressed to R. Taboada-Puig at [email protected] 668

form Mn3þ chelates, which can then non-specifically oxidize organic molecules.2,5,6 Formation of complexes with dicarboxylic acids alters the potential of Mn3þ. The more strongly that Mn3þ is chelated, the more stable it is. Increased stability inhibits disproportionation to Mn2þ and Mn4þ but also makes Mn3þ a weaker oxidant.7 Mn3þ-chelates have several advantages over proteins when used as oxidants. They are more tolerant to protein-denaturing conditions such as extreme temperatures, low and high pH, oxidants, detergents, and proteases. Such chelates may also penetrate microporous barriers that prevent the passage of proteins. The main drawback related to the application of enzymatic systems is that there are consumption and destabilization of the enzyme in the process, which renders low degradation productivity and limits the applicability of the enzymatic process.8,9 A more efficient enzymatic system would accomplish two main objectives: maximizing Mn3þ-chelate production and minimizing consumption and deactivation of the enzyme. In this sense, separation of the catalytic cycle of the enzyme and the degradation of the target recalcitrant compound by Mn3þ reactive species may be useful to accomplish both goals. In this study, three different groups of compounds were considered as target substances for oxidation: an industrial azo dye (Orange II), natural and synthetic estrogens (estrone, 17b-estradiol, and 17a-ethinylestradiol) that act as endocrine disruptors, and a polycyclic aromatic hydrocarbon (anthracene). These compounds are considered to be persistent and recalcitrant pollutants and represent diverse chemical structures and physico-chemical properties. The development of a two-stage process was carried out in consideration of the C 2011 American Institute of Chemical Engineers V

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influence of the following specific factors: (a) parameters that may directly affect the VP catalytic cycle (type and concentration of organic acids, Mn2þ, H2O2 as well as the influence of temperature and pH on the stability of the complex) and (b) parameters that may affect the degradation of the target xenobiotic in a batch reaction. Finally, both the enzymatic and degradation reactors were coupled in a continuous operation to bleach the dye.

Materials and Methods Versatile peroxidase production VP was obtained from cultures of Bjerkandera adusta strain BOS55 (ATCC 90940). A pre-inoculum was generated by adding five agar plugs (from 7-day-old colonized plates) to a 1-L Fernbach flask containing 100 mL of N-limited medium of the following composition: 10 g/L glucose (Panreac, Castellar del Valle`s, Spain), 2 g/L ammonium tartrate (Sigma-Aldrich, St. Louis, MO), 500 lM MnSO4 (Panreac, Castellar del Valle`s, Spain), and 100 mL BIII mineral salts10 in 20 mM sodium acetate buffer (Panreac, Castellar del Valle`s, Spain). The pre-inoculum was incubated at 30 C for 5 days in static culture. Thereafter, the fungal culture was blended in a Waring blender and added to 250-mL Erlenmeyer flasks containing 90 mL of the culture medium to a final concentration of 10% (v/v). The flasks were incubated in an orbital shaker (New Brunswick Scientific, Innova 4000) at 30 C and 150 rpm. After 3–4 days, pellets were transferred to a 10-L BIOSTAT-E bioreactor (Braun Biotech) in a proportion of approximately 10% (v/v). Skimmed cheese whey (25 g/L) was used as the C source in the fermenter instead of glucose. Extracellular culture broth was collected at the point of highest VP activity (typically after 4–6 days) and filtered through Whatman No. 1 paper filters to remove biomass. The filtered culture broth containing the enzyme was concentrated by ultrafiltration (Filtron Minisette System, 10 kDa cut-off) and kept at 21 C until future use. Palma et al.11 purified two different peroxidases (BOS1 and BOS2) differing in the degree of glycosylation from cultures of B. adusta strain BOS55 (ATCC 90940) using N-limited medium. These isoenzymes presented molecular weights of 45 and 40 kDa and pIs of 3.45 and 3.40, respectively. Determination of the manganese-dependent activity of VP and measurement of the Mn31-chelate Manganese-dependent VP activity was measured by spectrophotometrically monitoring the oxidation of 2,6-dimethoxyphenol (DMP, Fluka, Buchs, Switzerland) at 468 nm at a temperature of 30 C (e468 ¼ 49.6 mM1 cm1) (Shimadzu UV-Visible Spectrophotometer. UV-1603, Japan). The reaction mixture contained 50 mM sodium malonate (pH 4.5), 1 mM DMP, 1 mM MnSO4, and up to 600 lL of supernatant in a total volume of 1 mL. The reaction was initiated by adding 0.4 mM H2O2 (Sigma-Aldrich, St. Louis, MO). An enzyme unit was defined as the amount of enzyme producing 1 lmol of coerulignone per minute.12 The production of Mn3þ-chelate was determined spectrophotometrically in a quartz cell with a volume of 1 mL in a Shimadzu spectrophotometer. Finally, the concentration of Mn3þ-chelate was determined using extinction coefficients of e238 ¼ 6.5 mM1 cm1, e270 ¼ 11.59 mM1 cm1, e290 ¼ 5.89 mM1 cm1, and e270 ¼ 5.6 mM1 cm1 for Mn3þ-tartrate, -malonate, -lactate and -oxalate, respectively.13–15

