Laccase from Trametes sp. I-62: Production, Characterization, and

Article pubs.acs.org/IECR

Laccase from Trametes sp. I‑62: Production, Characterization, and Application as a New Laccase for Eucalyptus globulus Kraft Pulp Biobleaching Raquel Martin-Sampedro, Jesus Miranda, Juan C. Villar, and Maria E. Eugenio* Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA, Carretera de la Coruña, km 7.5, 28040 Madrid, Spain ABSTRACT: The production of laccase from Trametes sp. I-62 was optimized, and then it was applied for the first time in biobleaching in order to contribute to solve some problems associated with pulp bleaching. When wheat straw and copper sulfate were used in combination as inductors, the maximum laccase activity was obtained. The resulting enzymatic fluid was applied at optimum pH and temperature jointly with acetosyringone in a pretreatment stage (L) aimed to enhance the subsequent bleaching consisting of an alkaline extraction (E) and a hydrogen peroxide stage (P). Results showed that, even when a significantly lower amount of hydrogen peroxide was consumed in the LEP sequence (41.9% vs 89.9% for EP), higher delignification (40.6% vs 33.4%) and brightness (63.6 vs 56.3% ISO) were achieved. After accelerated aging, the optical properties of the biobleached pulp were found to be less stable but still remained higher than those of control pulps.

1. INTRODUCTION Bleaching is the most pollutant step in bleached cellulose pulp production. Traditionally, molecular chlorine, hypochlorite, or chlorine dioxide has been used as bleaching chemicals. The presence of hazardous chlorolignins in the bleaching effluents has fostered the replacement of chlorine compounds with less pollutant compounds, such as oxygen derivatives (molecular oxygen, ozone, or hydrogen peroxide), which brought about the widely known TCF (totally chlorine free) processes. However, these new processes are not as lignin-selective as those based on chlorine, and some of them are expensive.1 These are some of the reasons why the TCF processes have not been completely substituted and ECF (elemental chlorine free) sequences are also used frequently. On the other hand, biotechnology can contribute to solve some of the environmental problems of pulp bleaching. For example, ligninolytic enzymes, such as laccases, have been widely tested2−8 as pretreatment in conventional bleaching, producing pulps of the same quality but using less chemicals in subsequent bleaching steps. The importance of the use of laccase in this field stems from its capacity to oxidize a wide variety of lignin derivatives and phenolic compounds requiring only molecular oxygen and the presence of low molecular weight compounds called mediators. These mediators, once oxidized by the laccases, become stable radicals that may continue oxidizing other compounds not used as direct substrates by the enzyme.9 The use of both laccase and mediators is commonly known as a laccase−mediator system (LMS).9 However, restrictions, such as the high cost of the enzymes and/or the toxicity of the synthetic mediators, prevent a full industrial application of the LMS in the bleaching process. To solve these problems, nontoxic mediators and new laccases with specific physicochemical characteristics for biobleaching purposes are the object of intense research. In the search for new laccases, Trametes sp. I-62 has been previously studied as a laccase producer.10−15 Mansur et al.14,15 have detected seven laccase isozymes secreted by this fungus, © 2013 American Chemical Society

and they have identified and cloned three genes which codified these laccases. Their ability to degrade complex substrates such as alkali-lignins, polymeric dyes, commercial humic acid, and synthetic melanoidins has been proved. Based on this ability, different authors have applied Trametes sp. I-62 to the bioremediation of molasses-based distillery wastewater.16−19 However, this fungus has hardly been applied to the pulp and paper industry, for bioremediation of lignosulphonates20 and biopulping.21 These last authors have found that Trametes sp. I62, applied as a pretreatment prior to kraft pulping, can reduce the κ number (9.3%) and increase the viscosity (3.4%), but with consumption of more chemical reagents (5.9% Na2S). These authors also applied a commercial laccase pretreatment instead of the fungus pretreatment; however, the enzymatic crude enriched in laccase obtained from Trametes sp I-62 has not been previously used in this application. Although Silva22 has studied different medium composition and laccase inductors, the maximum laccase activity achieved by this author was 950 mUA/mL (using Kirk medium and adding HBT as inductor on the seventh day of incubation). In order to increase this enzymatic activity, several laccase inductors different from those used by Silva22 could be assayed. For instance, copper sulfate, ethanol, wheat straw, etc. successfully induced the laccase production of different fungi.23−25 Moreover, no studies using various laccase inductors simultaneously in order to obtain a hypothetical synergic effect have been reported for this fungus. Therefore, the goal of the present study was first to optimize the laccase production from Trametes sp. I-62 using different inductors (individually and in combination) and second to evaluate the role of the optimal enzymatic crude produced by Trametes sp. I-62 in biobleaching of Eucalyptus globulus kraft Received: Revised: Accepted: Published: 15533

