Effective Decolorization and Detoxification of Single and Mixed Dyes with Crude Laccase Preparation from a White-Rot Fungus Strain Pleurotus eryngii

To fully harness the potential of laccase in the efficient decolorization and detoxification of single and mixed dyes with diverse chemical structures, we carried out a systematic study on the decolorization and detoxification of single and mixed dyes using a crude laccase preparation obtained from a white-rot fungus strain, Pleurotus eryngii. The crude laccase preparation showed efficient decolorization of azo, anthraquinone, triphenylmethane, and indigo dyes, and the reaction rate constants followed the order Remazol Brilliant Blue R > Bromophenol blue > Indigo carmine > New Coccine > Reactive Blue 4 > Reactive Black 5 > Acid Orange 7 > Methyl green. This laccase preparation exhibited notable tolerance to SO42− salts such as MnSO4, MgSO4, ZnSO4, Na2SO4, K2SO4, and CdSO4 during the decolorization of various types of dyes, but was significantly inhibited by Cl− salts. Additionally, this laccase preparation demonstrated strong tolerance to some organic solvents such as glycerol, ethylene glycol, propanediol, and butanediol. The crude laccase preparation demonstrated the efficient decolorization of dye mixtures, including azo + azo, azo + anthraquinone, azo + triphenylmethane, anthraquinone + indigo, anthraquinone + triphenylmethane, and indigo + triphenylmethane dyes. The decolorization kinetics of mixed dyes provided preliminary insight into the interactions between dyes in the decolorization process of mixed dyes, and the underlying reasons and mechanisms were discussed. Importantly, the crude laccase from Pleurotus eryngii showed efficient repeated-batch decolorization of single-, two-, and four-dye mixtures. This crude laccase demonstrated high stability and reusability in repeated-batch decolorization. Furthermore, this crude laccase was efficient in the detoxification of different types of single dyes and mixed dyes containing different types of dyes, and the phytotoxicity of decolorized dyes (single and mixed dyes) was significantly reduced. The crude laccase efficiently eliminated phytotoxicity associated with single and mixed dyes. Consequently, the crude laccase from Pleurotus eryngii offers significant potential for practical applications in the efficient decolorization and management of single and mixed dye pollutants with different chemical structures.


