Discoloration of dyes by Trametes spp

The dyes used in the textile industry contribute signicantly to pollution of water sources as they are disposed, most of the time, without proper treatment. The objective of this work was to test three strains of two species of the genus Trametes collected in Brazil against the ability to discolor the indigo carmine dye and to detect the activity of the enzymes laccase, lignin peroxidase and manganese peroxidase. The experiment was carried out in Kirk medium under static, non-sterile condition, at ±28 °C for 120 h. Trametes lactinea (URM8350) discolored 81.40% of the indigo carmine dye, T. lactinea (URM8354) 85.09% and T. villosa (URM8022) 96.11%. Laccase was detected in all specimens. Manganese peroxidase was detected in T. villosa (URM8022) and T. lactinea (URM8354), while lignin peroxidase was not detected in any of the isolates under the conditions of the experiment. The discoloration rates observed demonstrate the ability of the strains to discolor carmine indigo and the promising use in the discoloration processes in wastewater from the textile segment.

Subsequently, the dyes are accumulated intracellularly in the living cells through a process known as bioaccumulation. The biodegradation process involves the breakdown of dye molecules by enzymes produced by microbial cells, where complete eradication of dyes is possible (Jasińska et al. 2015;Adenan et al. 2020). Mycorremediation emerges as an economically viable and ecologically effective biological alternative, as fungi can adapt to various pH and temperature ranges, in addition to producing extracellular lignolytic enzymes such as laccase (Lac, EC 1.10.3.2), lignin peroxidase (LiP, EC 1.11.1.14) and manganese peroxidase (MnP, EC 1.11.1.13), which can mineralize xenobiotic and recalcitrant compounds (Tien and Kirk 1984;Ellouze and Sayadi 2016;Sen et al. 2016;Singh 2017;Akhtar and Mannan 2020). White rot fungi, mainly Agaricomycetes, have been identi ed as a potentially e cient biological tool in the removal of synthetic dyes from textile e uents (Wesenberg et al. 2003;Ali 2010). Some studies have demonstrated the e cacy of Trametes species in the degradation of phenolic compounds in e uents from the paper industry, degradation of pentachlorophenol and synthetic dyes in textile e uents (Pinedo-Rivilla et al. 2009;Rodrigues-Couto 2009;Pandey et al. 2017). However, in Brazil, which has a high mycodiversity (Forzza et al. 2010;Maia et al. 2015), little is known about the potential for degradation and discoloration of the species collected in the country (Balan e Monteiro 2001;Lyra et al. 2009;Motato-Vasquéz et al. 2016;Araújo et al. 2020). Thus, the aim of the present study was to test three strains of two species of Trametes collected in Northeast Brazil for the ability to remove the indigo carmine used in the customization of denim and to quantify lignolytic enzymes (Lac, LiP and MnP) produced after the experiment.

Material And Methods
2.1 Microorganism: collection and cultivation conditions Specimens of Trametes lactinea (Berk.) Sacc. were collected on the campus of the Universidade Federal de Pernambuco (08°03'07"S 34°56'59"O, Atlantic Forest domain) in November 2019, while T. villosa (Sw.) Kreisel was collected in the Chapada Diamantina National Park (13°14'31"S, 41°40'7" O, Caatinga domain) in March 2015. For culture, three fragments with diameter of 5 mm were removed from the basidiomata and transferred to Petri dishes containing 2% malt extract supplemented with chloramphenicol (20 mg L-1). The plates were kept at 28 °C for 7 days or until mycelial development (Cavalcanti 1972;Stalpers 1978;Motato-Vásquez et al. 2016). The cultures obtained were deposited in the Collection of Cultures Micoteca URM of the Department of Mycology of the Center Biosciences of the Federal University of Pernambuco under registration numbers URM8350 (T. lactinea), URM8354 (T. lactinea) and URM8022 (T. villosa).

Microorganism: identi cation
The morphological identi cation of the basidioma and DNA analyses followed the usual for this group (Gomes-Silva et al. 2010;Verma et al. 2018;Xavier et al. 2020). The resulting ITS and LSU sequences were subjected to BLASTn search in NCBI to verify the closest identi cation match.

Qualitative tests for phenoloxidases
The qualitative analysis of phenoloxidase activity was veri ed using the Bavendamm method, which allows observing the production of cellular oxidase such as laccase, lignin peroxidase and manganese peroxidase, in addition to tyrosine and catechol oxidase (Nobles 1965;Melo and Azevedo 2008). In our assay, an agar block with diameter of 5 mm was removed from colonies with 7 days of cultivation and transferred to the center of the Petri dishes with diameter of 90 mm containing solid malt agar medium plus tannic acid (0.5%). The control was prepared under the same conditions without tannic acid. All procedures were performed under aseptic conditions. After 3 days of incubation, the formation of a brown halo was observed in the colony reverse, indicating a positive reaction to produce phenoloxidases. These halos were measured with the aid of a digital caliper. The enzyme index was measured through the relationship between the average diameter of the degradation zone and the average diameter of the colony, expressed in millimeter (Hankin and Anagnostakis 1975;Silva et al.2019).