July 8, 2013 September 18, 2013 October 7, 2013 October 7, 2013 dx.doi.org/10.1021/ie402160p | Ind. Eng. Chem. Res. 2013, 52, 15533−15540

Industrial & Engineering Chemistry Research

Article

2.4. Volumetric Laccase Activity. The laccase activity was determined daily in all samples of extracellular fluid from the fungal cultures, using ABTS as substrate at pH 5 and 25 °C.26 The reaction mixture consisted of 1:1 (v/v) culture fluid and substrate. One unit of laccase activity was defined as the amount of enzyme needed to obtain 1 μmol of product per minute under the assay conditions. Results were expressed in relative terms. 2.5. Effect of pH and Temperature on Laccase Activity. The enzymatic extracellular fluid that showed the maximum laccase activity was isolated from the fungus mycelium by filtration. In the resulting liquid, the laccase activity was measured at 25 °C and different pH values within the range pH 2.2 to pH 7, and using citric acid-disodium hydrogen phosphate buffer (200 mM). After establishing the optimum pH, the laccase activity was determined at different temperatures that ranged between 30 and 90 °C. These results determined the optimal pH and temperature that brought the maximum activity. However, the enzyme stability should also be taken into account when selecting the optimal operational conditions for biobleaching. To this end, the enzymatic extracellular fluid was kept under several different pH (2 to 7) and temperature (25, 40, 50, 60, and 70 °C) conditions during at least 3 h and the laccase activity was measured according to section 2.4 at different times. 2.6. Enzymatic Pretreatment. Assays were performed using 50 g of an industrial E. globulus kraft pulp (κ number, 14; brightness, 34.4% ISO; viscosity, 1136 mL/g; 3.21 of hexenuronic acid as κ number contribution) which was introduced into polyethylene bags where it was intensively mixed with the enzymatic fluid and chemicals before being submerged in a thermostatic bath. Consistency, reaction time, laccase dose, and mediator concentration were fixed at 10%, 1 h, 2.4 U/g, and 0.05 mmol/g, respectively. Acetosyringone was used as mediator. A few drops of 0.05% (v/v) Tween 80 were added to all assays in order to improve the interaction between enzyme and substrate. Temperature and pH were also fixed at the optimal values obtained from the study of the effect of pH and temperature on laccase activity (section 2.5): pH 3, 40 °C. Controls were included in the experiment. In two of the controls (called EP and P bleaching), the laccase pretreatment was omitted so that conditions would be consistent with a classical peroxide bleaching sequence with or without a previous alkali extraction. In another control (called ControlT), the laccase and the mediator were also omitted but the pulp was treated with an aqueous solution (buffer) with the same pH, time, and temperature of the laccase−mediator treatment. This control was included to distinguish the contributions of the LMS and of the buffer to the biobleaching process. All experiments were done twice. 2.7. Alkaline Extraction and Hydrogen Peroxide Treatments. After the enzymatic pretreatment, an alkaline extraction was carried out by applying 1.5% NaOH at 90 °C for 120 min, with a 5% of pulp consistency. Following this treatment, pulps were washed, and a hydrogen peroxide bleaching stage ensued under these conditions: 3% H2O2, 1.5% NaOH, 1% DTPA, 0.2% MgSO4, and 5% of pulp consistency, at 90 °C for 90 min. Residual hydrogen peroxide was analyzed in the bleaching effluent by standard titration. 2.8. Pulp Characterization. Treated pulps were characterized in terms of their κ number, brightness, and viscosity according to the ISO 302, ISO 2470, and ISO 5351-2 standards, respectively. Moreover, the hexenuronic acid

pulp. For this last purpose, the stability at different pH values and temperatures of the laccase produced when optimal inductors were used has been studied. Additionally, other parameters were analyzed, including delignification degree, brightness, pulp viscosity, and hexenuronic acid content of the bleached pulps. Finally, hydrogen peroxide consumption and stability of the optical properties of the resulting paper sheets were also determined.