Introduction
Laccase is one of the important ligninolytic enzymes produced by white-rot fungi.Laccase, known for its ability to catalyze the oxidation of phenols, polyphenols, anilines, lignin, and polycyclic aromatic hydrocarbons, consists of a copper-containing polyphenol oxidase.This enzymatic process occurs in the presence of molecular oxygen, resulting in the formation of corresponding free radicals and the reduction of molecular oxygen to water.The catalytic center of laccase contains four copper atoms, which can be classified into three types based on their spectral and magnetic properties, namely, type I copper (T1), type II copper (T2), and type III copper (T3).T1 is located in the substrate-binding site, with T2 proximal to T3, forming a trinuclear copper center essential for O 2 reduction [1,2].The laccase-catalyzed oxidation reaction occurs in three steps.First, T1 extracts an electron from the reduced substrate, resulting in oxidization to a free radical, and the redox potential of T1 must exceed that of the substrate for this reaction to occur.Second, through the Cys-His bridge, the electron will be transferred from T1 to the trinuclear copper center, causing sequential reduction of T3 and T2.Finally, within the catalytic center, molecular oxygen will undergo reduction into water, mediated by superoxide formation.Water consists of the sole byproduct of laccase-catalyzed oxidation [3].Laccase has broad application in the efficient degradation of various organic pollutants in the environment due to its outstanding characteristics, such as a broad substrate spectrum, molecular oxygen as the terminal electron acceptor, unique oxidation mechanism without the need for cofactors or peroxides, mild reaction conditions, and environmental friendliness [4,5].Extensive research has shown that laccase can effectively degrade a variety of recalcitrant environmental pollutants, including industrial synthetic dyes [6,7], olefins [8,9], chlorophenols [10,11], polycyclic aromatic hydrocarbons [12,13], pesticides [14,15], and endocrine disruptors [16,17].As a result, the ability of laccase to degrade various environmental pollutants has received increasing attention.
Dyes consist of a class of organic compounds known for imparting bright and durable colors to various materials.With the rapid development of modern industry, synthetic dyes have found application in photography, textile, papermaking, pharmacy, food, and cosmetics.Due to their extensive color range, color brightness, exceptional functionality, and broad suitability, dyes can serve the market demands for fibers and clothing materials.As a result, the textile industry has emerged as the primary consumer of synthetic dyes.Commercially, azo, triphenylmethane, and anthraquinone dyes are widely used in the textile sector [18,19].Synthetic dyes can be categorized into five groups based on their chromophores, namely, azo, anthraquinone, indigo, triphenylmethane, and phthalocyanine dyes.Of these, azo, anthraquinone, and triphenylmethane are the most commonly used commercial dyes, with azo dyes constituting approximately 70% of all dyes used in industrial processes [20].Anthraquinone dyes represent the second largest category of textile dyes due to their excellent colorfastness, lightfastness, and a diverse color spectrum.These dyes have typically been employed in the dyeing of cellulose-based fabrics, wool, and polyamide fibers [21].Although dyes have found widespread application in industries, their extensive use and consequential discharge have led to significant environmental contamination, resulting in adverse impacts on both ecological ecosystems and human health [22].During the dyeing process, approximately 10% to 15% of dyes are discharged into wastewater, causing serious environmental and health problems.Dyes at relatively low concentrations can significantly increase the chromaticity of water bodies, diminishing water transparency and self-purification.The presence of dyes in aquatic ecosystems will hinder light penetration into deeper layers, reducing photosynthetic processes in aquatic organisms, deteriorating the water quality, and posing a direct threat to the survival of aquatic organisms [23,24].The discharge of dye-contaminated wastewater into environments such as lakes and rivers can cause severe damage to aquatic organisms, potentially inducing mutagenic and teratogenic effects [24].Other than their ecological implications, dye pollutants can also pose a significant threat to human health.Azo, anthraquinone, and triphenylmethane dyes have carcinogenic properties, manifesting through direct contact with the human body and through the production of carcinogenic compounds as a result of dye degradation [25].One carcinogenic degradation product of azo dyes is p-diaminobiphenyl.Within its molecular structure, -N=N-can undergo reduction by a reducing agent, generating aromatic amines in humans or animals, contributing to the development of liver, bladder, breast, and colon cancers [26].Moreover, specific active groups in azo dyes can covalently bind to proteins or amino acids in skin and induce skin allergies, and certain substituents in anthraquinone dyes may also trigger skin sensitization in humans [27].Some anthraquinone-based disperse dyes also contain one or more primary amines capable of inserting into the base pairs of the DNA helix, thus, posing a carcinogenic risk [28].Several triphenylmethane dyes also have carcinogenic properties and can be highly irritating to the eyes, with various synthetic dyes, including acid dyes, basic dyes, and direct dyes, exhibiting carcinogenic effects.Reactive dyes such as reactive red and reactive blue have been associated with occupational asthma, while disperse dyes can lead to dermatological allergies [29].Overall, the presence of dye pollutants represents a significant risk to the ecological environment and human health, making it imperative to adopt effective methods for the degradation of dye pollutants and mitigation of their toxic effects.Hence, the exploration of dye pollutant degradation and detoxification has scientific importance and practical implications for eliminating or mitigating the considerable damage caused to the ecosystem and human health.
Recent research has revealed that crude laccase preparations and purified laccases hold substantial promise for the efficient degradation of dyes with diverse structures [30].Azo dyes contain -N=N-in their molecules and represent the most intensively and widely used synthetic dyes (>70%).Zeng et al. utilized Trametes trogii crude laccase preparation for the decolorization of azo dyes Acid Red 1 and Reactive Black 5, demonstrating significant enhancement in decolorization with a 1 mM redox mediator (1-hydroxybenzotriazole) [31].Pan et al. employed a crude laccase preparation from Phanerochaete chrysosporium for the decolorization and degradation of the azo dye Reactive Turquoise Blue KGL, achieving a decolorization efficiency of up to 80% under optimal conditions within 20 min [32].Yanto et al. explored the degradation of various dyes using crude laccase preparation derived from Trametes hirsuta.The study demonstrated variations in the decolorization efficacy associated with the chemical structures of dyes, and the efficient decolorization of the azo dye Reactive Black 5 was achieved with violuric acid as a mediator [33].Couto et al. assessed the performance of crude laccase from T. hirsuta in the decolorization of various industrial azo dyes, demonstrating that the azo dye SSB 4GL exhibited a high level of decolorization within 4 h (73%), followed by DC NBS (67%).However, the azo dyes DP 5GL and SSY 4GL displayed a high resistance to decolorization, potentially due to the nature and position of the dye substituents [34].Jasi ńska et al. studied the degradation of azo dyes using a crude laccase preparation from Myrothecium roridum.The introduction of a synthetic or natural mediator to a reaction mixture containing 1 U/mL laccase and the azo dye AB 113 resulted in a significant enhancement of the decolorization efficiency, indicating that appropriate mediator supplementation could promote the degradation of substrates that may be less amenable to laccase-mediated degradation [35].Anthraquinone dyes contain an anthraquinone unit in their molecules, including anthraquinone derivatives and fused-ring ketone dyes produced from anthraquinone derivatives.Anthraquinone dyes have found extensive application in the dyeing and textile industries, primarily due to their vibrant colors, high fixation rate, and excellent colorfastness.Zeng et al. observed that a crude laccase preparation of T. trogii provided efficient decolorization of the anthraquinone dyes Remazol Brilliant Blue R (RBBR), Reactive Blue 4 (RB4), and Acid Blue 129 without a mediator [36].Hou et al. highlighted the efficient decolorization of anthraquinone dyes through a crude laccase preparation derived from Pleurotus ostreatus, with the addition of an appropriate concentration of the redox mediator 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which further enhanced the degradation efficiency [37].Wong et al. found that anthraquinone dyes served as substrates for laccase-mediated first-order reactions, and anthraquinone dyes, as excellent substrates of laccase, showed increased decolonization with increasing enzyme activity [38].Yadav et al. reported on the efficient decolorization of the anthraquinone dye RBBR with laccase from Arthrographis kalrae, even in the absence of a redox mediator.Decolorization was influenced by parameters such as the enzyme dosage, pH, treatment duration, and initial dye concentration [39].Osma et al. investigated the decolorization of RBBR using a crude laccase prepared from Trametes pubescens.The study found that the formation of enzyme inhibitors during the decolorization process led to a significant reduction in the decolorization rate after a certain period [40].Following azo and anthraquinone dyes, triphenylmethane dyes represent the third-most commonly used synthetic dyes in the industry.These dyes feature a distinctive molecular structure with three benzene rings attached to a carbon atom, with each carrying different side chains.The inherent stability of this chemical structure results in resistance to degradation by common microorganisms.
Consequently, triphenylmethane dyes with high toxicity and bio-degradation resistance may cause serious environmental pollution when discharged into wastewater.Grassi et al. employed a crude laccase preparation from T. trogii to degrade the triphenylmethane dyes malachite green, bromophenol blue, and crystal violet, indicating that the decolorization efficiency was higher than 85% within one day [41].Li et al. utilized a crude laccase preparation from Pycnoporus sanguineus to decolorize crystal violet.However, the study noted that crystal violet was not a direct substrate and required the addition of the mediator ABTS to facilitate decolorization, resulting in a moderate decolorization efficiency of 35% within 180 min [42].Hadibarata et al. reported that crude laccase prepared from Armillaria sp.F022 displayed a limited degradation of the triphenylmethane dye brilliant green, and the degradation efficiency was less than 40% after 96 h of treatment [43].In addition, Núria et al. used T. versicolor laccase to decolorize the triphenylmethane dyes brilliant green 1, methyl green (MG), and acid fuchsin (AF).Brilliant green 1 and MG were completely decolorized within 24 h, and only 28% of AF was decolorized within 48 h [44].These variations were attributed to the differences in chemical structures of the triphenylmethane dyes.The degradation of triphenylmethane dyes with laccase was influenced by the dye structures, likely due to variations in the redox potential of laccases from different sources.Indigo dyes contain a C=C bond connecting two heterocyclic rings with carbonyl groups, forming a chromophore comprised of a conjugated system with four atoms in the carbonyl-double bonded-carbonyl groups.Indigo dyes are primarily used in denim production, and the substantial consumption of denim has resulted in the production of substantial amounts of indigo-containing wastewater.Indigo dyes consist of aromatic reducing dyes with a stable conjugated structure and poor biodegradability, posing significant environmental risks.Given the structural stability of indigo dyes, small-molecule electron-transfer mediators with diverse structures have been incorporated to facilitate their oxidation and degradation.Hu et al. utilized 1-hydroxybenzotriazole as a mediator for indigo dye decolorization with a crude laccase prepared from T. versicolor, achieving up to 90.1% decolorization efficiency within 80 min.The study showed nearly complete decolorization (90-100%) of IC within 1 h using phloroglucinol, thymol, and violuric acids as laccase mediators [45].
Prior research on laccase-mediated dye degradation has focused on assessing the decolorization of a specific dye with laccase from a specific source, with limited information on the degradation of a broad spectrum of dyes containing various structures.Furthermore, industrial dye wastewater typically consists of a complex mixture containing various dyes.Therefore, the investigation of laccase-mediated decolorization of dye mixtures holds substantial practical and theoretical value for the efficient treatment of industrial dye wastewater.Past studies, however, have focused on single-dye degradation with laccase, with little exploration of multi-dye degradation, and interaction between various dyes in the laccase-mediated degradation of dye mixtures has remained largely unexplored.The ability of laccase to detoxify dye pollutants during decolorization remains critical for evaluating its effectiveness in treating dye pollutants.However, previous research has mostly been limited to the detoxification effect of laccase on a single dye, and there is still a lack of reports on the detoxification ability of laccase on mixed dyes (mixtures of different types of dyes).There are almost no reports on the detoxification effect of laccase on different types of dye mixtures.Industrial dye wastewater often contains high levels of metal ions and organic solvents, which may influence laccase activity and, consequently, its effectiveness in treating dye wastewater.As a result, the comprehensive examination of laccase tolerance to various metal ions and organic solvents during dye decolorization holds practical value for the efficient treatment of dye wastewater with abundant metal ions and organic solvents.Nevertheless, systematic and in-depth research in this area remains limited.
In this study, we addressed the unresolved issues outlined above and obtained a laccase resource with enhanced applicability for the efficient decolorization and detoxification of both single and mixed dyes.To explore and utilize the potential of laccase in the efficient degradation and detoxification of single and mixed dyes, a crude laccase was prepared from the white-rot fungus strain Pleurotus eryngii.Subsequently, we systematically assessed the decolorization efficacy and reaction rates of single azo, anthraquinone, triphenylmethane, and indigo dyes.We also investigated the degradation kinetics and tolerance of this enzyme to varying concentrations of metal salts and organic solvents during the decolorization of four types of dyes.We evaluated the potential of this crude laccase for the treatment of dye wastewater containing diverse salts and organic solvents.Besides the single dyes, we also systematically examined the decolorization of dye mixtures and revealed their decolorization kinetics (azo + azo, azo + anthraquinone, azo + triphenylmethane, azo + indigo, anthraquinone + indigo, anthraquinone + triphenylmethane, and indigo + triphenylmethane).Accordingly, we systematically investigated the repeated-batch decolorization of single dyes and dye mixtures containing two and four dyes.The efficiency of repeated-batch decolorization of both single and mixed dyes was demonstrated, and we evaluated the reusability, stability, and efficiency of the crude laccase in repeated-batch decolorization.Finally, we assessed the elimination of phytotoxicity through the crude laccase for single and mixed dyes.Our assessment involved analyzing the impact of these dyes (single and mixed dyes) on the growth of crop seedlings before and after laccasemediated decolorization.Our findings revealed that the crude laccase from Pleurotus eryngii displayed distinct advantages and properties.These included the efficient decolorization of a wide range of dyes, high tolerance against salts and organic solvents, stable performance in repeated-batch decolorization of both single and mixed dyes, and simultaneous decolorization and detoxification for single dyes and mixtures of different types of dyes (mixed dyes).Therefore, the crude laccase obtained from this study offers significant potential in the efficient degradation and treatment of pollutants containing single or mixed dyes.

Preparation of the Crude Laccase from Pleurotus eryngii and Determination of Laccase Activity
The mycelia of Pleurotus eryngii were transferred from the PDB medium to GYP medium for laccase production.On day 3 of culturing in the GYP medium, CuSO 4 was added to induce laccase production.As shown in Figure 1, the laccase activity significantly increased after CuSO 4 addition and reached a maximum of 6868.33 U/L on day 7. Laccase activity was determined as described in the literature [46].The crude laccase preparation was stored at −20
As shown in Figure 2A,E,H, the crude laccase preparation demonstrated efficient de-

Kinetic Equations of Azo Dye Degradation
We further investigated the kinetic degradation equations of the azo dyes NC, AO7, and RB5 and calculated the reaction rate constant k.As shown in Figure 2L-N, degradation fit the linear regression models well, and the R 2 values were all above 0.95, indicating a high goodness of fit.Therefore, the degradation of NC, AO7, and RB5 followed first-order kinetics, and the k values of 100 mg/L NC, RB5, and AO7 were 1.84 × 10 −2 , 7.44 × 10 −3 , and 7.15 × 10 −3 min −1 , respectively (Figure 2L-N).The reaction rates of the three azo dyes followed the order NC > RB5 > AO7.The results of the degradation kinetics were consistent with the time courses of decolorization.

