Abstract

INTRODUCTION

Worldwide, about 10,000 different dyes and pigments are manufactured for commercialization, with annual production exceeding 7 x 10⁵ tons [¹1. Wong Y, Yu J. Laccase-catalyzed decolorization of synthetic dyes. Water Res. 1999; 33: 3512-3520.,²2. Forgacs E, Cserháti T, Oros G. Removal of synthetic dyes from wastewaters: a review. Environ Int. 2004; 30: 953- 971.,³3. Crini G. Non-conventional low-cost adsorbents for dye removal: a review. Bioresour Technol. 2006; 97: 1061-1085.,⁴4. Couto SR. Dye removal by immobilised fungi. Biotechnol Adv. 2009; 27: 227-235.]. Synthetic dyes are widely used for dyeing fabric, printing paper, colour photography and as additives in petroleum-based products. Based on the chemical structure of group chromophore, dyes are classified as anthraquinone, azo, triphenylmethane, indigo, heterocyclic and polymeric. Among these, azo, anthraquinone and triphenylmethane dyes are the most extensively produced and used in textile industries, the first group corresponding to approximately 50% of the total [⁵5. Rodríguez-Couto S. Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors. J Hazard Mat. 2011; 194: 297-302.].

Dyes are identified as the most problematic compounds present in textile effluents due to their high solubility in water and low degradability [⁶6. Khelifi E, Ayed L, Bouallagui H, Touhami Y, Hamdi M. Effect of nitrogen and carbon sources on Indigo and Congo red decolourization by Aspergillus alliaceus strain 121C. J Hazard Mat. 2009; 163: 1056-1062.]. It is estimated that the traditional textile finishing industry consumes about 100 L of water for 1 kg of textile material, and that 10 to 15% of the compounds used in dyeing processes are found in industrial effluents [⁷7. Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gübitz, GM. Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta. Appl Environ Microbiol. 2000; 66: 3357-3362.,⁸8. Wesenberg D, Kyriakides I, Agathos SN. White-rot fungi and enzymes for the treatment of industrial dye effluents. Biotechnol Adv. 2003; 22: 161-187.]. Dyes are usually stable to factors such as light and temperature, and visible in water even at relatively small concentrations (10 to 50 mg L^-1) [¹1. Wong Y, Yu J. Laccase-catalyzed decolorization of synthetic dyes. Water Res. 1999; 33: 3512-3520.]. Besides the visual impact, the presence of these compounds in water reduces its transparency, hindering the absorption of light and therefore affecting aquatic plants and algae [³3. Crini G. Non-conventional low-cost adsorbents for dye removal: a review. Bioresour Technol. 2006; 97: 1061-1085.,⁶6. Khelifi E, Ayed L, Bouallagui H, Touhami Y, Hamdi M. Effect of nitrogen and carbon sources on Indigo and Congo red decolourization by Aspergillus alliaceus strain 121C. J Hazard Mat. 2009; 163: 1056-1062.,⁹9. Couto SR, Toca-Herrera JL. Laccases in the textile industry. Biotechnol Mol Biol Rev. 2006; 1: 115-120.,¹⁰10. Zeng X, Cai Y, Liao X, Zeng X, Luo S, Zhang D. Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase. Process Biochem. 2012; 47: 160-163.]. Furthermore, the solubility of oxygen in water is also reduced, with adverse effects in terms of chemical oxygen demand (COD) and biological oxygen demand (BOD). In addition, dyes are potentially toxic, carcinogenic, mutagenic and allergenic compounds, and their efficient removal from industrial wastes is absolutely mandatory [⁵5. Rodríguez-Couto S. Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors. J Hazard Mat. 2011; 194: 297-302.,¹¹11. Rodríguez E, Pickard MA, Vazquez-Duhalt R. Industrial dye decolorization by laccases from ligninolytic fungi. Curr Microbiol. 1999; 38: 27-32.].

Because of their resistance to microbial attack, the elimination of these coloured substances from liquid effluents is mainly based on physical or chemical procedures, such as adsorption, coagulation, flocculation, membrane filtration, irradiation, concentration, and chemical transformation. However, these methods are expensive, what limits their applicability [²2. Forgacs E, Cserháti T, Oros G. Removal of synthetic dyes from wastewaters: a review. Environ Int. 2004; 30: 953- 971.,⁵5. Rodríguez-Couto S. Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors. J Hazard Mat. 2011; 194: 297-302.,⁸8. Wesenberg D, Kyriakides I, Agathos SN. White-rot fungi and enzymes for the treatment of industrial dye effluents. Biotechnol Adv. 2003; 22: 161-187.,¹²12. Niebisch CH, Malinowski AK, Schadeck R, Mitchell DA, Kava-Cordeiro V, Paba J. Decolorization and biodegradation of Reactive Blue 220 textile dye by Lentinus crinitus extracellular extract. J Hazard Mat. 2010; 180: 316-322.]. In this context, the development of unconventional processes for dye-containing effluents treatment is required.