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Influence of organic acids, Mn21, and H2O2 on Mn31-chelate production The influence of several parameters [VP activity, organic acid (type and concentration), levels of Mn2þ and H2O2 and pH] on the formation of Mn3þ-chelate was evaluated. The reactions were initiated by the addition of H2O2 and followed for 60 min in a Shimadzu spectrophotometer. Several organic acids, malonic (Sigma-Aldrich, St. Louis, MO), oxalic (Panreac, Montcada, Reixac, Spain), tartaric (Sigma-Aldrich, St. Louis, MO), and lactic (Panreac, Montcada, Reixac, Spain) acid, were evaluated at multiple concentrations from 5 to 50 mM to assay the production of Mn3þ-chelate in the presence of 2 mM MnSO4 and 400 lM H2O2 at pH 4.5. All acid solutions were prepared from their sodium salts. The effect of initial MnSO4 concentration between 1 and 4 mM was determined at pH 4.5 in the presence of 5, 10, 30, and 50 mM malonate with 400 lM H2O2. The effect of initial H2O2 concentration on the generation of the Mn3þ-chelate was evaluated in the range of 0.02–4 mM in the presence of 30 mM malonate and 2 mM MnSO4 at pH 4.5. The effect of the pH (ranging from 3 to 9) was evaluated in the presence of 30 mM malonate, 2 mM MnSO4, and 300 lM H2O2. Titers of VP activity were systematically changed from 5 to 100 U/L to maximize the production of the complex in the presence of 30 mM malonate, 2 mM MnSO4, and 300 mM H2O2 at pH 4.5. The stability of the Mn3þ-chelate was studied at three temperatures: 18 C, 4 C, and room temperature (25 C).

Batch production of Mn31-chelate and its application to the process of oxidizing several organic compounds The potential oxidation capacity of the Mn3þ-chelate was evaluated for different organic compounds after batch production of the Mn3þ-chelate to supply a sufficient amount of the complex. Batch production was conducted in a stirred Amicon ultrafiltration cell (8,200) with 150 mL of working volume and a 10 kDa cut-off polyethersulfone membrane (Millipore Corporation, Billerica, MA). Before batch production, the enzyme was dialyzed in 10 mM malonate buffer (pH 4.5). The standard composition of the reaction medium in the enzymatic reactor was: 100 U/L VP, 300 lM H2O2, 30 mM malonate, and 2 mM Mn2þ. After 15 min in the Amicon cell, N2 was used to flush the system to assure a head space pressure of 2 bar and allow output of the complex at a rate of 2.9  0.5 mL/min for 30–45 min. The complex was then measured, and an average concentration of 350 lM Mn3þ-chelate was obtained, which was later applied in the batch oxidation assays. Three different groups of compounds: an azo dye (Orange II), natural and synthetic estrogens, estrone (E1, SigmaAldrich, St. Louis, MO), 17b-estradiol (E2, Sigma-Aldrich, St. Louis, MO), and 17a-ethinylestradiol (EE2, SigmaAldrich, St. Louis, MO), and a polycyclic aromatic hydrocarbon, anthracene (Janssen Chimica, NJ), were selected as target compounds for the oxidation reaction. The experiments were performed in batches on a small scale, from 1-mL cuvettes to 100-mL Erlenmeyer flasks. Control assays in the absence of the complex were also conducted in parallel. Oxidation of the Orange II dye was performed in a 2 mL quartz spectrophotometer cell with 1 mL of reaction mixture for 10 min. Decolorization was measured spectrophotometrically at 480 nm16 by adding a stock solution of the dye (100 mg/L) and