2. EXPERIMENTAL SECTION 2.1. Chemicals. 2,2′-Azino-bis-3-ethylbenzthiazoline-6-sulfonate (ABTS) was purchased from Roche (Madrid, Spain). Tween 80 was supplied by Panreac Quimica (Barcelona, Spain). All other chemicals were reagent-grade and obtained from Merck (Barcelona, Spain) or Sigma−Aldrich (Madrid, Spain). 2.2. Organism, Maintenance, and Growth Conditions. The basidiomycete Trametes sp. I-62 was obtained from the IJFM collection (Instituto Jaime Ferran de Microbiologia-CIB). The fungus culture was grown on malt extract agar slants (2% malt extract, 2% Bacto Agar) at 28 °C for 10 days and stored at 4 °C. A plug (1 cm2) of fungus was transferred from the slants to the middle of a Petri dish containing malt extract agar and grown at 28 °C for 7 days. Media plugs overgrown with mycelium were used to start a preinoculum in 200 mL of sterilized liquid medium. The liquid medium used was the same optimized by Silva22 for this fungus with a different carbon source (maltose instead of glucose). This second medium proved to yield maximum laccase activity from Trametes sp. I-62 (data not shown). Once the preinoculum was prepared, it was incubated at 28 °C on a rotary shaker at 100 rpm for 2 days. 2.3. Trametes sp. I-62 Cultures with Laccase Inductors. 500 mL Erlenmeyer culture flasks were filled with 180 mL of the medium described in section 2.2 and then sterilized. Five grams of wheat straw (a potential laccase inductor), after grinding and sieving (0.71−0.4 mm), were added to half of the flasks under sterile conditions.25 Then, all flasks were inoculated with 20 mL of the culture resulting from the preinoculum described in section 2.2, placed in an orbital shaker at 120 rpm, and incubated at 28 °C. Based on control sample, the sixth day of inoculation was chosen to add ethanol and/or copper sulfate (the other potential laccase inductors) because it was the first day in which a significant laccase activity was detected. Details of the inductor addition are described in Table 1. All tests were done twice. The volumetric laccase activity was determined in the extracellular fluid on a daily basis over the 20 days of the study. Table 1. Inductors Added in Each Experiment experiments flask flask flask flask flask flask flask flask

1 2 3 4 5 6 7 8

(without WS)a (without WS) (without WS) (without WS) (with WS) (with WS) (with WS) (with WS)

copper sulfate (% p/v)b

ethanol (% v/v)c

0.005 0.005

4.3 4.3

0.005 0.005

4.3 4.3

a

WS: wheat straw. bInductors concentration as it is described by Collins and Dobson.23 cInductors concentration as it is described by Lomascolo et al.24 15534