The possibility of using white-rot fungi in biotreatment strategies comes from their capacity to produce a non-specific enzyme system able to metabolize a wide range of pollutants to CO₂ and H₂O [¹³13. Paszczynski A, Pasti-Grigsby MB, Goszczynski S, Crawford DL. Mineralization of sulfonated azo dyes and sulfonilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. Appl Environ Microbiol. 1992; 58: 3598-3604.,¹⁴14. Barr DP, Aust SD. Mechanism white rot fungi use to degrade pollutants. Environ Sci Technol. 1994; 28: 78-87.]. These fungi tolerate concentrations considerably high of pollutants and can transform polycyclic aromatic hydrocarbons (PAHs), polyphenols, dioxins, chlorinated pesticides, organophosphate insecticides, anilines and dyes, showing potential for use in industrial segments as processing of coal, oil refining, resins and plastics, electroplating, chemical, textile dyes, mining and pulp and paper industries [¹³13. Paszczynski A, Pasti-Grigsby MB, Goszczynski S, Crawford DL. Mineralization of sulfonated azo dyes and sulfonilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. Appl Environ Microbiol. 1992; 58: 3598-3604.,¹⁵15. Selvam K, Swaminathan K, Chae KS. Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp. Bioresour Technol. 2003; 88: 115-119.,¹⁶16. Wong DWS. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol. 2009; 157: 174-209.].

Fungi of the genus Pleurotus represent a cosmopolitan group of mushrooms that have great nutritional value, therapeutic, medicinal properties and several environmental and biotechnological applications, due to their enzyme complex [¹⁷17. Cohen R, Persky L, Hadar Y. Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol. 2002; 58: 582-594.]. The ligninolytic enzymes produced by Pleurotus and other Basidiomycetes include manganese peroxidases (MnP), lignin peroxidases (LiP) and laccases (Lac), which are secreted to the growth medium in response to low levels of nutrients [¹⁸18. Gill PK, Arora DS. Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi. J Ind Microbiol Biotechnol. 2003; 30: 28-33.]. Some fungi produce Lac, MnP and LiP, while others produce only one or two of these enzymes [¹⁹19. Boer CG, Obici L, Souza CGM, Peralta RM. Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme. Bioresour Technol. 2004; 94: 107-112.]. These organisms are enzymatically equipped to oxidize compounds with similar structures to lignin, breaking the aromatic ring and forming compounds that may suffer further degradation, being mineralised [¹⁷17. Cohen R, Persky L, Hadar Y. Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol. 2002; 58: 582-594.].

Laccases are multi-copper polyphenol oxidases, which oxidize phenolic compounds reducing oxygen to water by removing an electron from the aromatic substrate [¹⁶16. Wong DWS. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol. 2009; 157: 174-209.]. They are present in most white-rot fungi and also found in other types of fungi, plants, some bacteria and insects [²⁰20. Thurston CF. The structure and function of fungal laccases. Microbiol. 1994; 140: 19-26.,²¹21. Majeau JA, Brar SK, Tyagi RD. Laccases for removal of recalcitrant and emerging pollutants. Bioresour Technol. 2010; 101: 2331-2350.]. Fungal laccases are of particular interest due to of their capability to oxidize a wide range of industrially relevant substrates as phenolic and aromatic amines. Thus, these enzymes represent a promising alternative for biotechnological processes of environmental interest such as bleaching and delignification of cellulose pulp, decolourisation of textile dyes, oxidation of PAHs, phenol removal, detoxification of effluent and environmental pollutants, and degradation of recalcitrant compounds [⁸8. Wesenberg D, Kyriakides I, Agathos SN. White-rot fungi and enzymes for the treatment of industrial dye effluents. Biotechnol Adv. 2003; 22: 161-187.,¹⁶16. Wong DWS. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol. 2009; 157: 174-209.,²¹21. Majeau JA, Brar SK, Tyagi RD. Laccases for removal of recalcitrant and emerging pollutants. Bioresour Technol. 2010; 101: 2331-2350.,²²22. Couto SR, Herrera JLT. Industrial and biotechnological applications of laccases: a review. Biotechnol Adv. 2006; 24: 500-513.,²³23. Munari FM, Gaio TA, Calloni R, Dillon AJP. Decolorization of textile dyes by enzymatic extract and submerged cultures of Pleurotus sajor-caju. World J Microbiol Biotechnol. 2008; 24: 1383-1392.,²⁴24. Schmitt S, Souza R, Bettin F, Dillon AJP, Valle JAB, Andreaus J. Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases. Biocatal Biotransform. 2012; 30: 48-56.]. Laccases can also be used in cosmetics, chemical, pharmaceutical, food and beverage industries, as biosensor of phenolic compounds in environmental, pharmaceutical and industrial areas, in nanobiotechnology, synthetic chemistry and soil bioremediation [²²22. Couto SR, Herrera JLT. Industrial and biotechnological applications of laccases: a review. Biotechnol Adv. 2006; 24: 500-513.,²⁵25. Durán N, Esposito E. Potential applications of oxidase enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Appl Catal B: Environ. 2000; 28: 83-99.,²⁶26. Dhawan S, Lal R, Hanspal M, Kuhad RC. Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus. Bioresour Technol. 2005; 96: 1415-1418.,²⁷27. Brondani D, Scheeren CW, Dupont J, Vieira IC. Biosensor based on platinum nanoparticles dispersed in ionic liquid and laccase for determination of adrenaline. Sens Actuators B: Chem. 2009; 140: 252-259.,²⁸28. Antonopoulou I, Varriale S, Topakas E, Rova U, Christakopoulos P, Faraco V. Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application. Appl Microbiol Biotechnol. 2016; 100: 6519-6543.].