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Figure 1. Schematic diagram of the two-stage system of Mn31chelate formation.

Mn3þ-chelate. Different initial concentrations of Orange II, from 10 to 27 mg/L, and Mn3þ-chelate, from 70 to 200 lM, were evaluated. Transformation of the estrogen compounds was performed in 100-mL Erlenmeyer flasks with a reaction volume of 20 mL. The three estrogens, E1, E2, and EE2, were transformed independently. The reaction mixture consisted of 200 lL of estrogen solution (250 mg/L) and 19.8 mL of the Mn3þ complex. Oxidation of anthracene was carried out in 25-mL Erlenmeyer flasks with a reaction volume of 10 mL with magnetic stirring at room temperature (25 C) in the dark. The reaction mixture consisted of 4 mL of anthracene stock solution in acetone (12.5 mg/L) and 6 mL of the Mn3þ complex. Flasks containing Mn2þ, malonate, and H2O2 (Control 1) and Mn2þ and malonate (Control 2) at the same concentrations used in the Amicon Cell, instead of the retentate mixture, were also set up as control experiments. Samples were taken after 4 h and 2, 3, and 6 days. Two-stage process for the continuous production of the Mn31-complex and the decolorization of the azo dye The enzymatic system used for the continuous production of Mn3þ-malonate is presented in Figure 1. This system consists of a stirred tank reactor (200-mL working volume) operated in continuous mode coupled to a 10 kDa cut-off ultrafiltration membrane (Prep/Scale-TFF Millipore Corporation, Billerica, MA), which permits the recycling of the enzyme to the reaction vessel. The additional volume of the ultrafiltration unit and the interconnecting tubing was 65 mL, making the total volume of the reaction system 265 mL. Cofactors were added to the reactor from two stock solutions: H2O2 (13 lM) and a mixture of Mn2þ and sodium malonate (1 and 20 mM, respectively) at pH 4.5. These two stock solutions were fed into the reactor by independent variable-speed peristaltic pumps. The operational parameters were: initial VP activity, 100 U/L; H2O2 feed rate, 5 lM/ min; Na-malonate feed rate, 330 lM/min; MnSO4 feed rate, 16 lM/min; and hydraulic retention time (HRT), 60 min. The enzyme was recycled at a recycling:feed flow ratio of 12:1. The enzyme solution was provided as a single addition of VP at the beginning of the experiment. VP activity was measured by monitoring the amount of the Mn3þ-malonate complex (268 nm) in the outflow of the enzymatic reactor. Samples from the filtrate were taken and measured for Mn3þ-malonate concentration. The Mn3þ-malonate generated in the enzymatic reactor was used in a second stage for bleaching the azo dye Orange

Figure 2. Time course of Mn31-malonate formation by VP. Conditions were: malonate, 30 mM; Mn2þ, 2 mM; H2O2, 0.3 mM; pH, 4.5.