Decolorization Efficiency of Anthraquinone Dyes at Different Concentrations
The crude laccase preparation demonstrated excellent decolorization of the anthraquinone dyes RBBR and RB4 at different concentrations.As shown in Figure S1A,B, the 24 h decolorization efficiencies were 98.73% and 95.52% for RBBR and RB4, respectively, at 100 mg/L, 90.70% and 87.26% at 200 mg/L, and 90.25% and 80.66% at 800 mg/L.Therefore, the decolorization efficiency remained high for RBBR and RB4 at high concentrations.

Time Courses of Decolorization of Anthraquinone Dyes at Different Concentrations
As shown in Figure S1C,D, the crude laccase preparation demonstrated very rapid degradation of RBBR at different concentrations.The decolorization efficiencies of 100 mg/L RBBR at 1, 2, and 3 h were 88.03%, 95.97%, and 98.97%, respectively.The crude laccase preparation also showed rapid decolorization of RB4, with decolorization efficiencies of 55.88%, 72.43%, and 81.54% for 100 mg/L RB4 at 0.5, 1, and 2 h, respectively.These results indicated that the crude laccase preparation could rapidly decolorize anthraquinone dyes, and the decolorization rate followed the order RBBR > RB4.

Kinetic Equations of Anthraquinone Dye Degradation
The degradation kinetic equations of 100 mg/L anthraquinone dyes RBBR and RB4 were plotted and the k values were determined.As shown in Figure S1E,F, the degradation of these anthraquinone dyes fit the linear regression models well, with R 2 values above 0.95, indicating a high goodness of fit.Therefore, the degradation of RBBR and RB4 followed first-order kinetics, and the k values of 100 mg/L RBBR and RB4 were 6.05 × 10 −2 and 1.81 × 10 −2 min −1 , respectively (Figure S1E,F).These results showed that the crude laccase preparation could rapidly degrade both RBBR and RB4, and the reaction rate was higher for RBBR than RB4.The degradation kinetics results were consistent with the time courses of decolorization.

Decolorization Efficiency of Triphenylmethane Dyes at Different Concentrations
The crude laccase preparation demonstrated different decolorization efficiencies for the triphenylmethane dyes.The decolorization efficiency was excellent for MG and BB at different concentrations, good for CR, and poor for AF.As shown in Figure S2A,B, the 24 h decolorization efficiencies were 89.42% and 99.50% for MG and BB, respectively, at 50 mg/L, 94.27% and 99.50% at 200 mg/L, and 84.04% and 99.02% at 800 mg/L, indicating very efficient decolorization.The 24 h decolorization efficiencies of 50, 100, 200, 400, and 800 mg/L CR were 52.00%, 58.84%, 56.36%, 53.37%, and 50.12%, respectively (Figure S2C), indicating efficient decolorization.The 24 h decolorization efficiencies of AF at different concentrations were all below 10% (Figure S2D), indicating inefficient decolorization.In summary, the decolorization efficiency of triphenylmethane dyes followed the order BB > MG > CR > AF.

Kinetic Equations of Triphenylmethane Dye Degradation
The degradation kinetic equations of 100 mg/L triphenylmethane dyes BB and MG were investigated and the k values were determined.As shown in Figure S2G,H, the degradation of these dyes fit well with the linear regression models and the R 2 values were above 0.95, indicating a high goodness of fit.Therefore, the degradation of BB and MG followed first-order kinetics.The k values of 100 mg/L BB and MG were 4.344 × 10 −2 and 7.232 × 10 −4 min −1 , respectively (Figure S2G,H), and these results showed that the crude laccase preparation could rapidly degrade BB and the reaction rate of BB was significantly higher than that of MG.

Kinetic Equations of Indigo Dye Degradation
As shown in Figure S3C, the degradation of IC fit well with the linear regression model, with an R 2 value above 0.95, indicating a high goodness of fit.Therefore, the degradation of IC followed first-order kinetics.The k value of 100 mg/L IC was 3.05 × 10 −3 min −1 , indicating rapid degradation of IC by the crude laccase preparation.

Decolorization of Azo + Anthraquinone Dyes
The crude laccase preparation demonstrated efficient decolorization of the azo + anthraquinone dyes at different concentrations.For NC + RB4, the decolorization efficiency was approximately 85% for NC at different concentrations.With an increase in dye concentration, the decolorization efficiency of RB4 gradually decreased.But the decolorization efficiency of RB4 in the mixture remained high at different concentrations (Figure 3B).For NC + RBBR, the 24 h decolorization efficiencies were 87.24% and 91.25% for NC and RBBR, respectively, at 50 mg/L, 86.93% and 80.65% at 100 mg/L, 80.72% and 79.97% at 400 mg/L, and 78.20% and 77.89% at 800 mg/L (Figure 3B).Thus, the crude laccase preparation also demonstrated efficient decolorization of NC + RBBR dyes.

Decolorization of Azo + Triphenylmethane Dyes
The crude laccase preparation showed efficient decolorization of the azo + triphenylmethane dyes.For NC + MG, the decolorization efficiency was approximately 85% for NC at different concentrations, and the efficiency was slightly higher for MG (approximately 90%).Compared to single dyes, the decolorization efficiency decreased for NC, but increased for MG (Figure 3C).For NC + BB, the 24 h decolorization efficiencies were 89.58% and 94.55% for NC and BB, respectively, at 50 mg/L, 92.21% and 97.76% at 100 mg/L, 93.12% and 98.37% at 400 mg/L, and 89.49% and 97.44% at 800 mg/L (Figure 3C).For RB5 + BB, the 24 h decolorization efficiencies were 98.26% and 98.03% for 400 mg/L RB5 and BB, respectively, and 98.01% and 98.19% for 800 mg/L RB5 and BB, respectively (Figure 3C).Thus, the crude laccase preparation demonstrated efficient decolorization of the NC + BB and RB5 + BB dyes.

Decolorization of Triphenylmethane + Indigo Dyes
The crude laccase preparation showed very efficient decolorization of the triphenylmethane + indigo dyes (BB + IC), and the experiment demonstrated similar 24 h decolorization efficiencies (>90%) for BB and IC at different concentrations, with no significant differences in decolorization efficiency compared to the single dyes.The 24 h decolorization efficiencies were 98.62% and 98.77% for BB and IC, respectively, at 200 mg/L, 99.13% and 99.47% at 400 mg/L, and 97.33% and 96.16% at 800 mg/L (Figure 3F).The decolorization kinetics of the azo + azo dyes (100 mg/L NC + 100 mg/L RB5) were investigated with 100 mg/L NC or 100 mg/L RB5 as the controls.The ln c-t curves were plotted for the mixed and single dyes.
As shown in Figure S4A, the k value of NC decreased from 1.84 × 10 −2 min −1 in the control (decolorization of single dye) to 6.04 × 10 −3 min −1 in NC + RB5 (decolorization of mix dyes), and the k value of RB5 decreased from 7.44 × 10 −3 min −1 in the control to 2.76 × 10 −3 min −1 in NC + RB5.Therefore, in NC + RB5, the k values (decolorization rates) of both dyes decreased compared to the single dyes, with NC and RB5 showing mutual inhibition during decolorization of the two azo dyes.

Decolorization Kinetics of Azo + Anthraquinone Dyes
The degradation kinetics of the azo + anthraquinone dyes (100 mg/L NC + 100 mg/L RBBR and 100 mg/L NC + 100 mg/L RB4) were investigated using single dyes as the controls (100 mg/L NC, 100 mg/L RBBR, and 100 mg/L RB4).The ln c-t curves were plotted for the mixed and single dyes.
As shown in Figure S4B, the k value of NC increased from 1.84 × 10 −2 min −1 in the control to 2.00 × 10 −2 min −1 in NC + RB4, while the k value of RB4 decreased from 1.81 × 10 −2 min −1 in the control to 1.28 × 10 −2 min −1 in NC + RB4.As shown in Figure S4C, the k value of NC increased from 1.84 × 10 −2 min −1 in the control to 2.28 × 10 −2 min −1 in NC + RBBR, while the k value of RBBR decreased from 6.05 × 10 −2 min −1 in the control to 2.68 × 10 −2 min −1 in NC + RBBR.These results indicated that during the decolorization of azo + anthraquinone dyes, the presence of the anthraquinone dye significantly enhanced the decolorization rate of the azo dye, while the decolorization rate of the anthraquinone dye was inhibited.

Decolorization Kinetics of Azo + Indigo Dyes
As shown in Figure S4D, the k value of NC significantly decreased from 1.84 × 10 −2 min −1 in the control to 7.58 × 10 −4 min −1 in NC + IC, while the k value of IC increased from 3.05 × 10 −3 min −1 in the control to 4.62 × 10 −3 min −1 in NC + IC.These results indicated that in the decolorization of azo + indigo dyes, the decolorization rate of the indigo dye increased while the decolorization rate of the azo dye significantly decreased compared to the single dyes.In NC + IC, the azo dye promoted the decolorization of the indigo dye, while the indigo dye inhibited the decolorization of the azo dye.