Development of aerobic bacteria to be used for the decolourisation of dyes often results in strains with ability limited to attack a single chemical structure. Many azo dyes can be broken down in potentially mutagenic and/or carcinogenic amines in anaerobiosis, due to the action of very specific azo-reductases [¹1. Wong Y, Yu J. Laccase-catalyzed decolorization of synthetic dyes. Water Res. 1999; 33: 3512-3520.]. However, laccases act by oxidation and are less specific with respect to the substrate [¹⁶16. Wong DWS. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol. 2009; 157: 174-209.,²⁰20. Thurston CF. The structure and function of fungal laccases. Microbiol. 1994; 140: 19-26.]. Due to the fact of using synthetic dyes with a wide variety of chemical structures, it is interesting the development of biocatalytic processes able to act on this diversity [²⁹29. Yang FC, Yu JT. Development of a bioreactor system using an immobilized white rot fungus for decolorization. Bioprocess Eng. 1996; 15: 307-310.]. The use of laccases for developing enzyme-based treatment processes is particularly interesting because it can be produced with less demanding induction conditions than those observed for LiP and MnP [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.].

Given this, the aim of this work was to evaluate the decolourisation of twenty-two dyes belonging to the chromophore groups anthraquinone, azo and triphenylmethane, using laccase-containing preparation produced by Pleurotus sajor-caju PS-2001 in submerged process, with respect to the influence of the parameters pH, temperature and agitation.

MATERIAL AND METHODS

Organism and Culture Conditions

Pleurotus sajor-caju strain PS-2001, from the microorganism culture collection of the Institute of Biotechnology of the University of Caxias do Sul (Brazil), was grown and maintained in a medium containing (per litre): 20 g Pinus spp. sawdust, 20 g wheat bran, 2.0 g CaCO₃, and 20 g agar-agar. Petri dishes with the fungus were incubated at 28°C until complete mycelial growth and subsequently stored at 4°C [³¹31. Bettin F, Montanari Q, Calloni R, Gaio TA, Silveira MM, Dillon AJP. Production of laccases in submerged process by Pleurotus sajor-caju PS-2001 in relation to carbon and organic nitrogen sources, antifoams and Tween 80. J Ind Microbiol Biotechnol. 2009; 36: 1-9.].

The inoculum for bioreactor cultivations were prepared in 500-mL Erlenmeyer flasks containing 100 mL of medium containing (per litre): 5 g glucose, 1.5 g pure casein (Synth^®) and 100 mL of a mineral solution (composition per litre: 20 g KH₂PO₄, 14 g (NH₄)₂SO₄, 3.0 g MgSO₄.7H₂O, 3 g urea, 3.0 g CaCl₂, 15.6 g MnSO₄.H₂O, 50 mg FeSO₄, 14 mg ZnSO₄, and 20 mg CoCl₂) [³¹31. Bettin F, Montanari Q, Calloni R, Gaio TA, Silveira MM, Dillon AJP. Production of laccases in submerged process by Pleurotus sajor-caju PS-2001 in relation to carbon and organic nitrogen sources, antifoams and Tween 80. J Ind Microbiol Biotechnol. 2009; 36: 1-9.]. After autoclaving at 1 atm for 15 minutes, three mycelial disks with 1.5 cm in diameter were scraped from the stored Petri dishes containing P. sajor-caju mycelium and added to the flasks [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.]. Inoculum growth occurred under reciprocal agitation of 180 min^-1, at 28±2^oC, for 6 days. Volumes of 400 mL of inoculum suspension (10% v/v) were used to start bioreactor cultivations.

For the production of laccase-containing enzyme broth, the following cultivation medium was used (per litre): glucose, 5.0 g; pure casein (Synth^®), 1.5 g; CuSO₄, 100 mg; benzoic acid, 100 mg; mineral solution, 100 mL [32]. The cultivations were carried out in a stirred-tank bioreactor B. Braun Biotech model Biostat^®B with 4.0 L of working volume. The bioreactor containing the cultivation medium was autoclaved for 20 minutes at 1.5 prior to inoculation. During the initial hours of cultivation the bioreactor was kept at an impeller speed of 200 min^-1 and an air flow rate of 2 L min^-1). Afterwards, when the dissolved oxygen concentration (DO) decreased to about 30% of saturation, DO was maintained at this level by automatically varying the air flow rate [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.]. Before inoculation, under the initial aeration and agitation conditions, the volumetric oxygen transfer coefficient (K_La) was determined by the method described by Moo-Young and Blanch [33] as 12 h^-1. Silicone-based antifoam was used when necessary and the pH was automatically controlled at 6.5 by adding 2 mol L^-1 NH₄OH or 2 mol L^-1 H₂SO₄ at 28±1^oC [³¹31. Bettin F, Montanari Q, Calloni R, Gaio TA, Silveira MM, Dillon AJP. Production of laccases in submerged process by Pleurotus sajor-caju PS-2001 in relation to carbon and organic nitrogen sources, antifoams and Tween 80. J Ind Microbiol Biotechnol. 2009; 36: 1-9.].

Dye decolourisation assays

In decolourisation assays, a total of twenty-two dyes were assayed as follows: ten from the azo chromophore group, four from the anthraquinone group, and eight from the triphenylmethane group (Table 1). The basic chemical structures of the different groups of dyes used in this work are presented in Figure 1.