dx.doi.org/10.1021/ie402160p | Ind. Eng. Chem. Res. 2013, 52, 15533−15540


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of incubation (1048 U/mL). Therefore, these results show that wheat straw has a very strong influence on laccase activity during the first days of incubation before the addition of other inductors. Pérez-Leblic et al.25 have also demonstrated that wheat straw is an efficient laccase inductor using Pleurotus eryngii. The laccase induction produced by wheat straw could be explained by the high p-coumaric acid content of this raw material, since Terrón et al.11 have observed that this compound, among the different aromatic compounds assayed, was the one which produced the highest laccase induction using Trametes sp. I-62. Regarding the other inductors tested, when wheat straw was not present (Figure 1a), the sample containing only copper sulfate showed the highest laccase activity on the 15th day of incubation (984 U/mL), probably due to the presence of a centered copper ion in the laccase. This activity was similar to that observed when only wheat straw was added as inductor (1048 U/mL) (Figure 1b), but the induction effect was observed later (on the 15th day of incubation with copper sulfate, as against the fifth day with wheat straw). So, copper sulfate seems to be a laccase inductor for Trametes sp. I-62 as good as wheat straw is, although its effect was observed later. When wheat straw was present in combination with copper sulfate, the highest laccase activity was observed among all assayed experiments (1599 U/mL) and also occurred on the 15th day of incubation, as expected. On the other hand, the experiment containing only ethanol showed lower laccase activity than the control. Also, when ethanol was used in combination with copper sulfate, regardless of the addition of wheat straw, laccase activity was observed to be similar to that of the control. These results confirm that ethanol seems not to be a laccase inductor for Trametes sp. I-62. Eugenio et al.29 have ́ also reported this effect with Pycnoporus sanguineus using the 24 same conditions. However, Lomascolo et al. have found a 155-fold increase in laccase activity comparing ethanol-induced cultures with noninduced cultures using the same ethanol concentration but a different fungus (Pycnoporus cinnabarinus). All these results suggest that, using wheat straw in combination with copper sulfate as inductors, maximum laccase activity was obtained from Trametes sp. I-62 (1599 U/L). This combination also was the most effective when Pycnoporus ́ sanguineus was used by Eugenio et al.29 However, the last authors observed a synergic effect reaching an activity of 3250 U/L, while using wheat straw and copper sulfate independently the activities were 1090 and 720 U/L, respectively. Therefore, it can be concluded that the intensity of the inductor’s effect on laccase activity strongly depends on the fungi that are being studied. 3.2. Effect of pH and Temperature on the Laccase Activity of the Extracellular Enzymatic Fluid Produced from Trametes sp. I-62. The extracellular enzymatic fluid which showed the highest laccase activity was isolated from the fungus mycelium, and the variations in its volumetric laccase activity was studied under several different pH and temperature values (Figure 2). It can be observed from Figure 2a that the enzymatic fluid reached the highest volumetric laccase activity at pH 3. Similar results have been reported by Rancano et al.,30 with lacasse produced by Trametes versicolor, and by Eugenio et al.,29 with ́ laccase obtained from Pycnoporus sanguineus. Laccase activity gradually decreased as the pH increased, and only approximately 29% of the maximum laccase activity remained at a pH of 5. On the other hand, Figure 2b demonstrates that, in the

(HexA) content was measured with the method of Gellerstedt and Li27 and its contribution to the pulp κ number was calculated using the molar oxidation equivalent of 8.6:11.6 μmol of HexA in 1 g of pulp corresponds approximately to 1 κ number unit.28 2.9. Accelerated Aging. The bleached and the unbleached pulps were subjected to accelerated aging to analyze the evolution of their optical properties. This accelerated aging was carried out in a climatic test cabinet CTS (model C-20/250/S) and consisted in moist heating at 80 °C and 65% relative humidity for 6 days, according to the ISO 5630-3 standard. Bleached pulps before and after accelerated aging were characterized in terms of their CIE L*a*b* and CIE L*C* coordinates (T 527) using a spectrophotometer ELREPHO 070 (Lorentze and Wettre).

3. RESULTS AND DISCUSSION 3.1. Effect of the Inductors on Trametes sp. I-62 Laccase Activity. The influence of different inductors (copper sulfate, wheat straw, and ethanol) on laccase production by Trametes sp. I-62 was examined. Figure 1 shows the volumetric laccase activity determined in the extracellular fluid on a daily basis. As can be observed, in all experiments without wheat straw, almost no laccase activity was observed during the first five days of incubation, while, in most of the experiments with wheat straw, high laccase activity was found, peaking on the fifth day

Figure 1. Volumetric laccase activity of Trametes sp. I-62 using either ethanol (ET) and copper sulfate (CS) (a) or ethanol (ET), copper sulfate (CS), and wheat straw (WS) (b) as inductors. C is the control. Data shown are the average of two repetitions with standard deviation lower than 8%. 15535



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Figure 2. Variation of the volumetric laccase activity in the extracellular enzymatic fluid, induced with wheat straw and copper sulfate at different values of pH (a) and temperature (b). Data shown are the average of two repetitions with standard deviation lower than 5%.