II. The decolorization reactor (200-mL working volume) was fed with 2.5 mL/min of Mn3þ-malonate and 2.5 mL/min of a stock solution of the dye (50 mg/L). The reaction was performed at room temperature and 150 rpm. Finally, Mn2þ was recovered and reintroduced into the enzymatic reactor using a reverse osmosis system. Determination of the concentrations of Orange II dye, estrone, 17b-estradiol, 17a-ethinylestradiol, and anthracene Decolorization of the Orange II dye was measured spectrophotometrically at 480 nm and monitored for 15 min.16 The determination of E1, E2, and EE2 concentrations was made using a HP 1090 HPLC equipped with a diode array detector for monitoring absorbance at 220 nm, a Lichrosphere100 C18 reversed-phase column (250  4.6 mm, particle size: 5 lm; LiChrocart, Merck, Germany) and an HP ChemStation data processor. The injection volume was set at 200 lL, and the isocratic eluent (acetonitrile: 50 mM phosphate, 60:40; pH 4.5) was pumped in at 0.8 mL/min. A calibration curve was plotted to correlate the area and the concentration of the stock solutions, which ranged between 0.1 and 5 mg/L. Determination of anthracene concentration was performed as follows, with an HP 1090 HPLC, equipped with a diode array detector for monitoring absorbance at 253 nm, a 4.6 mm  200 mm Spherisorb ODS2 reverse-phase column (5 lm; Waters) and an HP ChemStation data processor. The injection volume was set at 10 lL, and the isocratic eluent (80% acetonitrile and 20% water) was pumped in at 0.4 mL/min. A calibration curve was plotted to correlate the area and the concentration of the stock solutions, which ranged between 0.1 and 10 mg/L.

Results 3þ

Generation of the Mn -chelate in batch mode was monitored by spectrophotometry for 11 h. The reaction medium was similar to that used for the determination of Mn-dependent activity, except for the lack of a phenolic substrate (2,6dimethoxyphenol), allowing direct monitoring of Mn3þ-chelate formation. Figure 2 shows a typical time course of Mn3þ-chelate generation by VP.14 A steady slope in the generation of the Mn3þ-chelate from the beginning of the experiment was observed, reaching a maximum of 240

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Figure 3. Influence of different parameters on the production of Mn31-chelate. (a) Type and concentration of organic acid, (n) malonic acid, (h) oxalic acid, (~) tartaric acid, (D) lactic acid; (b) concentration of hydrogen peroxide; (c) pH; (d) initial VP activity.

lM. After the maximum, a slight decay was detected at very slow rate in the incubation period between 1.5 and 11 h, which was indicative of substantial stability over the evaluation period. The kinetics of Mn3þ-chelate formation requires the evaluation of the effect of certain major parameters such as the type and concentration of organic acids, levels of Mn2þ and H2O2, pH, VP activity, temperature, and storage time. Two types of factors were studied: (a) those related to the production of the complex and (b) those related to its stability after being produced.

Table 1. Effect of MnSO4 and Sodium Malonate Concentrations on the Formation of Mn31-chelate (lM) Mn3þ-chelate formed (lM) [Mn2þ] (mM) 1

2

4

64 111 169 157

80 140 212 180

89 133 186 163

[Na-malonate] (mM) 5 10 30 50

Effect of MnSO4 concentration Effects of the type and concentration of organic acid Organic acids such as oxalic and malonic acid are secreted by fungi.15 Organic acids play an important role in Mn-dependent enzymatic reactions by facilitating the release of Mn3þ from the active site of the enzyme and stabilizing the metal ion by chelation.15 In this study, the type and concentration of organic acid dramatically affected both the rate of generation of the Mn3þ-chelate and the peak levels achieved (Figure 3a). Low levels of chelate were found for tartaric and lactic acid and intermediate levels were detected when oxalic acid was used whereas the highest titers were achieved in the presence of malonic acid. According to these results, malonate enhanced the production of the Mn3þ-chelate. Taking this into account, this compound was considered the best organic acid to be used in the production of the Mn3þ-chelate.