Figure 1
Basic chemical structures of dyes of chromophore groups anthraquinone (A), azo (B) and triphenylmethane (C).

Decolourisation assays were done in 25-mL test tubes containing 7.0 mL of 50 mg L^-1 dye solution, 7.0 mL of Mc'Ilvaine buffer (sodium hydrogen phosphate and citric acid), at an adequate pH value according to the condition evaluated, and 7.0 mL of crude P. sajor-caju cultivation broth with 30 U mL^-1 laccases, corresponding to 10 U mL^-1 in the reaction medium as defined by Schmitt et al. [²⁴24. Schmitt S, Souza R, Bettin F, Dillon AJP, Valle JAB, Andreaus J. Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases. Biocatal Biotransform. 2012; 30: 48-56.]. The tubes containing the mixtures were kept in a thermostatic bath, and 0.4 mL samples were collected in triplicate each 24 hours, during 168 or 240 hours of reaction. The absorbance of samples was read in spectrophotometer at a wavelength between 350 to 750 nm, as previously defined for each particular dye (Table 1).

The decolourisation reaction was evaluated at pH values of 2.4, 3.2, 4.4 and 5.0, at 30°C and without agitation [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.].

For the assessment of the effect of temperature on decolourisation, the reaction tubes were kept at 35, 40, 45, and 50^oC in a bath without agitation [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.].

Decolourisation assays under agitation were performed at pH 3.2 and temperatures of 30 and 35^oC, under reciprocal agitation of 100 min^-1.

Determination of Laccases Activity

Laccases (Lac) activity was determined at 25°C using 0.45 mmol L^-1 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS - Sigma^®) as substrate in reaction mixtures containing 90 mmol L^-1 of pH 5.0 sodium acetate buffer solution and an appropriate amount of culture supernatant. ABTS oxidation was estimated by measuring increase in absorbance at 420 nm ((₄₂₀ = 3.6 x 10⁴ cm^-1 mol^-1) for 90 seconds [³⁴34. Wolfenden BS, Willson RL. Radical-cations as reference chromogens in the kinetic studies of one-electron transfer reactions: pulse radiolysis studies of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate). J Chem Soc, Perkin Trans II. 1982; 02: 805-812.]. One enzyme unit corresponds to the quantity in (mol of product released per minute per mL of sample.

Determination of Degree of Decolourisation

Decolourisation degree was calculated by comparing the initial absorbance of the reaction mixture and those at the different assay times, the results being expressed in terms of percentage of reduction in absorbance [²⁴24. Schmitt S, Souza R, Bettin F, Dillon AJP, Valle JAB, Andreaus J. Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases. Biocatal Biotransform. 2012; 30: 48-56.].

RESULTS

Dye decolourisation assays were carried out under different pH values at 30^oC for up to 168 hours. The pH values evaluated were 2.4, 3.2, 4.4, and 5.0. In a previous work of our group [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.], these pH values were defined as the optimum for the activity of three possible laccase isoforms which were identified in P. sajor-caju cultivation broth. The results obtained in the decolourisation assays with the twenty-two dyes evaluated are shown in Table 2. From these data, one can be seen that the best results in terms of percentage of decolourisation were mostly obtained at pH 3.2, the only condition under which all dyes were affected by the enzymatic treatment. The highest decolourisation levels were observed for the dyes Acid Blue 80, Acid Green 28, Brilliant Green, Bromocresol Green, Coomassie Brilliant Blue G-250, Disperse Blue 79 and Reactive Red 198, showing percentages of decolourisation between 23% (Disperse Blue 79) and 46% (Brilliant Green) after different reaction times. Among these dyes, three of them belong to the triphenylmethane group, two to the azo group and two to the anthraquinone group, as shown in Table 1.

At pH 2.4, only five of the dyes assayed showed no decolourisation after 168 hours (Bromocresol Green, Congo Red, Disperse Red 324, Levafix Brilliant Red E-4BA and Reactive Blue 220), three of them belonging to the azo group (Table 1). Treatments of solution of Acid Blue 80, Acid Green 28, Brilliant Green and Reactive Red 198 presented removal percentages above 20%.

In contrast to the results obtained at pH 3.2 and 2.4, pH 4.4 and 5.0 have shown to be ineffective with regard to enzymatic decolourisation of dyes under the conditions evaluated. Only thirteen dyes had decrease in colour after 168 hours at pH 4.4, with results above 20% of decolourisation for Acid Blue 80 and Reactive Blue 220 (anthraquinone), Congo Red (azo) and Gentian Violet (triphenylmethane). The less satisfactory results were obtained at pH 5.0, since only six dyes showed decolourisation after the treatment (Acid Blue 80, Brilliant Green, Gentian Violet, Malachite Green, Methyl Violet and Reactive Blue 220), four of them belonging to the triphenylmethane group and two to anthraquinone group (Table 1).

In the previous assays, pH 3.2 was identified as the most suitable for the decolourisation of dyes at 30^oC. Therefore, the set of experiments carried out to assess the influence of the reaction temperature (35, 40, 45 and 50^oC) on the decolourisation of the dyes under evaluation were done at this pH value. Such temperature values were defined from previous studies that have shown increasing laccases activities from 30 to 50°C [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.]. The results are presented in Table 3.