Decolorization Kinetics of Azo + Triphenylmethane Dyes
As shown in Figure S4E, the k value of NC decreased from 1.84 × 10 −2 min −1 in the control to 9.25 × 10 −3 min −1 in NC + MG, while the k value of MG increased from 7.23 × 10 −4 min −1 in the control to 3.30 × 10 −3 min −1 in NC + MG.These results indicated that in the decolorization of azo + triphenylmethane dyes, the azo dye NC promoted the decolorization rate of the triphenylmethane dye MG, while MG inhibited the decolorization rate of NC.

Decolorization Kinetics of Anthraquinone + Indigo Dyes
As shown in Figure S4F, the k value of RB4 increased from 1.81 × 10 −2 min −1 in the control to 7.01 × 10 −2 min −1 in RB4 + IC, and the k value of IC increased from 3.05 × 10 −3 min −1 in the control to 6.17 × 10 −2 min −1 in RB4 + IC.The reaction rate of IC greatly increased.These results indicated that in the decolorization of anthraquinone + indigo dyes, the anthraquinone dye RB4 significantly enhanced the decolorization rate of the indigo dye IC.

Decolorization Kinetics of Indigo + Triphenylmethane Dyes
As shown in Figure S5A, the k value of CR decreased from 1.62 × 10 −2 min −1 in the control to 1.00 × 10 −2 min −1 in IC + CR, while the k value of IC increased from 3.05 × 10 −3 min −1 in the control to 1.55 × 10 −1 min −1 in IC + CR.The improvement effect was very obvious.These results indicated that in the decolorization of indigo + triphenylmethane dyes, the triphenylmethane dye CR significantly promoted the decolorization rate of the indigo dye IC, while IC inhibited the decolorization rate of CR.

Decolorization Kinetics of Anthraquinone + Triphenylmethane Dyes
As shown in Figure S5B, the k value of RBBR decreased from 6.05 × 10 −2 min −1 in the control to 3.46 × 10 −2 min −1 in RBBR + CR, while the k value of CR increased from 1.62 × 10 −2 min −1 in the control to 1.81 × 10 −2 min −1 in RBBR + CR.These results indicated that in the decolorization of anthraquinone + triphenylmethane dyes, the decolorization rate of the triphenylmethane dye CR was promoted, but the decolorization rate of the anthraquinone dye RBBR was inhibited compared to the single dyes.In RBBR + CR, RBBR promoted the decolorization rate of CR, while CR inhibited the decolorization rate of RBBR.

Decolorization Kinetics of Triphenylmethane + Triphenylmethane Dyes
As shown in Figure S5C, in the single and mixed dyes, the concentrations of both MG and BB were 100 mg/L.The k value of MG increased from 7.23 × 10 −4 min −1 in the control to 5.78 × 10 −3 min −1 in MG + BB, while the k value of BB decreased from 4.34 × 10 −2 min −1 in the control to 3.34 × 10 −3 min −1 in MG + BB.As shown in Figure S5D, the k value of MG increased significantly from 7.23 × 10 −4 min −1 in the control to 1.48 × 10 −2 min −1 in MG + CR, while the k value of CR decreased from 1.62 × 10 −2 min −1 in the control to 1.11 × 10 −2 min −1 in MG + CR.In addition, the k value of AF increased from 3.87 × 10 −4 min −1 in the control to 7.8 × 10 −3 min −1 in BB + AF, while the k value of BB decreased from 4.34 × 10 −2 min −1 in the control to 1.59 × 10 −2 min −1 in BB + AF (Figure S5E).These results indicated that in the decolorization of triphenylmethane + triphenylmethane dyes, the decolorization rate of one triphenylmethane dye was significantly enhanced, while the decolorization rate of the other triphenylmethane dye was inhibited.In a mixture containing two triphenylmethane dyes, the first dye significantly promoted the decolorization rate of the second dye, while the second dye inhibited the decolorization rate of the first dye.
The crude laccase preparation showed a low tolerance to Cl − salts during NC decolorization.With 50 mM Cl − salts, the enzyme preparation was tolerant to NaCl, KCl, NiCl, and MgCl 2 , and the 24 h decolorization efficiencies were 59.60%, 55.09%, 57.22%, and 56.68%, respectively.With 100 mM Cl − salts, the tolerance was greatly reduced, while with ≥200 mM Cl − salts, the 24 h decolorization efficiency of NC was almost zero.
Compared to the SO 4 2− salts, the Cl − salts demonstrated significantly stronger inhibition for NC degradation.With 100 mM MnSO 4 and MgSO 4 , the 24 h decolorization efficiencies were 93.25% and 91.59%, respectively, and NC decolorization was slightly affected.With 100 mM MnCl 2 and MgCl 2 , the decolorization efficiencies were only 26.44% and 1.65%, respectively.Compared to the 100 mM SO 4 2− salts, the 100 mM Cl − salts significantly inhibited NC degradation (Figure 4B).These results indicated that MnCl 2 and MgCl 2 exhibited significant inhibition of the decolorization of NC.S6).The crude laccase preparation demonstrated the highest tolerance to Na 2 SO 4 during RBBR degradation, and with 800 mM Na 2 SO 4 , the 24 h decolorization efficiency of RBBR still reached 88.72%, which was close to the control without salts (Figure S6F).
The crude laccase preparation demonstrated a low tolerance to the Cl − salts during RBBR degradation.With 400 mM Cl − salts, RBBR decolorization was significantly inhibited, except for CdCl 2 and LiCl, and the 24 h decolorization efficiency was below 10%.
Compared to the SO 4 2− salts, the Cl − salts showed a significantly stronger inhibition of RBBR degradation.With 100 mM MnSO 4 and MgSO 4 , the 24 h decolorization efficiencies were 100.00% and 68.94%, respectively, and with 100 mM MnCl 2 and MgCl 2 , the 24 h decolorization efficiencies decreased to 24.81% and 21.43%, respectively (Figure S6B).Therefore, the Cl − salts were stronger inhibitors of RBBR degradation compared to the SO 4 2− salts.

Effects of Metal Salts at Different Concentrations on the Degradation of the Triphenylmethane Dye BB
The crude laccase preparation showed a high tolerance to SO  S7), and decolorization remained efficient.These results indicated that the crude laccase preparation was tolerant to high concentrations of SO 4 2− salts during the degradation of BB.The crude laccase preparation showed a low tolerance to Cl − salts during the degradation of BB.With ≥600 mM Cl − salts, BB degradation was significantly inhibited and the decolorization efficiency was almost zero.

Effects of Metal Salts at Different Concentrations on the Degradation of the Indigo Dye IC
The crude laccase preparation showed a moderate tolerance to SO 4 2− salts such as MnSO 4 , MgSO 4 , ZnSO 4 , Na 2 SO 4 , and CdSO 4 , as well as a low tolerance to Cl − salts during IC degradation (Figure S8).With 50 mM Cl − salts, IC degradation was significantly inhibited, except for CdCl 2 and NiCl 2 , while for ≥200 mM Cl − salts, stronger inhibition was recorded.Compared to the SO 4 2− salts, Cl − salts demonstrated stronger inhibition of IC decolorization (Figure S8).As shown in Figure 5A, the degradation of NC was slightly affected by 10% (v/v) glycerol, butanediol, propanediol, ethylene glycol, methanol, ethanol, acetonitrile, isopropanol, and acetone, while 10% (v/v) DMSO and DMF significantly inhibited NC decolorization.The degradation of NC was slightly affected by 20% (v/v) glycerol, butanediol, propanediol, ethylene glycol, methanol, ethanol, and isopropanol, though 20% (v/v) acetonitrile and acetone significantly inhibited the decolorization of NC with the crude laccase (Figure 5B).The crude laccase preparation showed a strong tolerance to 40% (v/v) glycerol, ethylene glycol, propanediol, and butanediol during NC degradation, and the 24 h decolorization efficiencies were 91.53%, 93.94%, 68.50%, and 44.44%, respectively.However, 40% (v/v) of other organic solvents almost completely inhibited NC decolorization and the decolorization efficiency was almost zero (Figure 5C).The enzyme preparation was still tolerant to 50% (v/v) ethylene glycol, glycerol, and propanediol, and the 24 h decolorization efficiencies were 91.80%, 20.72%, and 14.87%, respectively (Figure 5D).At 70% (v/v), all organic solvents demonstrated significant inhibitory effects, and the 24 h decolorization efficiencies were below 10% (Figure 5E).In summary, among the 11 organic solvents, the crude laccase preparation showed high tolerance to ethylene glycol, glycerol, propanediol, and butanediol, with the highest tolerance to ethylene glycol during NC degradation.In contrast, DMSO and DMF demonstrated strong inhibitory effects for NC decolorization (low tolerance to DMSO and DMF).