Figure 3. Volumetric laccase activity at different pH values after 1, 2, and 3 h (a) and temperatures each 30 min during 3.5 h (b). Data shown are the average of two repetitions with standard deviation lower than 3%.

a lacasse obtained from the same fungus and by Eugenio et al.29 ́ with that obtained from Pycnoporus sanguineus. The stability studies indicated that, although laccase activity peaked at 60 °C, the optimum temperature seems to be 40 °C, as this was the highest temperature with almost no laccase inactivation. 3.3. Effect of the Enzymatic Pretreatment on Bleached Pulp and Hydrogen Peroxide Consumption. The LMS consisting of the optimal enzymatic fluid obtained from Trametes sp. I-62 and acetosyringone as mediator was applied to the biobleaching of an E. globulus kraft pulp. Figure 4 represents the evolution of the κ number, brightness, and pulp viscosity after the laccase-mediator stage (L), alkali extraction (E), and hydrogen peroxide bleaching (P) in the different sequences assayed. Eucalyptus kraft pulps are known to contain significant amounts of hexenuronic acids after cooking, in addition to the residual lignin. Like lignin, these acids increase κ number result.28 They also consume chemicals in pulp bleaching and must be removed to avoid brightness reversion.31 There are other nonlignin substances which also increase the κ number, but their significance is minor. For this reason, the κ number is shown in Figure 4 as the contribution of only hexenuronic acids and lignin. Figure 4a shows that the highest reduction in κ number achieved at the end of the sequence was found in the experiment where a laccase mediator treatment was used (LTrametes experiment) with a reduction of 40.6% compared with 34.0%, 33.4%, and 30.1% for control-T, EP, and P

temperature range 30−60 °C, higher temperatures are associated with greater volumetric laccase activity. However, with temperatures above 60 °C, a gradual inactivation of the laccase can be observed. Stability studies of the enzymatic fluid at different pH values (2−7) and at temperatures within the range 25−70 °C are plotted in Figure 3. These studies are necessary for selecting the best operational conditions of the enzymatic fluid obtained from Trametes sp. I-62 to be used in the subsequent biobleaching process. From Figure 3a it can be observed that stability was higher at higher pH values. The most outstanding deactivation was observed at a pH of 2, followed by a pH of 3, although the latter showed deactivation only at the end of the study. Therefore, taking into consideration that a pH of 3 was associated with a peak in laccase activity (Figure 2a) and that laccase inactivation was only observed after 2 h (time required in biobleaching experiments), a pH value of 3 seems to be the optimum pH for bleaching application. Figure 3b shows that, at moderate temperatures (25−40 °C), the volumetric laccase activity remained relatively constant for at least 3.5 h. When the temperature was raised to 50 °C, the laccase activity fell to 68% within 1 h, and it reached close to half its initial value (48%) after 3.5 h. Finally, when the enzymatic fluid was kept at 60 °C, the volumetric laccase activity became residual (16%) within the first hour and dropped to undetectable levels at 70 °C with a much faster rate of decline. A similar behavior has been observed by Silva22 with 15536