The data obtained at pH 3.2 and 30^oC showed decolourisation in different percentage levels for all dyes in test (Table 2). With regard to the results obtained under the further temperatures evaluated (Table 3), it was observed that at 35^oC the percentage of decolourisation was generally higher than those achieved with the other temperatures tested (30, 40, 45 and 50^oC). At 35^oC and pH 3.2, only three dyes, belonging to triphenylmethane chromophore group showed no reduction in colour (Gentian Violet, Malachite Green and Methyl Violet). With the exception of Phenol Red, all other dyes showed percentages of decolourisation above 20%, and some of them reached values above 60% (Acid Blue 80, Congo Red, Disperse Blue 79, Disperse Red 324 and Orange G). Among the temperatures evaluated (Table 3), 40^oC has shown to be inadequate for this treatment, given that only ten dyes showed decolourisation under this condition, although in some cases decolourisation has been greater than 30%. At 45^oC, fourteen dyes responded positively to the enzymatic treatment, some of them showing relatively high percentage of decolourisation. However, only nine dyes were decolourised at 50^oC, with reduction in colour lower than those obtained under the other conditions.

Figure 2 depicts the profile of the absorbance readings, performed every 24 hours, of the enzymatic treatment of the thirteen dyes which showed more than 50% decolourisation at pH 3.2 and 35^oC (Table 3), comprising eight dyes belonging to the azo group, three to the anthraquinone group, and two to the triphenylmethane group. It is interesting to remark that all acid dyes (Acid Blue 80, Acid Green 28 and Acid Red 315) and all disperse dyes (Disperse Blue 79, Disperse Orange 30 and Disperse Red 324) evaluated are included among these thirteen dyes.

Figure 2
Decolourisation of dyes as a function of time during enzymatic treatment with laccase-containing Pleurotus sajor-caju PS-2001 cultivation broth at pH 3.2 and 35^oC. Dyes of chromophore groups anthraquinone (A), azo (B) and triphenylmethane (C).

As shown in Tables 2 and 3, pH 3.2 and temperatures of 30 and 35^oC were adequate conditions for decolourisation of dyes of different chromophore groups. Thus, the assays under reciprocal agitation were performed using this value of pH in both temperatures during 240 hours of incubation. In Table 4, the percentages of decolourisation of the dyes studied obtained in experiments performed under reciprocal agitation of 100 rpm are presented. The data indicate that the use of agitation led to a remarkable effect on the decolourisation of dyes. At 30°C, only three dyes showed decrease in colour (Brilliant Green, Malachite Green and Methyl Violet), all from the triphenylmethane group. At 35^oC, decolourisation was observed for five dyes, being one from the anthraquinone group (Acid Blue 80) and four from triphenylmethane group (Brilliant Green, Gentian Violet, Malachite Green and Methyl Violet). In general, the percentages of removal observed under agitation (Table 4) were much lower than those obtained in static conditions (Tables 2 and 3) using the same values of pH and temperature. In fact, only Gentian Violet solution, at 35°C and after 240 hours of incubation, was decolourised in a level over 40%.

Considering the development of an enzymatic technology for dye decolourisation to be applied to large-scale effluent treatment systems, the results of this set of experiments are quite interesting, since the use of agitation implies in a high demand for energy, which would raise the costs of the process.

DISCUSSION

The data obtained in the present study have shown that is possible to attain significant decolourisation of dyes of different chromophore groups by using laccase-containing preparations from P. sajor-caju PS-2001 under controlled conditions. In general, as occurs in any enzymatic process, decolourisation of dyes is affected by parameters such as characteristics of the substrate, pH, temperature, agitation, and incubation time, as shown in this work, together with other factors like dye and enzyme concentrations, and use of mediators that have also been described by different authors [²⁴24. Schmitt S, Souza R, Bettin F, Dillon AJP, Valle JAB, Andreaus J. Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases. Biocatal Biotransform. 2012; 30: 48-56.,³⁵35. Yesilada O, Asma D, Cing S. Decolorization of textile dyes by fungal pellets. Process Biochem. 2003; 38: 933-938.,³⁶36. Kaushik P, Malik A. Fungal dye decolourization: recent advances and future potential. Environ Int. 2009; 35: 127-141.,³⁷37. Van der Zee FP, Cervantes FJ. Impact and application of electron shuttles on the redox (bio)transformation of contaminants: a review. Biotechnol Adv. 2009; 27: 256-277.]. These and other works found in the specialised literature, which may serve to corroborate in some extent our findings, are focused in the sequence.

With respect to the effect of the temperature on the decolourisation of the different dyes, it was observed a negative effect on the process by temperatures over 35°C, despite the fact that laccases from P. sajor-caju PS-2001 present increasing activities up to 50°C [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.], as already mentioned. The explanation for this behaviour lies in the stability of these enzymes as a function of temperature and time. In studies on the laccases of P. sajor-caju PS-2001, these same authors have shown that enzyme activities were practically unaltered after pre-incubation at 20°C for 168 hours, and 30^oC for 72 hours. At 40^oC, however, less than 20% of the original activity remained after 72 hours of pre-incubation, whereas at 50 and 60^oC, laccases were almost totally inactivated after approximately 10 hours [³⁰30. Bettin F, Rosa LO, Montanari Q, Calloni R, Gaio TA, Malvessi E, Silveira MM, Dillon AJP. Growth kinetics, production, and characterization of extracellular laccases from Pleurotus sajor-caju PS-2001. Process Biochem. 2011; 46: 758-764.].