Effects of Organic Solvents at Different Concentrations on the Degradation of the Triphenylmethane Dye BB
As shown in Figure S10, among the 11 organic solvents, the crude laccase preparation demonstrated the highest tolerance to glycerol for BB degradation.With 10%, 20%, 40%, 50%, and 70% glycerol, the 24 h decolorization efficiencies were 99.66%, 100.00%, 100.00%, 100.00%, and 76.20%, respectively, and with 80% (v/v) glycerol, the 24 h decolorization efficiencies could still reach 50.8% (Figure S10).In addition to glycerol, the crude laccase preparation demonstrated moderate tolerance to ethylene glycol and propanediol during BB degradation.The organic solvents used in this study were glycerol, butanediol, propylene glycol, ethylene glycol, methanol, ethanol, isopropanol, acetonitrile, acetone, DMSO, and DMF.The control group consisted of a 1 mL decolorization system without metal salts.(A) The final concentration of organic solvent was 10% (v/v).(B) The final concentration of organic solvent was 20% (v/v).(C) The final concentration of organic solvent was 40% (v/v).(D) The final concentration of organic solvent was 50% (v/v).(E) The final concentration of organic solvent was 70% (v/v).(F) The final concentration of organic solvent was 80% (v/v).Red color: control, no organic solvents added.Black color: different organic solvents added.

Effects of Organic Solvents at Different Concentrations on the Degradation of the Indigo Dye IC
As shown in Figure S11, among the 11 organic solvents, the crude laccase preparation showed the highest tolerance to glycerol and ethylene glycol during the degradation of IC.With 10%, 20%, 40%, 50%, and 70% glycerol, the 24 h decolorization efficiencies were 99.74%, 100%, 78.83%, 58.51%, and 33.87%, respectively, and with 10%, 20%, 40%, 50%, and 70% ethylene glycol, the 24 h decolorization efficiencies were 99.17%, 98.55%, 71.06%, 61.41%, and 32.14%, respectively (Figure S11).The inhibition of decolorization increased with increasing organic solvent concentration, and at 80% (v/v), all organic solvents demonstrated significant inhibition of IC decolorization, and the decolorization efficiency of IC was below 20% (Figure S11F).We investigated the repeated-batch decolorization of the azo dyes NC and RB5.The decolorization efficiency of the first batch was set to 100%, and the relative decolorization efficiency of the following batches (relative to the first batch) was calculated, with the results shown in Figure 6A.The crude laccase preparation showed a high efficiency in the repeated-batch decolorization of the azo dye NC, and in batch 5, the relative decolorization efficiency of NC was 84.85%, while in batch 7, the relative decolorization efficiency of NC was 74.65%, indicating efficient decolorization (Figure 6A).The crude laccase preparation showed a less-efficient repeated-batch decolorization of the azo dye RB5, while in batches 5 and 7, the relative decolorization efficiencies of RB5 were 48.02% and 33.36%, respectively (Figure 6A).These results indicated that the crude laccase demonstrated a significantly higher efficiency in repeated-batch decolorization of NC compared to RB5.
We investigated the repeated-batch decolorization of the anthraquinone dyes RBBR and RB4, with the results shown in Figure 6B.The crude laccase preparation showed efficient repeated-batch decolorization of the anthraquinone dye RBBR, and the relative decolorization efficiencies of batches 2, 3, 4, and 5 were 98.47%, 92.73%, 96.54%, and 93.21%, respectively (Figure 6B).No significant changes in decolorization efficiency were observed in these batches relative to batch 1 (90.46%), and the relative decolorization efficiency of batch 6 was 58.10%, which was significantly lower than batch 1.The crude laccase preparation also showed efficient repeated-batch decolorization of the anthraquinone dye RB4, and the relative decolorization efficiencies in batches 2, 3, and 4 were 94.62%, 98.24%, and 95.24%, indicating no significant changes relative to batch 1 (Figure 6B).The relative decolorization efficiencies of batches 5 and 6 were 76.29% and 24.23%, which were significantly lower than batch 1.These results indicated that repeated-batch decolorization was more efficient for RBBR compared to RB4.
We investigated the repeated-batch decolorization of the indigo dye IC, and the results are shown in Figure 6D.The crude laccase preparation showed highly efficient decolorization of IC with repeated batches.The relative decolorization efficiencies of batches 2, 3, 4, and 5 were 100.00%,100.00%, 99.82%, and 97.23%, respectively.After five consecutive decolorization batches, the relative decolorization efficiency of IC was 97.23%, which was almost unchanged compared to batch 1.In batch 7, the relative decolorization efficiency of IC was 74.42%, which was slightly lower than in batch 1, but the decolorization still remained efficient (Figure 6D).We investigated the repeated-batch decolorization of the anthraquinone dyes RBBR and RB4, with the results shown in Figure 6B.The crude laccase preparation showed efficient repeated-batch decolorization of the anthraquinone dye RBBR, and the relative decolorization efficiencies of batches 2, 3, 4, and 5 were 98.47%, 92.73%, 96.54%, and 93.21%, respectively (Figure 6B).No significant changes in decolorization efficiency were observed in these batches relative to batch 1 (90.46%), and the relative decolorization efficiency of batch 6 was 58.10%, which was significantly lower than batch 1.The crude laccase preparation also showed efficient repeated-batch decolorization of the anthraquinone dye RB4, and the relative decolorization efficiencies in batches 2, 3, and 4 were 94.62%, 98.24%, and 95.24%, indicating no significant changes relative to batch 1 (Figure 6B).The relative decolorization efficiencies of batches 5 and 6 were 76.29% and 24.23%, which were significantly lower than batch 1.These results indicated that repeated-batch decolorization was more efficient for RBBR compared to RB4.
We also investigated the repeated-batch decolorization of the triphenylmethane dyes MG and BB, with the results shown in Figure 6C.The crude laccase preparation showed inefficient repeated-batch decolorization of the triphenylmethane dyes MG and BB.With repeated batches, the relative decolorization efficiency of MG and BB decreased considerably.For BB, the relative decolorization efficiencies of batches 2, 3, 4, 5, and 6 were 99.25%, 93.27%, 59.26%, 33.73%, and 20.51%, respectively, while for MG, the relative decolorization efficiencies of batches 2, 3, 4, 5, and 6 were 53.34%, 35.25%, 37.13%, 23.32%, and 20.86%, respectively (Figure 6C).Repeated-batch decolorization was more efficient for BB than for MG.We also tested the laccase activity changes in each round during the repeated-batch decolorization of single dyes (Figure S13).The initial laccase activity of the crude laccase (cycle 0) was set to 100%, and the relative laccase activity of each cycle (%) was calculated.As shown in Figure S13, the overall trend of laccase activity decreased, but it still maintained a certain level during seven consecutive rounds of degradation.As shown in Figure S13A, during the repeated-batch decolorization of the azo dye NC, using the initial laccase activity (cycle 0) as the control (100%), the relative laccase activities of the first, second, third, fourth, fifth, sixth, and seventh cycles were 65.39%, 44.33%, 42.86%, 38.91%, 39.16%, 36.45%, and 27.34%, respectively.As shown in Figure S13F, during the repeated-batch decolorization of the indigo dye IC, the relative laccase activities of the first, second, third, fourth, fifth, sixth, and seventh cycles were 87.44%, 79.44%, 96.81%, 99.63%, 95.96%, 89.27%, and 78.54%, respectively.As shown in Figure S13G, during the repeated-batch decolorization of triphenylmethane dye MG, the relative laccase activities of the first, second, third, fourth, fifth, sixth, and seventh cycles were 93.10%, 79.06%, 82.02%, 74.87%, 67.24%, 52.95%, and 47.78%, respectively.The above results indicated that during the repeated-batch decolorization of single dyes, the laccase activity of the crude enzyme exhibited good stability.Especially during the repeated-batch decolorization of IC, the laccase activity of the crude laccase remained at a very high level throughout the entire seven rounds of reaction.

The Change in Laccase Activity during the Repeated-Batch Decolorization of Two-Dye Mixtures
As shown in Figure S14D, during the repeated-batch decolorization of the RBBR + MG mixture, using the initial laccase activity (cycle 0) as the control (100%), the relative laccase activities of the first, second, third, fourth, fifth, sixth, and seventh cycles were 64.53%, 64.78%, 62.31%, 48.27%, 38.91%, 32.51%, and 31.77%,respectively.As shown in Figure S14F, during the repeated-batch decolorization of the RBBR + IC mixture, the relative laccase activities of the first, second, third, fourth, fifth, sixth, and seventh cycles were 53.45%, 50.24%, 43.35%, 35.96%, 26.35%, 23.15%, and 24.14%, respectively.The above results indicated that during the repeated-batch decolorization of two-dye mixtures, the laccase activity of the crude enzyme also exhibited good stability.
In summary, the crude laccase preparation showed high efficiency in the repeatedbatch decolorization of four-dye mixtures (azo + anthraquinone + triphenylmethane + indigo), and the crude laccase was stable in the repeated-batch degradation of dye mixtures.
2.12.Detoxification of Single and Mixed Dyes 2.12.1.Detoxification of Single Dyes Azo Dye NC We investigated the toxicity of the untreated and decolorized azo dye NC to rice seedling shoot and root growth.
As shown in Figure 8A,B, untreated NC showed strong inhibition of rice seedling shoot and root growth.The shoot lengths of the control (buffer without dye) were 1.24-and 1.26-fold those of the 200 and 600 mg/L untreated NC (200-0 and 600-0), respectively, while the root lengths of the control (buffer without dye) were 1.41-and 1.18-fold those of the 200 and 600 mg/L untreated NC (200-0 and 600-0), respectively.These results indicated that untreated NC was highly toxic to rice seedling shoot and root growth.
As shown in Figure 8A, the shoot lengths with 200 and 600 mg/L decolorized NC (200-1 and 600-1) were 1.38-and 1.24-fold those of 200 and 600 mg/L untreated NC (200-0 and 600-0), respectively, and the differences were highly significant (p < 0.01).Thus, the shoot growth of 200 and 600 mg/L decolorized NC was significantly better than untreated NC, and these results indicated that decolorized NC demonstrated lower toxicity to rice seedling shoot growth compared to untreated NC.Degradation with the crude laccase preparation could reduce the toxicity of the azo dye NC to rice seedling shoot growth.
As shown in Figure 8B, the root lengths of 200 and 600 mg/L decolorized NC (200-1 and 600-1) were 1.41-and 1.40-fold those of 200 and 600 mg/L untreated NC (200-0 and 600-0), respectively.These results indicated that decolorized NC demonstrated lower toxicity to rice seedling root growth compared to untreated NC.Thus, degradation with the crude laccase preparation could reduce the toxicity of azo dye NC to rice seedling root growth.
In summary, the untreated azo dye NC was highly toxic to the root and shoot growth of rice seedlings, while the toxicity of NC was significantly reduced after decolorization with the crude laccase.Decolorization with the crude laccase preparation efficiently reduced the phytotoxicity of the azo dye NC.
seedling shoot and root growth.