The effect of initial MnSO4 concentrations in the range of 1–4 mM was evaluated in the presence of 5, 10, 30, and 50 mM malonate. The highest peak titer corresponded to a concentration of 2 mM Mn2þ, and no further increase was observed at a higher concentration of Mn2þ. Decay of the Mn3þ-chelate after the maximum level was reached was significantly faster at higher concentrations of MnSO4 (Table 1). Effect of H2O2 concentration The effect of initial H2O2 concentration on the generation of the Mn3þ-chelate was evaluated in the presence of 30 mM malonate. As shown in Figure 3b, the enzymatic production of Mn3þ-malonate reached its peak at a H2O2 concentration of 0.4 mM.

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Figure 4. Influence of temperature on Mn31-malonate stability. (n) Room temperature, (~) T ¼ 4 C, (l) T ¼ 18 C.

Effect of pH With the objective of evaluating the influence of pH on the production of the Mn3þ-chelate, pH was adjusted from 3 to 9. As shown in Figure 3c, the highest concentration of the complex was obtained at pH 4–4.5, whereas at more basic or acid pH, the production dropped drastically due to enzyme deactivation. Effect of VP activity The influence of VP activity was analyzed in experiments performed with variable titers of VP, ranging from 5 to 100 U/L. These experiments were carried out in a stirred Amicon ultrafiltration cell (8,200) with 150 mL of working volume and a 10 kDa cut-off polyethersulfone membrane. As shown in Figure 3d, the amount of initial VP activity had a significant effect on the production of the Mn3þ-chelate. VP activity of 100 U/L led to the highest levels of the Mn3þ-chelate out of the range tested. Further increases had no significant effect on the production of the Mn3þ-chelate. Taking this into account, it was considered that the best initial activity for the production of Mn3þ-malonate was 100 U/L. Effect of temperature on the stability of the Mn31-malonate complex The stability of Mn3þ-malonate at different temperatures was studied by incubating the complex at 18 C, 4 C, and room temperature in 20 mM malonate (Figure 4). The halflife of the complex at room temperature was found to be 3.75 h. This value was increased at low temperatures, especially when the Mn3þ-chelate was kept frozen at 18 C. According to these results, the application of the complex should be conducted immediately after its production, and its storage is only recommended at a temperature that will keep it frozen. Application of the Mn31-chelate for oxidizing recalcitrant compounds The Mn3þ-chelate was produced in an Amicon cell in similar conditions to those described above in the optimization experiments and was later used for the oxidation of three different groups of compounds: an azo dye (Orange II), natural

Figure 5. Batch degradation of the Orange II dye using Mn31malonate. (a) Influence of the initial concentration of the dye on the extent of degradation, (n) 27 mg/L, (h) 21 mg/L, (~) 16 mg/ L, (D) 11 mg/L; (b) kinetics of degradation of Orange II with different initial concentrations of Mn3þ-malonate, (n) 200 lM, (h) 165 lM, (~) 110 lM, (D) 70 lM.

and synthetic estrogens (E1, E2, and EE2), and a polycyclic aromatic hydrocarbon (anthracene). The oxidation of these compounds was performed in batches at room temperature (25 C). Control assays in the absence of the complex were also conducted in parallel. Azo Dye (Orange II). The capability of the complex to decolorize Orange II was evaluated with different initial concentrations of the dye, ranging from 10 to 27 mg/L. The initial concentration of the complex was 165 lM. In all of the cases, the complex was able to oxidize the dye, reaching the same percentage of decolorization (80–85%). However, as shown in Figure 5a, when the initial concentration was 27 mg/L, the final concentration was 5.5 mg/L after 10 min (i.e., 21.5 mg/L of Orange II was decolorized), while when the initial concentration was 11 mg/L, 8.1 mg/L of Orange II was decolorized. Different concentrations of the Mn3þ-chelate, ranging from 70 to 200 lM, were evaluated to maximize the extent and rate of decolorization. Results are shown in Figure 5b. It was observed that the higher the initial concentration of the complex, the higher the amount of oxidation. Thus, when the concentration of the oxidizing agent was 200 lM, the percentage of decolorized Orange II was 90%, whereas when the initial concentration of oxidizing agent was 70 lM, the decolorization percentage was 42.5%.