Unlike the methodology adopted in the present work, which used crude enzymatic broth produced in stirred-tank bioreactor, Munari et al. [²³23. Munari FM, Gaio TA, Calloni R, Dillon AJP. Decolorization of textile dyes by enzymatic extract and submerged cultures of Pleurotus sajor-caju. World J Microbiol Biotechnol. 2008; 24: 1383-1392.] studied the decolourisation of various dyes during the growth of P. sajor-caju PS-2001 in solid and liquid cultures. In that work, satisfactory results were noticed for the decolourisation of dyes belonging to the anthraquinone group (Acid Blue 80 and Reactive Blue 220), whereas azo dyes presented resistance to degradation under the assayed conditions. Schmitt et al. [²⁴24. Schmitt S, Souza R, Bettin F, Dillon AJP, Valle JAB, Andreaus J. Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases. Biocatal Biotransform. 2012; 30: 48-56.] evaluated the potential of three oxidoreductases, including laccase of P. sajor-caju PS-2001 and two peroxidases, for the decolourisation of textile disperse dyes (Disperse Red 343, Disperse Red 167 and Disperse Blue 148). In this study, which was carried out in aqueous solutions, the influence of different concentrations of dyes and enzymes, pH, temperature, and addition of mediators - hydroxybenzotriazole (HBT) and syringaldazine (SYR) - was assessed. Tests with Disperse Red 167 and Disperse Blue 148 showed percentages of decolourisation of 15% and 25%, respectively, and under the conditions evaluated, the use of only 10 U mL^-1 laccases of P. sajor-caju PS-2001 has shown to be more efficient in colour removal than horseradish and microbial peroxidases.

Similarly to P. sajor-caju, other species of the genus Pleurotus are also able to discolour industrial dyes. According to Rodríguez et al. [11], this ability in Pleurotus ostreatus is related to the activity of laccases. Zilly et al. [³⁸38. Zilly A, Souza CGM, Barbosa-Tessmann IP, Peralta RM. Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme. Folia Microbiol. 2002; 47: 272-277.] have shown that the fungus Pleurotus pulmonarius, which produces only laccases, can discolour synthetic dyes of azo and triphenylmethane groups. Pleurotus calyptratus was applied to the decolourisation of anthraquinone (Remazol Brilliant Blue R) and azo (Orange G) dyes, but showed limited capacity for degrading triphenylmethane dyes, as Crystal Violet and Malachite Green [³⁹39. Eichlerová I, Homolka L, Lisá L, Nerud F. Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461. Process Biochem. 2006; 41: 941-946.].

By using laccases from Paraconiothyrium variabile, Ashrafi et al. [⁴⁰40. Ashrafi SD, Rezaei S, Forootanfar H, Mahvi AH. The enzymatic decolorization and detoxification of synthetic dyes by the laccase from a soil-isolated ascomycete, Paraconiothyrium variabile. Int Biodeterior Biodegrad. 2013; 85: 173-181.] achieved 60% of decolourisation of the dye Reactive Red 120, after 30 minutes of treatment, and 90% of decolourisation of Disperse Blue 56, after 60 minutes, at pH 5.0 and 40°C. However, in this case, purified laccases were used, while in the present work we have used a crude enzyme preparation without any purification procedure. Yang et al. [⁴¹41. Yang XQ, Zhao XX, Liu CY, Zheng Y, Qian SJ. Decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated Trametes sp. SQ01 and its laccase. Process Biochem. 2009; 44: 1185-1189.] studied a strain of Trametes able to discolour efficiently a variety of synthetic dyes, including some from azo, triphenylmethane and anthraquinone groups, after 5 days of cultivation. The same dyes were also treated with the purified laccases produced by the fungus, in the absence of redox mediators, but few of them have been completely decoloured. Superior results were obtained by Lu et al. [⁴²42. Lu L, Zhao M, Zhang BB, Yu SY. Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme. Appl Microbiol Biotechnol. 2007; 74: 1232-1239.] that achieved 80% of decolourisation of Remazol Brilliant Blue R by using 5 U mL^-1 purified laccase from Pycnoporus sanguineus at pH 3.0 and 40°C.

In addition to oxidation-reduction reactions, Durán and Esposito [²⁵25. Durán N, Esposito E. Potential applications of oxidase enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Appl Catal B: Environ. 2000; 28: 83-99.] reported that laccases also participate in polymerization of compounds. This was also observed by Moldes et al. [⁴³43. Moldes D, Lorenzo M, Sanromán MA. Degradation or polymerisation of Phenol Red dye depending to the catalyst system used. Process Biochem. 2004; 39: 1811-1815.] in studies on the decolourisation of dyes in the absence of mediators. The occurrence of that kind of reaction could be an explanation for the poor results obtained in assays under agitation (Table 4). In fact, in these experiments, few dyes were decolourised and, in most of the cases, it was observed an increase in absorbance of the reaction media, suggesting that the enzymes present in the crude broth have catalysed polymerization reactions of dyes as a consequence of the intensification of the mixing conditions. In this work, in which a crude laccase-containing preparation was used, agitation caused negative effects on the decolourisation of dyes. On the other hand, Kaushik and Malik [³⁶36. Kaushik P, Malik A. Fungal dye decolourization: recent advances and future potential. Environ Int. 2009; 35: 127-141.] observed increasing dye decolourisation when the process was carried out simultaneously with fungal growth in flasks under agitation in comparison to a stationary culture, a result that was surely associated to the more intense oxygen transfer and nutrient distribution in the first case.