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mediators and commercial laccases) have been reported by Camarero et al.,2 by Babot et al.33 (both with eucalyptus pulp), and by Fillat et al.32 (with flax pulp). This reduction could be due to the oxidative degradation of lignin by LMS. However, results found by Martin-Sampedro et al.5,6 (using olive tree and oil palm empty fruit bunches, respectively), Aracri et al.34 (using sisal pulp), and Cadena et al.35 (using flax pulp) have described increases in κ number after LMS stages consisting of natural mediators and commercial laccases. These authors attributed such κ number increases to grafting of the mediator to the fibers. However, Barneto et al.36 have recently suggested that a κ number increase can be a result not only of mediator− fiber grafting but also of their polymerization, and that the magnitude of these two processes depends on the type of mediator and fibers used. Based on these findings, it can be concluded that, depending on the type and structure of the mediator, the type of laccase used in the laccase−mediator system, and the type of lignin (raw materials), the grafting/ polymerization reactions and the oxidative degradation of lignin will occur with more or less intensity during the enzymatic process. The evolution of hexenuronic acid (HexA) contents in the proposed bleaching sequences is shown in Figure 4, where it is expressed as a contribution to the κ number. Conventional bleaching (EP) decreased the HexA content from 3.2 to 2.6, being the alkaline extraction responsible for most of the HexA removal. This finding is likely due to the result of the extraction of HexA bound to xylan−lignin complexes under alkaline conditions as Li et al.37 and Costa and Colodette38 have reported. When an enzymatic stage was introduced as a pretreatment in the EP sequence, no significant additional reduction was observed at the end of the sequence. Thus, after the pretreatment, similar HexA reductions with L-Trametes and Control-T were observed. Taking into account that acid washing of the pulps can remove HexA,39,40 the reduction in hexenuronic acids observed in these two pretreatments must be attributed to the acidic treatment and not to the direct action of the LMS. In agreement, Valls et al.8 have observed that the laccase mediator system consisting of Myceliophthora thermophile laccase and methyl syringate as mediator was unable to remove HexA from fibers. Contrarily, Valls and Roncero41 and Eugenio et al.,3,4 who pretreated E. globulus kraft pulps with different LMS systems, have reported a reduction in HexA likely due to the oxidative elimination of HexA directly accessible to the laccase mediator system.32,35,42 Therefore, it can be concluded that some of LMS are able to remove both HexA and lignin while others only remove lignin, such as the one studied in this work. Figure 4b shows brightness evolution along the LEP sequences assayed. An increase in brightness can be observed after most of the bleaching stages, especially after the hydrogen peroxide treatment. So, the variations in pulp brightness with the different treatments are consistent, in most cases, with the evolution of the κ number. Therefore, the highest brightness (63.6% ISO) was reached in the same experiment where the minimum κ number was found (L-Trametes experiment). This brightness was 4.9, 7.2, and 8.6 points % ISO higher than those found in the control (Control-T experiment), and the two conventional bleaching experiments (EP and P), respectively. Reductions of around 8−9 points % ISO were also found by Camarero et al.,2 Eugenio et al.,3 and Babot et al.,33 who biobleached E. globulus pulp with a laccase from Pycnoporus

Figure 4. Evolution of pulp properties after each stage in the biobleaching sequences: (a) the κ number is divided in lignin and hexenuronic acid contributions; (b) brightness; (c) viscosity. Unbleached refers to the unbleached pulp; L, laccase stage; E, alkaline extraction stage; P, hydrogen peroxide stage; Control-T, experiment without addition of LMS; L-Trametes, experiment with addition of LMS.

experiments, respectively. The same effect has been previously reported using different laccases (fungal and bacterial) and different raw materials.3−6,32 This finding can be explained by changes induced by laccase rendering lignin easier to be removed in the subsequent chemical bleaching. It can also be observed in Figure 4a that each stage of the biobleaching sequence, including the enzymatic stage, led to a κ number reduction. Similar findings (always with natural 15537



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cinnabarinus, Pycnoporus sanguineus, and Mytheliophthora thermophila, respectively. It is necessary to point out that, after the pretreatment, the brightness of L-Trametes pulp was lower than that of ControlT pulp (35.0% ISO against 37.3% ISO). This finding can be explained considering the different reactions that can take place between the natural mediators and the fibers during an enzymatic treatment. Thus, as Barneto et al.36 have reported, mediators such as acetosiringone can polymerize during the enzymatic stage, producing a decrease in brightness due to the formation of chromophores. Moreover, with this type of mediators, polymerization is more prominent than grafting; therefore, the κ number variation is not as significant as the brightness decrease, as our findings have confirmed. However, after alkaline extraction, brightness was higher for the LTrametes experiment compared to the control, which appears to confirm that the alkaline extraction can oxidize and dissolve the chromophoric species and lignin degradation products produced during the enzymatic step, enhancing the subsequent chemical bleaching. Figure 4c shows the viscosity after all stages of the LEP sequences assayed. It can be observed that viscosities were reduced (between 5 and 9%) at the end of the sequences. Comparing the different sequences, no significant differences in viscosity were observed. The hydrogen peroxide bleaching was ́ the most influential stage in viscosity reduction, as MartinSampedro et al.5,21 have reported previously. These results are consistent with the greatest reduction in κ number observed after the P stage, in which some carbohydrates have been likely degraded. The enzymatic treatment significantly reduced hydrogen peroxide consumption compared to conventional bleaching. This finding can be explained if we consider that a logical consequence of lignin removal in the enzymatic treatment is a major reduction of chemicals used in the subsequent bleaching. Moreover, lignin remaining in the pulp is partially oxidized by the enzymatic treatment, and therefore, fewer chemical reagents are needed in the following steps to remove it. Thus, while the conventional sequence consumed 89.9% of the initial hydrogen peroxide load, the introduction of the laccase treatment (which produced the pulp with the lowest lignin content) reduced the consumption to 41.9%. The reduction in chemical found in this study after the enzymatic treatment (53.4%) was much higher than those reported by Eugenio et al.3,4 (7% and 9.6%, respectively) using laccase from the fungus Pycnoporus sanguineus and the bacteria Streptomyces ipomoea, respectively. Contrarily, other authors have reported an increase in bleaching chemical consumption when a LMS pretreatment was assayed,5,6 albeit using different raw materials, pulping processes, and laccases. 3.4. Effect of the Enzymatic Pretreatment on Accelerated Aging of the Bleached Pulps. Accelerated aging of bleached pulps was carried out to evaluate the influence of the enzymatic treatment on the stability of optical paper properties. Table 2 and Figure 5 show changes in brightness and color coordinates, respectively, after accelerated aging. As can be observed in Table 2, after accelerated aging, the pulp enzymatically pretreated showed greater reduction in brightness at the end of the bleaching sequence than the control pulp and the pulps which were not pretreated (EP or P ́ bleaching). Similar results have been found by Martin5,6 Sampedro et al., who evaluated the enzymatic treatment