Discoloration tests
The indigo carmine dye was purchased from Sigma-Aldrich Corporation, St. Louis, Missouri, USA and used at a concentration of 50 mg L-1. The experiment was carried out in Erlenmeyer asks (250 mL) containing 50 mL of Kirk medium without sterilization (Kirk and Farrell 1987) plus 5 disks of the fungal mycelium with diameter of 5 mm grown in 2% malt extract after 7 days. The vials were kept for 120 h at ± 28 °C under static condition; 2 mL aliquots were removed from the broth and centrifuged at 1500 rpm for 15 min at 4 °C. The percentage of discoloration of the supernatant was measured in a spectrophotometer (Hitachi U.5100) at a wavelength of 610 nm, obtained from scanning the dye, where the highest absorbance peak was observed at 610 nm. As a control, Kirk medium was used with the dye without fungal inoculum. The experiments were carried out in triplicate. The percentage of discoloration was calculated according to equation: D % = percentage of discoloration, Abscontrol = absorbance of the control at 610 nm and Abstest =absorbance with fungal treatment at 610 nm. The discolored broth was used to quantify the production of the enzymes laccase, manganese peroxidase and lignin peroxidase.

Enzymatic assays
The enzymatic activity of the laccase was veri ed by measuring the oxidation of ABTS (2,2′-azino-bis (3ethylbenzthiazoline-6-sulphonic acid) 0.5 mM in 100 mM sodium acetate buffer (pH 5) plus the enzyme broth. The nal volume of the reaction was 1 mL (800 μL of ABTS + 100 μL of sodium acetate buffer + 100 μL of crude extract). Activity was calculated based on ABTS molar absorptivity (ε420nm = 36,000 M-1. Cm-1) (Bourbonnais et al. 1997;Boran 2019). The activity of lignin peroxidase was veri ed through the oxidation of the mixture composed of 375 μL of 0.25 M sodium tartrate buffer at pH 3.0; 125 μL of 10 mM veratryl alcohol; 50 μL of 2 mM hydrogen peroxide and 500 μL of enzymatic extract. The reaction was monitored on a spectrophotometer at a length of 310 nm (ε310nm = 9,300 M-1. Cm-1) (Tien and Kirk 1984). The reaction mixture for manganese peroxidase (1mL) was composed of 100 µL of phenol red (0.01%), 100 µL of sodium lactate (25 mM), 50 µL of MnS04 (100 mM), 200 µL of egg albumin (0.5%), 50 µL of H202 (100 µM) in 20 mM sodium succinate buffer (pH 4.5) and 500 µL of enzymatic extract. The reactions were carried out at 30 °C for 5 minutes and stopped with the addition of 40µL of 2N NaOH. The absorbance was monitored at 610 nm (Kuwahara et al. 1984). A unit of enzymatic activity was de ned as 1 µmol of the product formed per minute. All tests were performed in triplicate.

Statistical analysis
The decolorization test was carried out in triplicate. The data were analyzed using analysis of variance (ANOVA) with the software Statistical Package for the Social Sciences (SPSS) version 24.0. The Tukey-Kramer multiple comparison test (honestly signi cant difference, HSD, P <0.05) or paired t test (P <0.05) was also performed to obtain statistical signi cance between the mean values.

Detection of phenoloxidases
All strains tested showed a dark amber halo in three days of the experiment, evidenced by the degradation of tannic acid and the production of phenoloxidases: diameter of 80 mm for T. villosa (URM8022), diameter of 90 mm for T. lactinea (URM8350) and diameter of 80 mm for T. lactinea (URM8354). According to Bavendamm (1928), these amber-colored diffusion zones around the fungal colony are the result of the oxidation of phenolic acid produced by extracellular phenoloxidases. The detection of phenoloxidases in microorganisms is used as a way to select promising strains with the potential for degradation of complex compounds to be used in studies of degradation of recalcitrant compounds. The production of phenoloxidase complex enzymes is associated with the discoloration of synthetic dyes due to the similarity in the chemical structure of the dyes and the components of lignin (Melo and Azevedo 2008;Arora and Sharma 2010;Sen et al. 2016;Singh 2017).

Discoloration of indigo carmine
The percentages of discoloration detected were 81.40% for T. lactinea (URM 8350), 85.09% for T. lactinea (URM8354) and 96.11% for T. villosa (URM8022) (Fig. 1). Our results were better that those of Lopes et al. . However, T. lactinea has not been tested before for discoloration of dyes, while T. villosa has not been tested for discoloration of indigo carmine.Also, studies of discoloration of this dye using species, not only of Trametes, collected in Brazil are scarce. Balan and Monteiro (2001) tested Phellinus gilvus and Pycnoporus sanguineus, probably collected in the Atlantic forest, Phanerochaete chrysosporium ATCC24725, and Pleurotus sajor-caju, commercially produced, to remove the indigo carmine in liquid culture medium. Phellinus gilvus discolored 100% of the dye after 4 days of experiment, followed by Pl. sajor-caju (94%), Py.sanguineus (91%) and Ph. chrysosporium (70-75%). Lyra et al. (2009) found that T. membranacea collected in the Atlantic Forest was able to discolor 99.2% of the bromophenol blue and 71.8% of the methylene blue in 10 days, while Ortiz-Monsalve et al. (2019) observed that T. villosa, also collected in the Atlantic Forest, discolored 93.8% of acid orange 142 in 264 h of incubation. To date, our study is the rst report of discoloration of indigo carmine and quanti cation of lignolytic enzymes using species of Trametes collected in Brazil.