Besides the already cited fungi, several other species produce different isoforms of extracellular laccases. Champagne et al. [⁴⁴44. Champagne PP, Nesheim ME, Ramsay JA. Effect of a non-ionic surfactant, Merpol, on dye decolorization of Reactive Blue 19 by laccase. Enzyme Microb Technol. 2010: 46: 147-152.] showed that laccases of Trametes versicolor were able to remove colour of the anthraquinone dye Reactive Blue 19. Niebisch et al. [¹²12. Niebisch CH, Malinowski AK, Schadeck R, Mitchell DA, Kava-Cordeiro V, Paba J. Decolorization and biodegradation of Reactive Blue 220 textile dye by Lentinus crinitus extracellular extract. J Hazard Mat. 2010; 180: 316-322.] applied laccases of Lentinus crinitus for degrading the textile dye Reactive Blue 19. Khelifi et al. [⁶6. Khelifi E, Ayed L, Bouallagui H, Touhami Y, Hamdi M. Effect of nitrogen and carbon sources on Indigo and Congo red decolourization by Aspergillus alliaceus strain 121C. J Hazard Mat. 2009; 163: 1056-1062.] reported the ability of the fungus Aspergillus alliaceus to discolour Indigo and Congo Red dyes during their growth in liquid media, with activities of laccases, manganese peroxidises, and lignin peroxidises being detected in the cultivation broth. Cantele et al. [⁴⁵45. Cantele C, Vilasboa J, Reis EE, Fontana RC, Camassola M, Dillon AJP. Synthetic dye decolorization by Marasmiellus palmivorus: simultaneous cultivation and high laccase-crude broth treatment. Biocatal Agric Biotechnol. 2017; 12: 314-322.], using 10 U mL^-1 laccases from Marasmiellus palmivorus in the reaction medium, the same activity used in the present work, have shown that these enzymes were able to efficiently discolour only Acid Blue 80 and Reactive Blue 220 among eleven different dyes. Similar results were observed even when the enzyme activity was increased up three times.

According to Ashrafi et al. [⁴⁰40. Ashrafi SD, Rezaei S, Forootanfar H, Mahvi AH. The enzymatic decolorization and detoxification of synthetic dyes by the laccase from a soil-isolated ascomycete, Paraconiothyrium variabile. Int Biodeterior Biodegrad. 2013; 85: 173-181.], azo dyes are recalcitrant compounds, less susceptible to enzymatic action, while those of the anthraquinone group are more easily oxidised by laccases. This was also reported by Eichlerová et al. [³⁹39. Eichlerová I, Homolka L, Lisá L, Nerud F. Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461. Process Biochem. 2006; 41: 941-946.] after evaluating results of assays conducted with P. calyptratus, suggesting that azo dyes are more resistant to decolourisation due to its chemical structure. For the biodegradation of reactive dyes of anthraquinone and azo classes, Forss and Welander [⁴⁶46. Forss J, Welander U. Biodegradation of azo and anthraquinone dyes in continuous systems. Int Biodeterior Biodegrad. 2011; 65: 227-237.] evaluated the use of a continuous system, observing colour removal for the dyes Reactive Black 5, Reactive Red 2 and Reactive Blue 4.

In a study of Jarosz-Wilkolazka et al. [⁴⁷47. Jarosz-Wilkolazka A, Kochmanska-Rdest J, Malarczyk E, Wardas W. Leonowicz, A. Fungi and their ability to decolourize azo and anthraquinonic dyes. Enzyme Microb Technol. 2002; 30: 566-572.], 115 fungus strains were compared regarding the ability of removing colour from anthraquinone and azo dye solutions. White-rot fungi were the fastest in the colour removal, and Acid Red 183 proved to be more resistant to decolourisation. Among the species tested, sixty-nine showed ability to decolourise the dye Basic Blue 22 (anthraquinone) and only sixteen discoloured Acid Red 183 (azo). Chagas and Durrant [⁴⁸48. Chagas EP, Durrant LR. Decolorization of azo dyes by Phanerochaete chrysosporium and Pleurotus sajorcaju. Enzyme Microb Technol. 2001; 29: 473-477.] stated that the white-rot fungi Phanerochaete chrysosporium and P. sajor-caju could be used in bioprocesses for removing colour from industrial effluents. P. sajor-caju bleached 50% of the azo dye Orange G, indicating that laccases have greater participation in the process of decolourisation than MnP. Laccases also showed ability to decolourise Remazol Brilliant Blue R during the growth of Trametes pubescens in bioreactor with decolourisation of 55% in 4 hours and 70% in 24 hours, without the addition of redox mediators [⁵5. Rodríguez-Couto S. Production of laccase and decolouration of the textile dye Remazol Brilliant Blue R in temporary immersion bioreactors. J Hazard Mat. 2011; 194: 297-302.].