Table 2. Brightness before and after Accelerated Ageing brightness (% ISO) stage

experiment

original

aged

brightness reduction

L

Control-T L-Trametes Control-T L-Trametes E Control-T L-Trametes EP P

37.3 35.0 37.2 38.5 37.4 58.7 63.6 56.3 55.0

31.8 30.2 36.7 37.1 36.4 52.4 55.1 52.1 48.4

5.6 4.8 0.6 1.4 1.0 6.4 8.4 4.3 6.6

LE

LEP

with a commercial laccase in pulps obtained from oil palm empty fruit bunches and from olive tree pruning residues, respectively. However, Cadena et al.43 and Eugenio et al.4 have reported the opposite results when E. globulus kraft pulp was enzymatically pretreated. These authors attributed the lower brightness reversion to the smaller hexenuronic acid content of the biobleached pulps. However, as has been mentioned above, the LMS used in this study did not remove HexA. Furthermore, these acids are not the only ones responsible for the brightness reversion of the pulps; lignin and hemicellulose also play a significant role.28 In addition, attention must be drawn to the fact that, in this study, an enzymatic liquid obtained from Trametes sp. I-62 was used instead of a purified enzyme, which means that there could be other compounds which are also playing a role during the enzymatic pretreatment and influencing the optical properties of the bleached pulps. The above discussion relates to brightness stability at the end of the bleaching sequence. However, it is worth mentioning that, after the enzymatic treatment, brightness reversion was lower in the enzymatically pretreated pulp. This improvement was not observed after the alkaline extraction nor, as has been mentioned above, after bleaching with hydrogen peroxide. Figure 5a shows that, after accelerated aging the a* coordinate shifted to the right (from green to red) and the b* coordinate increased (from blue to yellow) in all bleached pulps. Figure 5b shows that accelerated aging resulted in an increase in color (C*) and a reduction in lightness (L*) in all pulps, with a greater effect in the laccase-pretreated pulp. Nevertheless, the final color coordinates for pretreated pulp were better than those of the control pulp and the conventionally bleached pulps. To sum up, it can be concluded that, although less stable, enzymatically pretreated pulps show the highest final brightness and the best CIE L*a*b* and CIE L*C* color coordinates among all experiments performed, even after accelerated aging.