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Figure 6. Chromatograms of the estrogens E1 (a), E2 (b), and EE2 (c) after 1 min of reaction time. A chromatogram of a standard solution containing the initial concentration of each estrogen is shown in the upper right corner.

Natural and Synthetic Estrogens. The capacity of the complex to oxidize three different estrogens, estrone, 17b-estradiol, and 17a-ethinylestradiol, was evaluated by the batch method. The initial concentration of the complex was 350 lM, and the concentration of each estrogen was 2.5 mg/L. Samples were taken after 1 and 15 min and 1, 6, and 12 h and analyzed for estrogen concentration. As shown in Figure 6, the transformation of the three estrogens was immediately achieved. Anthracene. The ability of the complex to oxidize a solution containing anthracene was also evaluated. The low solubility of anthracene in water was countered by the addition of acetone to a final concentration of 36% (v/v), which permitted an increase in its concentration from 0.07 to 10 mg/L. The final transformation percentage after 6 days was 90%.

A continuous process for production of the Mn31-chelate and decolorization of the Orange II dye in two stages Continuous production of the Mn3þ-malonate complex was carried out in a stirred reactor coupled with an external ultrafiltration membrane. Conditions were set based on the previous results obtained in batch reactions. The differences from the previous experiments presented in this study include the ultrafiltration system used, the optimized conditions for Mn3þ-malonate generation and the scale of the ultrafiltration system (1 mL cuvette cell, 150 mL Amicon cell, and 265 mL Prep/Scale Millipore System). In the second system used, the enzyme is retained within the cell, and the application of N2 pressure allows low molecular compounds to flow through the membranes. In the third system, the enzyme is recirculated using peristaltic pumps, retaining

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defined as the relation between the Mn in the recirculation line and the feeding line, was 96%.

Discussion

Figure 7. Profile of production of Mn31-malonate and degradation of Orange II. (n) Mn3þ-malonate produced in the enzymatic reactor. This value reflects enzymatic activity, (~) Mn3þ-malonate introduced in the oxidation reactor, and (*) decolorization of Orange II.

the enzyme within the two-stage system. In both systems, mixing in the reactors is carried out by magnetic stirring. During the continuous operation of the two-stage system, the optimal concentrations of the cofactors in terms of enzyme activity were similar to those found to be optimal during the batch experiments. In the batch operation, a ratio of malonate/Mn2þ/H2O2 of 75/5/1 (30, 2, and 0.4 mM) was optimal for production of the complex. In the continuous system, a ratio of 50/2.5/1 (66, 3.33, and 1.3 lmol/min) was used. It is important to note that scaling up the amounts used in the batch operation to provide adequate feeding rates in the continuous mode is not straightforward. Optimal values reported for the one-stage operation of a similar system were taken into consideration for determining the H2O2 feeding rate.16 Thus, an H2O2 volumetric feeding rate of 8 lM/min (considering a reaction volume of 265 mL) was used as the initial value. Lo´pez et al.16 have found that volumetric feeding rates of H2O2 in the range of 5–15 lM/min caused no enzyme inactivation but, at the same time, limited the decolorization rate of the system significantly. In our case, a low peroxide feeding rate was sufficient to maintain high levels of production of the Mn3þ-malonate complex. The scale-up of the two-stage system from an optimized batch process to a continuous system increased the generation of the Mn3þ complex from 350 to 400 lM. The amount of Mn3þ-malonate produced increased during the first 7 h and then remained stable for the next 5 h; production reached a maximal level of 400 lM (Figure 7). The continuous reactor used for Mn3þ-malonate production was coupled with an oxidation system operating with the azo dye Orange II. Similar to previous results, this compound presented an intermediate oxidation rate, and spectrophotometric monitoring was simple and straightforward.9 Bleaching of Orange II was directly proportional to the concentration of the complex; at the beginning of the operation of the system, the level of decolorization was low but later increased to 60% as the concentration of the complex reached 400 lM. VP activity was maintained throughout the reaction because the concentration of the complex never dropped throughout the entire operation time. During the operation of the system, the reverse osmosis recovery,