Azo Dye RB5
We subsequently investigated the toxicity of the untreated and decolorized azo dye RB5 to rice seedling shoot growth.
As shown in Figure 8C, 600 and 800 mg/L of untreated RB5 showed a strong inhibition of rice seedling shoot growth, and the shoot lengths of the control (buffer without dye) were 1.5-and 1.1-fold those of 600 and 800 mg/L untreated RB5 (600-0 and 800-0), respectively.These results indicated that untreated RB5 was highly toxic to rice seedling shoot growth.
As shown in Figure 8C, the shoot lengths of 600 and 800 mg/L decolorized NC (600-1 and 800-1) were 1.70-and 1.15-fold those of 600 and 800 mg/L untreated RB5 (600-0 and 800-0), respectively, and the differences were highly significant (p < 0.01).These results indicated that decolorized RB5 demonstrated a lower toxicity to rice seedling shoot growth compared to untreated RB5.Decolorization with the crude laccase preparation could efficiently reduce the toxicity of RB5 to rice seedling shoot growth, and decolorization with the crude laccase preparation efficiently reduced the phytotoxicity of the azo dye RB5.

Triphenylmethane Dye MG
We investigated the toxicity of the untreated and decolorized triphenylmethane dye MG to rice seedling root growth.As shown in Figure 8D, 200 and 400 mg/L untreated MG showed strong inhibition of root growth, and inhibition increased with increasing dye concentration, with untreated 400 mg/L MG almost completely inhibiting root growth.The root lengths of the control (buffer without dye) were 1.89-and 7.48-fold those of 200 and 400 mg/L untreated MG (200-0 and 400-0), respectively.These results indicated that untreated MG was highly toxic to the root growth of rice seedlings.
As shown in Figure 8D, the rice seedling root lengths of 200 and 400 mg/L decolorized MG (200-1 and 400-1) were 2.19-and 4.00-fold those of 200 and 400 mg/L untreated MG (200-0 and 400-0), respectively, and the differences were highly significant (p < 0.01), indicating that the root growth of 200 and 400 mg/L decolorized MG was significantly better than that of untreated MG.These results demonstrated that decolorized MG showed a much lower toxicity to rice seedling root growth compared to untreated MG.Thus, decolorization with the crude laccase preparation could efficiently reduce the toxicity of MG to rice seedling root growth.
As shown in Figure 8E, 200 and 600 mg/L untreated MG inhibited wheat seedling root growth.The root lengths of the control (buffer without dye) were 1.09-and 1.71-fold those of 200 and 600 mg/L untreated MG (200-0 and 600-0), respectively, and these results indicated that untreated MG was toxic to wheat seedling root growth.
As shown in Figure 8E, the wheat seedling root lengths of 200 and 600 mg/L decolorized MG (200-1 and 600-1) were 1.22-and 2.55-fold those of 200 and 600 mg/L untreated MG (200-0 and 600-0), and the differences were highly significant (p < 0.01).These results indicated that decolorized MG demonstrated a lower toxicity to wheat seedling root growth compared to untreated MG.Thus, decolorization with the crude laccase preparation could also reduce the toxicity of MG to wheat seedling root growth.
In summary, the untreated triphenylmethane dye MG was highly toxic to the root growth of rice and wheat seedlings, and the toxicity of MG was reduced by decolorization.
Decolorization with the crude laccase preparation efficiently reduced the phytotoxicity of the triphenylmethane dye MG.

Anthraquinone Dye RBBR
We subsequently investigated the toxicity of the untreated and decolorized anthraquinone dye RBBR to rice and wheat seedling shoot growth.
As shown in Figure 9A, compared to the control (buffer without dye), untreated NC + RB5 + RB4 + RBBR showed significantly reduced wheat seedling shoot length, indicating that the untreated dye mixture inhibited the shoot growth of wheat seedlings.These results demonstrated that untreated NC + RB5 + RB4 + RBBR was highly toxic to wheat seedling shoot growth.As shown in Figure 9A, compared to the control (buffer without dye), untreated NC + RB5 + RB4 + RBBR showed significantly reduced wheat seedling shoot length, indicating that the untreated dye mixture inhibited the shoot growth of wheat seedlings.These results demonstrated that untreated NC+RB5+RB4+RBBR was highly toxic to wheat seedling shoot growth.
As shown in Figure 9C, the wheat seedling root lengths of 400 and 800 mg/L decolorized NC + RBBR + BB + IC (400-1 and 800-1) were 1.37-and 1.12-foldthose of 400 and 800 mg/L untreated NC + RBBR + BB + IC (400-0 and 800-0), respectively, and the differences were highly significant (p < 0.01).These results indicated that decolorized NC + RBBR + BB + IC demonstrated a lower toxicity to wheat seedling root growth compared to the untreated dye mixture.Thus, decolorization with the crude laccase preparation could also reduce the toxicity of the mixed dyes NC + RBBR + BB + IC to wheat seedling root growth.
As shown in Figure 9E, the rice seedling shoot length of 100 mg/L decolorized NC + RB5 + RBBR + RB4 + MG + BB + IC (100-1) was 1.32-fold that of the 100 mg/L untreated NC + RB5 + RBBR + RB4 + MG + BB + IC (100-0), and the differences were highly significant (p < 0.01).These results indicated that decolorization with the crude laccase preparation efficiently reduced the toxicity of the seven-dye mixture to the shoot growth of rice seedlings.