Laccases of T. versicolor play an important role in the attack on the structure of triphenylmethane dyes. However, the identification of degradation products is also a relevant issue, since the metabolites produced after processing can be highly toxic [⁴⁹49. Casas, N, Parella T, Vicent T, Caminal G, Sarrà M. Metabolites from the biodegradation of triphenylmethane dyes by Trametes versicolor or laccase. Chemosphere. 2009; 75: 1344-1349.]. Champagne and Ramsay [⁵⁰50. Champagne PP, Ramsay JA. Dye decolorization and detoxification by laccase immobilized on porous glass beads. Bioresour Technol. 2010; 101: 2230-2235.] used immobilised laccases for treating anthraquinone and azo dyes. Although anthraquinone dyes have been discoloured more quickly than azo dyes, their reaction derivatives have shown to be much more toxic than the original form of the dye, whereas azo dyes discolouration products did not show toxicity after enzymatic treatment. Selvam et al. [¹⁵15. Selvam K, Swaminathan K, Chae KS. Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp. Bioresour Technol. 2003; 88: 115-119.] reported that laccases of Thelephora sp. were able to discolour the azo dyes Orange G and Congo Red in the absence of redox mediators, showing greater efficiency than MnP and LiP enzymes in colour removal process. However, Murugesan et al. [⁵¹51. Murugesan K, Dhamija A, Nam IH, Kim YM, Chang YS. Decolourization of Reactive Black 5 by laccase: optimization by response surface methodology. Dyes Pigments. 2007; 75: 176-184.] observed that the presence of the mediator HBT was essential for the decolourization of the dye Reactive Black 5 by purified laccases from P. sajor-caju. In addition, Zeng et al. [¹⁰10. Zeng X, Cai Y, Liao X, Zeng X, Luo S, Zhang D. Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase. Process Biochem. 2012; 47: 160-163.] suggest that anthraquinone dyes can act as mediators in processes of decolourisation of azo dyes, using laccases of Trametes troggi.

Several studies have shown that white-rot fungi are able discolour a wide range of dyes with different chemical structures, such as anthraquinone, azo, triphenylmethane and heterocyclic, in which is also reported partial mineralization of dyes by enzymatic and non-enzymatic systems [⁵²52. Cripps C, Bumpus JA, Aust SD. Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl Environ Microbiol. 1990; 56: 1114-1118.,⁵³53. Heinfling A, Martínez MJ, Martínez AT, Bergbauer M, Szewzyk U. Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction. Appl Environ Microbiol. 1998; 64: 2788-2793.]. Spectrophotometric and microscopic analyses of Funalia trogii pellets showed that the process of decolourisation occurs due to microbial metabolism, but not by biosorption [³⁵35. Yesilada O, Asma D, Cing S. Decolorization of textile dyes by fungal pellets. Process Biochem. 2003; 38: 933-938.]. In addition to the factors already mentioned (pH, temperature, use of redox mediators, type and initial concentration of dye), ionic strength and redox potential also affect the decolourisation of dyes by fungi [³⁶36. Kaushik P, Malik A. Fungal dye decolourization: recent advances and future potential. Environ Int. 2009; 35: 127-141.,³⁷37. Van der Zee FP, Cervantes FJ. Impact and application of electron shuttles on the redox (bio)transformation of contaminants: a review. Biotechnol Adv. 2009; 27: 256-277.]. Laccases and peroxidases are eco-friendly biocatalysts for the removal of wide spectrum of textile and non-textile dyes [⁵⁴54. Kalsoom U, Bhatti HN, Asgher M. Characterization of plant peroxidases and their potential for degradation of dyes: a review. Appl Biochem Biotechnol. 2015; 176: 1529-1550.]. However, laccases from different sources exhibit a wide range of redox potentials, which interfere in its potential of application in decolourisation processes [¹⁶16. Wong DWS. Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol. 2009; 157: 174-209.].

Table 5 summarises some relevant works found in the specialised literature about the decolourisation of dyes by fungal enzymes under both in vivo and in vitro conditions.

The results of the present work as well as those found in the literature clearly indicate that the success of the enzymatic decolourisation of dyes is dependent on the particular characteristics of both coloured compound and enzyme preparation. As such, taking in account the large number of dyes used all over the world, a continuous search for new producing fungi and their enzymes might be done in order to make this technology widely applicable in the industrial activity.

CONCLUSION

From the data obtained in this work, it is possible to establish that the laccase-containing enzymatic preparation from P. sajor-caju PS-2001, produced in submerged process in bioreactor, is able to unspecifically oxidize a wide range of dyes with different chemical structures, including compounds from anthraquinone, azo, and triphenylmethane chromophore groups. For each dye, different incubation times for decolourisation are required, an aspect probably related to the affinity between enzyme and substrate, as well as to the higher or lower recalcitrance of the particular compound.

The results obtained in this study indicate that pH 3.2 and temperatures of 30 and 35^oC, without agitation, are adequate conditions for the decolourisation of aqueous solution of dyes belonging to different chromophore groups. The ideal values for temperature are related, below the best for laccases activity as reported in the literature, are dependent on the thermal stability of the enzymes. Agitation does not favour the removal of colour from the reactional mixture evaluated. On the contrary, increasing colour intensity is observed when the reaction is carried out under mixing, possibly due to the occurrence of polymerization reactions of dyes mediated by the laccases themselves.

The findings of this work are important because they reinforce the technical feasibility of using crude fungal enzyme extracts, without any procedure of purification. That possibility could lead to a significant decrease in the costs of the process envisaging future large-scale application of this biotreatment technology in different sectors of the industry.

Acknowledgments

This work was supported by grants from Universidade de Caxias do Sul (UCS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). F. Bettin was supported by post-doctoral fellowships from Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, Brazil) and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

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