4. CONCLUSIONS The enzymatic fluid produced by Trametes sp. I-62 has been optimized to increase the laccase production. The highest laccase activity was found when wheat straw and copper sulfate were used simultaneously as inductors. 40 °C and pH 3 were the optimal conditions for the laccase stability. The use of this enzymatic fluid supplemented with a natural mediator (acetosiringone) as pretreatment of conventional bleaching (EP) resulted in an increase of delignification, from 33.4% to 40.6%, and of brightness, from 56.3 to 63.6% ISO. Interestingly, to achieve these improvements in bleaching, lower hydrogen peroxide was consumed (41.9% vs 89.9%). This reagent savings was much higher than those found for other reported LMSs. 15538



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Figure 5. Influence of the accelerated aging treatment in the CIE L*a*b* (a) and CIE L*C* (b) color coordinates of the bleached pulps. Black and gray symbols represent pulps before and after the accelerating aging, respectively. Data shown are the average of two repetitions with standard deviation lower than 3%. (9) Bourbonnais, R.; Paice, M. G. Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2,2′azinobis-(3-ethylbenzthiazole-6-sulfonate). Appl. Microbiol. Biot. 1992, 36, 823−827. (10) Arana-Cuenca, A.; Roda, A.; Tellez, A.; Loera, O.; Carbajo, J. M.; Terrón, M. C.; González, A. E. Comparative analysis of laccaseisozymes patterns of several related Polyporaceae species under different culture conditions. J. Basic Microbiol. 2004, 44, 79−87. (11) Terrón, M. C.; González, T.; Carbajo, J. M.; Yague, S.; AranaCuenca, A.; Tellez, A.; Dobson, A. D. W.; González, A. E. Structural close-related aromatic compounds have different effects on laccase activity and on lcc gene expression in the ligninolytic fungus Trametes sp I-62. Fungal Genet. Biol. 2004, 41, 954−962. (12) González, T.; Terrón, M. C.; Zapico, E. J.; Tellez, A.; Yague, S.; Carbajo, J. M.; González, A. E. Use of multiplex reverse transcriptionPCR to study the expression of a laccase gene family in a basidiomycetous fungus. Appl. Environ. Microbiol. 2003, 69, 7083− 7090. (13) González, T.; Terrón, M. C.; Zapico, E.; Yague, S.; Tellez, A.; Junca, H.; González, A. E. Identification of a new laccase gene and confirmation of genomic predictions by cDNA sequences of Trametes sp I-62 laccase family. Mycol. Res. 2003, 107, 727−735. (14) Mansur, M.; Suarez, T.; Fernández-Larrea, J. B.; Brizuela, M. A.; González, A. E. Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Appl. Environ. Microbiol. 1997, 63, 2637−2646. (15) Mansur, M.; Suarez, T.; González, A. E. Differential gene expression in the laccase gene family from basidiomycete I-62 (CECT 20197). Appl. Environ. Microbiol. 1998, 64, 771−774. (16) González, T.; Terrón, M. C.; Yague, S.; Zapico, E.; Galletti, G. C.; González, A. E. Pyrolysis/gas chromatography/mass spectrometry monitoring of fungal-biotreated distillery wastewater using Trametes sp I-62 (CECT 20197). Rapid Commun. Mass. Spectrom. 2000, 14, 1417− 1424. (17) González, T.; Terrón, M. C.; Yague, S.; Junca, H.; Carbajo, J. M.; Zapico, E. J.; Silva, R.; Arana-Cuenca, A.; Tellez, A.; González, A. E. Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp I-62. Res. Microbiol. 2008, 159, 103−109. (18) Pant, D.; Adholeya, A. Nitrogen removal from biomethanated spentwash using hydroponic treatment followed by fungal decolorization. Environ. Eng. Sci. 2009, 26, 559−565. (19) Verma, A. K.; Raghukumar, C.; Naik, C. G. A novel hybrid technology for remediation of molasses-based raw effluents. Bioresour. Technol. 2011, 102, 2411−2418. (20) Eugenio, M. E.; Carbajo, J. M.; Terrón, M. C.; González, A. E.; Villar, J. C. Bioremediation of lignosulphonates by lignin-degrading basidiomycetous fungi. Bioresour. Technol. 2008, 99, 4929−4934.

Finally, pretreated bleached pulps showed better optical properties after accelerated aging, notwithstanding a greater brightness reversion.

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AUTHOR INFORMATION

Corresponding Author

*E-mail, [email protected]; phone number, (+34) 913473948; fax, (+34) 913476767. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the Spanish MINECO for funding this study via Project CTQ 2011-28503-C02-01 and Program PTA 2011-4857-I.

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REFERENCES

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Laccase from Trametes sp. I-62: Production, Characterization, and

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