Quanti cation of detected enzymes
In the present study, the production of enzymes was detected ( Table 1). The low enzyme indicesobserved for laccase may be related to the presence of the dye, as found by Novotny et al. (2001) in which the presence of dye decreased the detection rates of laccase and manganese peroxidase in alineage of Irpex lacteus, as well as the mycelial development of the fungus. Trombini and Obara-Doi(2012) obtained 99.97% of discoloration using Ganoderma sp. and low laccase indices, showing theaction of another enzyme or other mechanisms involved in the discoloration process. Dye discolorationprocess may involve the participation of enzymes as well as the association of other mechanisms suchas adsorption involved in the discoloration process (Novotný et al. 2001;Couto and Sanromán 2004;Srinivasan and Viraraghavan 2010). Several studies indicate that laccase acts as the enzymeresponsible for discoloration (Couto and Sanromán 2004;Rodríguez-Couto et al. 2006;Levin et al.2004;Zeng et al. 2011;Yuan et al. 2012;Younes et al. 2015;Orzechowski et al. 2018;Uribe-Arizmendi et al. 2020).
However, the participation of manganese peroxidase has also been observed in some discoloration studies (Eichlerová et al. 2007;Grassi et al. 2011;Li et al. 2015;Zhang et al. 2016 In the present study, the indices of discoloration of indigo carmine were well above the rate observed  Lyra et al. (2009) in 10 days (T. membranacea, 99.2% of bromophenol blue and 71.8% of methylene blue), and Zouari- Mechichi et al. (2006) after three weeks (T. trogii, 97% of the remazol brilliant blue). Generally, studies that report good results of dye discoloration in a shorter time are those that use optimization of the enzymes of interest with addition of the dye after enzymatic production, commonly laccase and/or manganese peroxidase (Campos et al. 2001;Rodrigues-Couto et al. 2001;Rodríguez-Couto et al. 2006;Li et al 2015;Wang et al. 2019;Xu et al. 2020). The chemical treatment processes of indigo carmine generate potentially dangerous by-products and sludge, causing serious environmental pollution.
The treatment with indigoids using the enzymatic arsenal of fungal species has been considered a promising strategy at an environmental and economic level (Nyanhongo et al. 2007;Mugdha and Usha 2011;Li et al. 2015). Species belonging to the genus Trametes can produce multiple isoforms of Lac and MnP expressed under different cultivation conditions. However, LiP, when observed, is produced in low quantities (Choi 2021). Lacase contains copper polyphenoloxidases, produces four free electrons that react with phenolic and non-phenolic molecules and is one of the few enzymes capable of catalyzing the reduction of four electrons of molecular oxygen to water and even produced in small quantities can act in the degradation of recalcitrant compounds. The catalytic e cacy of Lac and MnP in the removal of recalcitrant compounds is due to the high redox potential, activity and stability of these enzymes, whether in a raw or puri ed state. However, other enzymes may be involved in the discoloration process (Nyanhongo et al. 2007;Campos et al. 2016;Zheng et al. 2017;Xu et al. 2020;Choi 2021).

Conclusion
Species of Agaricomycetes that cause white rot have an arsenal of degradable lignolytic enzymes that can be used in bioremediation processes. These enzymes are expressed according to the composition of the substrate and the lineage used. The interest in identifying promising strains has been increasing as an effort to minimize and/or treat environments polluted or degraded by anthropic action. Knowing the enzymatic potential of neotropical species is essential in view of the fungal megadiversity in these still unexplored, but threatened environments. The strains T. lactinea (URM8350), T. lactinea (URM8354) and T. villosa (URM8022), collected in Northeastern Brazil, showed signi cant percentage of discoloration of indigo carmine in a short time and at a low cost and their Lac and MnP were e cient in discoloration of the dye. The present work allowed the identi cation of promising strains of the genus Trametes that can be used in the treatment of textile e uents, with T. lactinea being the rst study to report discoloration of a dye. Further studies will be necessary to verify the toxicity level of the discoloration product. statistical analysis, article review, supervision.
Con ict of interest: The authors declare that they have no con ict of interest.
Data availability statements: All data generated or analysed during this study are included in this published article (and its supplementary information les).
Featured article: The discoloration rates demonstrate the potential use of the tested strains in removing indigo carmine and as an ecologically correct and economically viable alternative, as well as show the paucity of studies with tropical species for this purpose. Thus, species of the genus Trametes, under suitable conditions, can be explored in the process of environmental bioremediation against several recalcitrant compounds, among which dyes.

Figure 1
Effect of discoloration of the indigo carmine dye (50 mg L-1) by fungal treatment.