On the basis of the production of Mn3þ-chelate, malonic acid was found to be the best organic acid. This result agrees with those obtained by Cui and Dolphin4 and Wariishi et al.,15 who reported that malonate presented the strongest chelating strength. Nevertheless, Glenn and Gold17 and Lopez et al.18 found that the acid with the strongest chelating strength was malic and oxalic acid, respectively. Considering the range of concentrations evaluated, 30 mM was optimal, with a maximum peak higher than 200 lM. At lower concentrations, the titer and generation rate of the Mn3þ-chelate were both lower. At concentrations higher than 30 mM, there was a slight decrease in the titers achieved. Mielgo et al.9 observed that the highest levels of decolorization of Orange II by MnP were obtained when no malonic acid was added whereas Kishi et al.5 reported that MnP activity (measured as oxidation of 2,6-DMP) increased with increasing malonate concentration, reaching a maximum near 50 mM. Thus, the type and concentration of the organic acid used is dependent on the application. The highest production of the complex corresponded to a concentration of 2 mM Mn2þ. The inactivation of VP in the absence of substrate (DMP) occurred during incubation of the enzyme with H2O2, with inactivation especially evident at high concentrations of H2O2. Moreover, higher concentrations of H2O2 did not correlate with a significant increase in the titers of the complex, suggesting an imbalance in the cyclic process of oxidation and reduction of the mediator and destabilization of the enzyme during the reaction. Inactivation of the peroxidase by H2O2 probably occurred due to formation of the catalytically inactive Compound III.6,15 Among the different parameters evaluated, the concentration of H2O2 was found to be crucial to the activity of the enzyme. The concentration of H2O2 needed to be strictly controlled, as an excessively concentrated reagent would cause the inactivation of the enzyme. On the contrary, an excessively low concentration would limit the reaction rate and extent. The highest concentration of the Mn3þ complex was obtained at pH 4–4.5. The same optimal pH value has been found when the inactivation of a similar VP enzyme was evaluated.19 Another aspect to be considered when evaluating the effect of pH on the generation of the Mn3þ complex is the chemical equilibrium of Mn3þ. It is known that Mn3þ is a strong oxidant, as observed from Eq. 1.

Mn3þ þ e $ Mn2þ

Eo ¼ 1:54 V

(1)

In an alkaline medium, the two cations precipitate, but the extent of Mn3þ precipitation is higher than that of Mn2þ due to differences of the solubility of the products Mn(OH)3 and Mn(OH)2 (Ksp, 1036 and 1013, respectively). Thus, if the pH of the medium is alkaline, the chemical equilibrium is described by Eq. 2.

MnðOHÞ3 þ e $ MnðOHÞ2 þ OH

Eo ¼ 0:1 V (2)

The oxidation potential of Mn3þ/Mn2þ in basic medium is very low compared to that achieved at acid pH.20 For this