Comparison of Crude Laccase from Pleurotus eryngii (Used in This Study) with Other Crude Laccases Reported Previously in Decolorization of Different Dyes
We compared the differences in degradation of the azo dyes between the crude laccase from Pleurotus eryngii and other laccases reported in previous studies.Enayatzamir et al. used a crude laccase preparation (500 U/L) from T. pubescens to decolorize 60 mg/L of RB5 at pH 4.5 and room temperature and achieved a 24 h decolorization efficiency of 22.7% [47].Zeng et al. used a crude laccase preparation from T. trogii to decolorize 18.3 mg/L of RB5 at 30 • C and pH 4.0, and the decolorization efficiency reached 65.4% at 30 min [36].In this study, the crude laccase from Pleurotus eryngii (1 U/mL) was used to degrade 100 mg/L of RB5 at 30 • C, and the 24 h decolorization efficiency reached 72.58%.Even when the concentration of RB5 was increased to 800 mg/L, the 24 h decolorization efficiency was still 50.82%.Compared to previously reported crude laccase preparations [36,47], the crude laccase from Pleurotus eryngii achieved rapid and efficient decolorization of higher azo dye RB5 concentrations with an equal (or lesser) enzyme dosage.Compared to previously reported crude laccase preparations, the crude laccase from Pleurotus eryngii was more efficient for the decolorization of the azo dye RB5, suggesting that the crude laccase from Pleurotus eryngii offers significant potential in the decolorization and degradation of azo dye pollutants.
We also compared the similarities and differences of the crude laccase from Pleurotus eryngii and other laccases reported in previous studies, specifically regarding the degradation of the anthraquinone dye RBBR.Zeng et al. used 2 U/mL of a crude laccase preparation from T. trogii to degrade 50 mg/L of the anthraquinone dye RBBR, and the 6-day decolorization efficiency was 89.4 ± 2.9% [31].In contrast, 1 U/mL of crude laccase from Pleurotus eryngii showed a 24 h decolorization efficiency of 98.73% for 100 mg/L of RBBR, and the dye was almost completely decolorized.Both the crude laccase preparation derived from T. trogii and the crude laccase from Pleurotus eryngii showed efficient RBBR decolorization.However, compared to the crude laccase preparation from T. trogii [31], the crude laccase from Pleurotus eryngii achieved near-complete decolorization (98.73%) with a higher concentration (100 mg/L) of RBBR in a shorter period (24 h) with a lower dosage (1 U/mL).Our results also demonstrated that even when the concentration of RBBR increased to 800 mg/L, the 24 h decolorization efficiency remained at 90.25%.The above comparisons indicated that the crude laccase from Pleurotus eryngii was more efficient in the decolorization of the anthraquinone dye RBBR compared to the crude laccase preparation from T. trogii [31].For the decolorization of the anthraquinone dye RBBR at high concentrations, the crude laccase from Pleurotus eryngii was superior to the crude laccase prepared from T. trogii [31].
For the decolorization of triphenylmethane dyes, Darvishi et al. used a 50 U/L crude laccase preparation from Yarrowia lipolytica to decolorize 33.33 mg/L BB, and the 24 h decolorization efficiency was 50%, while the 24 h decolorization efficiency was 35% for MG [48].Hadibarata et al. showed that a crude laccase preparation from Armillaria sp.F022 was inefficient in the degradation of brilliant green, and the 96 h decolorization efficiency was less than 40% [43].In our study, 1 U/mL crude laccase from Pleurotus eryngii demonstrated 24 h decolorization efficiencies of 99.50% and 85.79% for 100 mg/L BB and MG, respectively, while the 24 h decolorization efficiencies of higher concentrations of BB and MG (800 mg/L) remained at 99.02% and 84.04%, respectively, maintaining a high level of decolorization.These comparisons showed that compared to the Y. lipolytica crude laccase preparation [48], the crude laccase from Pleurotus eryngii was more efficient in the decolorization of the triphenylmethane dyes BB and MG, even at very high concentrations.
For the decolorization of the indigo dye IC, Domınguez et al. achieved nearly 100% decolorization of 150 µM IC after 24 h using 800 U/L crude laccase prepared from T. hirsuta [49].Grassi et al. also used a crude laccase preparation from T. hirsuta to achieve 96% decolorization efficiency of IC after 24 h.These results suggested excellent decolorization of IC with crude laccase preparations [41].In our study, the 24 h decolorization efficiencies of 50, 100, and 200 mg/L IC were 94.81%, 94.93%, and 97.24%, respectively, using 1 U/mL crude laccase from Pleurotus eryngii.These results suggested that the crude laccase from Pleurotus eryngii was very efficient for IC decolorization, which was consistent with the results of previous studies.However, previous studies did not document the decolorization efficiency of IC at higher concentrations with crude laccase preparations.Our study showed that the crude laccase from Pleurotus eryngii was very efficient in the decolorization of IC at high levels, and the 24 h decolorization efficiencies of 400 and 800 mg/L IC were 99.13% and 96.98%, respectively, with the dye nearly completely decolorized.

Possible Reasons for the Different Decolorization Efficiencies of Azo Dyes
Our results showed differences in the decolorization of azo dyes with the crude laccase from Pleurotus eryngii, with the highest decolorization efficiency achieved for NC and relatively low efficiency for RO16, DR81, and RBV5R.This could be explained by the changes in structures of the azo dyes via the laccase degradation of chromophores, where an electron was extracted from the phenolic/naphthol ring to produce a phenoxy radical.Then, a second electron was extracted to generate an aromatic cation, which was subjected to nucleophilic attack by water, resulting in the breakdown of the dye molecule [50,51].The formation of aromatic cations could be promoted by the electron-donating group on the aromatic ring.For azo dyes carrying strong electron-donating hydroxyl groups in the ortho and para positions of the azo bond, the best performance of biochemical decolorization would be achieved.Conversely, the presence of electron-accepting groups on the aromatic ring, such as halogen and nitro, would impede the formation of radical cations, consequently leading to inefficient dye degradation [52].
The chemical structure of a dye will affect its decolorization efficiency with laccase, i.e., decolorization will be affected by the molecular structure of the dye.Compared to dyes with substantial substitutions and high molecular weights, dyes with simple structures and low molecular weights will be amenable to decolorization [53].The high decolorization efficiency of NC relative to RO16, DR81, and RBV5R could be attributed to its simple structure and low molecular weight.
Differences in the electron distribution, charge density, and steric hindrance of the dyes will significantly influence the decolorization efficacy and reaction rate [53,54].The resistance (recalcitrance) of RBV5R to decolorization could be attributed to the presence of stable aromatic rings connected to the azo group.

Possible Reasons for the Different Decolorization Efficiencies of Triphenylmethane Dyes
Our results showed that the decolorization efficiency of the crude laccase from Pleurotus eryngii varied for triphenylmethane dyes with different structures, where the decolorization efficiencies of BB and MG were the highest, while the decolorization efficiency of AF was very low.This could be explained by the central carbon atom of triphenylmethane, which was chemically active and susceptible to replacement by metal atoms.Nevertheless, the arrangement of the three benzene rings in a non-coplanar, three-bladed propeller structure introduced substantial steric hindrance.This hindrance prevented the catalytic center of laccase from accessing the central methine group, thus inhibiting the reaction.Additionally, it was not feasible to achieve simultaneous electron delocalization across all three benzene rings, and electron delocalization with a benzene ring would only occur when the sp 2 hybrid orbital of the carbon anion was appropriately aligned with one of the aromatic systems.This arrangement reduced the catalytic decolorization efficiency of triphenylmethane dyes [55].The substrate structure serves as a key factor affecting degradation efficiency, and in MG and BB, simple groups such as -NH 2 or -OH were directly attached to the aromatic rings, consisting of electron-donating groups that can facilitate substrate degradation.Nevertheless, the high molecular weight of AF rendered it less amenable to binding with laccase, potentially accounting for its low decolorization efficiency.

Explanation of How the Decolorization Activity of Laccase Was Higher at Higher Concentrations of Dyes
Our results suggested that the decolorization activity of laccase was higher at higher concentrations of dyes.A possible explanation for this phenomenon is as follows.The intermediate products of laccase degradation of some dyes can serve as mediators for laccase reactions.Mediators can improve the catalytic reaction ability of laccase and ultimately improve the decolorization efficiency of dyes.Therefore, the higher the dye concentration, the higher the level of mediator produced by the reaction, leading to a higher decolorization activity of laccase and stronger dye decolorization.

Crude Laccase from Pleurotus eryngii Had a High Tolerance to Various Metal Salts and Organic Solvents in the Degradation of Dyes
Dye wastewater originating from the textile industry typically contains high levels of metal salts, which can affect the degradation process by modulating various factors associated with biodegradation.Consequently, the bioremediation of industrial dye wastewater must consider the inhibitory effect of metal salts on the degradation process [56].
Previous studies have predominantly investigated the effects of low concentrations of metal ions on the degradation of dyes with laccase [57,58].In this study, we systematically investigated the impacts of metal ions at high concentrations on the degradation of azo, anthraquinone, triphenylmethane, and indigo dyes through crude laccase from Pleurotus eryngii.Our results showed that the crude laccase was tolerant to high concentrations of SO 4 2− salts such as 800 mM MnSO 4 , MgSO 4 , ZnSO 4 , K 2 SO 4 , and Na 2 SO 4 during the decolorization of different types of dyes.Even at elevated concentrations, these salts did not exert significant impacts on the degradation of dyes.The crude laccase from Pleurotus eryngii demonstrated a high tolerance to a variety of SO 4 2− salts, even at extremely high concentrations, in the degradation of the triphenylmethane dye BB.These findings underscored the advantages of crude laccase from Pleurotus eryngii in the decolorization of dyes.This crude laccase preparation may hold great potential for the efficient treatment of dye wastewater containing metal ions at high concentrations.