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reason, it is much more convenient to use the complex at acid pH values, at which both Mn3þ and Mn2þ are soluble in the reaction medium. On the contrary, if a basic pH is used, insoluble hydroxides will form. Previous results have demonstrated the ability of ligninmodifying enzymes to oxidize estrone,21 17b-estradiol, and 17a-ethinylestradiol.22 The enzymatic transformation of these compounds was performed after 1 h of treatment, whereas in the present study, the complete disappearance of E1, E2, and EE2 was achieved immediately after being added to the enzymatic system. In previous studies, E1 was detected as the by-product of the biodegradation of E2 both in laboratoryscale activated-sludge and nitrifying activated sludge reactors.23,24 In both studies, E1 was partially degraded and converted into more polar metabolites. In this study, none of the peaks for the three estrogens in the effluent of the Mn3þ-reactor was observed (Figure 6). In particular, the oxidative treatment was able to transform E1 completely (Figure 6a). Accordingly, the system is believed to degrade not only the original compound E1 added in the assays but also the potential E1 obtained in the degradation of E2. In another study, the abiotic oxidation of EE2 with a similar oxidative system based on Mn3þ-acetate was shown to result in the dehydrogenation of ring, as determined using LC-MS and NMR, but not in the formation of E1.25 Therefore, we assume that E1 was not a degradation product of EE2. During the oxidation of anthracene, a rapid disappearance of the hydrocarbon was initially observed, followed by a slower rate of transformation. Considering that the half-life of the Mn3þ complex at room temperature is 4 h, the oxidation of anthracene may partially be attributed to the formation of reactive oxygen species, such as hydroxyl or phenoxy radicals that subsequently oxidize the polycyclic aromatic hydrocarbon (PAH). In this case, the Mn3þ complex would act as a direct oxidant during the first hours, oxidizing the PAH, as well as triggering an oxidative cascade through the formation of active oxidant intermediate compounds such as quinones. Considering that anthraquinone is the main metabolite produced during oxidation of anthracene by ligninolytic enzymes,26,27 one possible explanation for the behavior of the reaction stems from the autocatalytic effect of anthraquinone. In previous studies of enzymatic oxidation of anthracene with the same enzymatic system,28 the oxidation rate was observed to be significantly faster than that obtained in the two-stage system in this study. One possible application for the Mn3þ-malonate complex (for degrading compounds with low solubility such as anthracene) would be in a biphasic system like that proposed by Eibes et al.8 In such a system, replacement of the aqueous phase by fresh Mn3þchelate in each of several cycles would produce an increase in the anthracene oxidation rate. Mn3þ-chelates, being nonproteinaceous, can solve problems related to the use of organic solvents for increasing the solubility of certain compounds, as the solvent would not interfere with the enzyme and may have little effect on the stability of the complex. The oxidation rates of the three types of compounds using Mn3þ-malonate as an oxidizing agent were in agreement with those observed using the MnP enzyme in a one-stage process. The azo dye and the estrogens were easily transformed by Mn3þ-malonate, whereas the transformation of anthracene was slower. This broad degradation capacity sup-

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ports the use of the Mn3þ complex for the treatment of other xenobiotics. During the continuous production of the complex, a 400lM concentration of Mn3þ-malonate was achieved. The concentration of the complex in the oxidation reactor inlet was smaller than in the enzymatic reactor outlet because the complex was stored in an intermediate tank at room temperature. The continuous reactor used for Mn3þ-chelate production was coupled with an oxidation system operating with the azo dye Orange II. Continuous decolorization of the dye by MnP in an enzymatic membrane bioreactor was performed previously by Lo´pez et al.16 Fairly constant decolorization (to levels [90%) was reached over 150 min, but the average MnP consumption was 37 U/L/h. In this study, decolorization levels between 50% and 60% were maintained for 7 h without loss of enzyme activity. Previously, other systems for the generation and use of Mn3þ-chelates in a two-stage reactor system have been reported.29,30 Both of these studies proposed enzyme immobilization on different supports, the NH2-Emphaze polymer (Pierce Chemical, Rockford, IL) and FSM-16 (a mesoporous material), respectively. The Emphaze-MnP column system requires NaCl to prevent the adsorption of Mn3þ-chelate to the support matrix, and salt conditions must be controlled carefully. However, this study reports the continuous production of Mn3þ-chelate in reactions catalyzed by free VP from B. adusta in a stirred reactor coupled to an external ultrafiltration membrane. Hence, this system does not require enzyme immobilization or additives such as NaCl.

Acknowledgment This study was supported by the Spanish projects PGIDIT06PXIB265088PR, CTQ2007-66788, and CTQ201020258. R. Taboada expresses his gratitude to the Spanish Ministry of Science and Innovation for his financial support (BES2008-006977). T. Lu´-Chau and G. Eibes thank the Isabel Bar´ ngeles Albarin˜o programs, respectively, from the Gareto and A lician Government for the economic support provided during the development of this work.

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