Decolorization Kinetics of Mixed Dyes
The kinetics of azo + anthraquinone dye decolorization showed that for NC + RB4, the k value of the azo dye NC increased from 1.84 × 10 −2 min −1 in the control (single-dye decolorization) to 2.00 × 10 −2 min −1 in the mixture, while for NC + RBBR, the k value of the azo dye NC increased from 1.84 × 10 −2 min −1 in the control (single-dye decolorization) to 2.84 × 10 −2 min −1 in the mixture.These results demonstrated that the presence of an anthraquinone dye significantly increased the reaction rate of the azo dye, where an anthraquinone dye in the mixture promoted the decolorization rate of the azo dye.A potential explanation for this phenomenon is as follows.Zeng et al. used a T. trogii crude laccase preparation to decolorize azo and anthraquinone dyes without exogenous redox mediators.The study found that the enzyme could decolorize azo dyes only in the presence of a mediator, such as 20 µM anthraquinone dye, suggesting that anthraquinone dyes played a mediating role in azo dye degradation [31].This was also consistent with our results.During the degradation of mixed azo and anthraquinone dyes with crude laccase from Pleurotus eryngii, the anthraquinone dye promoted the decolorization of the azo dye (increased the decolorization rate of the azo dye).The anthraquinone dye appeared to act as a mediator rather than a catalyst, as it was gradually degraded.Notably, the degradation rate of the azo dye significantly decreased when the concentration of the anthraquinone dye was reduced to zero.This phenomenon could be attributed to the enzymatic oxidation of the dye molecule (M) (anthraquinone dye), which was a substrate of laccase, forming a radical cation (M).This reactive and unstable radical cation returned to its original state (M) via spontaneous degradation, generating enzymatic oxidation products (P + M ) or the oxidization of non-substrate dye molecules (D) (azo dye).The oxidized non-substrate dye molecules would be unstable and undergo degradation, yielding the final product (P D ).The electrons extracted by laccase would then be transferred to molecular oxygen, resulting in the formation of water.According to this reaction mechanism, dye molecules functioning as substrates for laccase could potentially act as mediators, facilitating the degradation of dyes that were not substrates for laccase.
The decolorization kinetics of the azo + indigo dyes (NC + IC) showed that the azo dye in the mixture increased the decolorization rate of the indigo dye; however, the indigo dye inhibited the decolorization rate of the azo dye.This was because the indigo dye was not a direct substrate of laccase, and low-molecular-weight mediators would be added in the degradation of indigo dye, forming highly active and relatively stable intermediates under the action of laccase.These active intermediates could transfer electrons from molecular oxygen to the indigo dye to facilitate its degradation [59].In a mixture of the azo dye NC and the indigo dye IC, NC could function as a low-molecular-weight mediator and promote the decolorization of IC due to its simple structure and the ease of binding to the active center of laccase.When an indigo dye is mixed with an azo dye, polymerization may occur, impeding the binding of the azo dye to the active center of laccase, subsequently reducing the rate of laccase-mediated decolorization of the azo dye.
The decolorization kinetics of the azo + triphenylmethane dyes (NC + MG) showed that the azo dye NC in the mixture promoted the decolorization rate of the triphenylmethane dye MG; however, the triphenylmethane dye inhibited the decolorization of the azo dye.A possible explanation for this phenomenon is as follows.Upon mixing of the azo dye NC and triphenylmethane dye MG, non-covalent interactions, such as van der Waals forces, hydrogen bonds, or π-π stacking, possibly facilitated their association.This association could lead to the formation of complexes or aggregates, complicating the structure of NC and reducing its binding to the active center of laccase, thus reducing the decolorization rate of NC.When NC was bound to the active center of laccase, it could induce conformational changes in the enzyme.These changes could expose the active center and facilitate the association of MG with laccase, thus increasing the decolorization rate of MG.
The kinetics of decolorization of anthraquinone + indigo dyes (RB4 + IC) showed that the anthraquinone dye RB4 greatly increased the decolorization rate of the indigo dye IC.A potential explanation for this phenomenon is as follows.Studies have shown that an anthraquinone dye capable of electron transfer can be directly degraded as a substrate of laccase [60].Furthermore, when laccase and a mediator combine to create a laccasemediator system, the decolorization and degradation of non-laccase substrates, such as indigo dye, could be significantly enhanced.When RB4 was mixed with IC, RB4 could act as a redox mediator to promote the decolorization of IC through the crude laccase preparation.
The decolorization kinetics of indigo + triphenylmethane dyes (IC + CR) showed that the triphenylmethane dye CR greatly promoted the decolorization rate of the indigo dye IC, but the indigo dye inhibited the decolorization rate of the triphenylmethane dye.This phenomenon could be explained by the following mechanism.In the process of laccase-mediated degradation of CR, electron donation through the hydroxyl group within CR led to the formation of a free radical.This free radical acted as an activator to facilitate the laccase-mediated degradation of IC.When laccase was engaged in the degradation of a mixture of indigo dye and triphenylmethane dye, the presence of both dyes collectively increased the overall dye concentration.Therefore, the degradation rate of the triphenylmethane dye in the mixture was significantly reduced due to the limited availability of laccase to target individual dye molecules.
The decolorization kinetics of anthraquinone + triphenylmethane dyes (RBBR + CR) showed that the anthraquinone dye RBBR promoted the decolorization rate of the triphenylmethane dye CR.A possible explanation for this phenomenon is as follows.Similar to the promotion of indigo dye decolorization by anthraquinone dye, the anthraquinone dye could also serve as a mediator in the laccase-mediated degradation of the triphenylmethane dye through electron transfer.When RBBR was mixed with CR, RBBR underwent reduction by the electrons from the medium and promptly transferred these electrons to CR to enhance its decolorization rate.
The decolorization kinetics of triphenylmethane + triphenylmethane dyes (MG + BB, MG + CR, and BB + AF) showed that the decolorization rate of one triphenylmethane dye significantly improved, while the decolorization rate of the other triphenylmethane dye was reduced.A possible explanation is as follows.The generally low degradation efficiency of triphenylmethane dyes with laccase could be attributed to the substantial size of triphenylmethane, which possibly impeded its access to the active center of laccase [61].When a triphenylmethane dye (A) with a relatively simple structure is bound to the active center of laccase, it could induce a conformational change in the enzyme, facilitating the subsequent binding of another triphenylmethane dye (B) with a more complex structure, thus improving the decolorization rate of B. However, competition for laccase between dyes B and A results in inhibited decolorization of dye A.
The decolorization kinetics of azo + azo dyes (NC + RB5) showed that the decolorization rate of both NC and RB5 in the mixture was reduced compared to the single dyes.Possible explanations for this phenomenon are elucidated as follows.In the degradation of NC + RB5, the competitive binding of two structurally similar azo dyes to the active center of laccase diminished the decolorization rate and reduced the k values of both dyes.In addition, Andrea et al. identified the generation of phenolic compounds and an abundance of polymerization products during the laccase-mediated degradation of azo dyes.These products contributed to maintaining the structural integrity of azo groups and participated in connecting bound and unbound dyes [62].The formation of these polymerization products constrained the mobility of dye molecules, subsequently limiting their accessibility to laccase and slowing down the decolorization process.

Possible Reasons for the Decrease in Toxicity of Dyes after Decolorization
Our results suggested that decolorization with the crude laccase preparation efficiently reduced the phytotoxicity of different dyes.The possible mechanism for reducing the toxicity of dyes to plants is as follows.Our results indicated that the untreated dye was highly toxic to plant seedling shoot and root growth.Dyes that have not been decolorized had strong toxicity to the germination and growth of plant seeds.Untreated dyes can have negative effects on various physiological functions of plant seeds.For example, when plant seeds absorb a large amount of azo dyes, the azo dyes will firstly be converted into corresponding aromatic amines through the reduction reaction of -N=N-bonds, and finally form N+ ions through the activation mechanism of aromatic amines, which can bind to the electronegative centers of biological macromolecules (such as N and O atoms in DNA bases and -SH and -OH groups in protein molecules).This can lead to severe inhibition of normal physiological functions such as respiration and nutrient transport in plant seeds.Therefore, untreated dyes had strong toxicity to the germination and growth of plant seeds.
The inhibitory effect of dyes decolorized with laccase on the normal physiological functions of plant seeds, such as respiration and nutrient transport, was weakened, thereby reducing the toxicity of dyes to plant seedling shoot and root growth.

Advantages and Practical Application Value of Crude Laccase Preparation for Decolorization and Detoxification of Dye Pollutants
Our findings highlighted the substantial potential of crude laccase from Pleurotus eryngii for the efficient decolorization and detoxification of both single and mixed dyes with varying structures.The use of a crude laccase preparation for the decolorization and detoxification of dye pollutants had the following advantages and practical applications.Zeng et al. showed that the degradation efficiency of a crude laccase preparation was higher than that of purified laccase, which could be explained by the fact that fungi secrete laccases along with redox mediators to facilitate the decolorization of dyes [36].A crude laccase preparation can efficiently decolorize different types of dyes without requiring additional mediators, as these molecules will be inherently present in the crude laccase preparation.Therefore, the use of a crude laccase preparation can substantially reduce the overall cost of laccase-mediated dye-pollutant degradation.Furthermore, compared to the purification of laccase, the preparation of crude laccase offers a simple process that eliminates the need for a series of complex purification steps.This approach will eliminate the requirement for methods and materials for laccase separation and purification.Consequently, the costs associated with using laccase in pollutant treatment will be greatly reduced.Our results showed that the crude laccase from Pleurotus eryngii could efficiently decolorize and detoxify single and mixed dyes, greatly enhancing the feasibility and practicality of large-scale laccase applications in dye-pollutant treatment.In summary, the direct use of a crude laccase preparation for dye wastewater treatment yielded satisfactory results while substantially reducing the overall cost.This approach offers significant promise for the practical application of laccase in pollutant bioremediation.
Furthermore, our investigation revealed the efficacy of crude laccase from Pleurotus eryngii in the repeated-batch decolorization of single and mixed dyes, such as azo + triphenylmethane dyes (RB5 + BB) and azo + indigo dyes (NC + IC and RB5 + IC).The crude laccase from Pleurotus eryngii had a strong continuous batch decolorization ability for single and mixed dyes.In the practical application of laccase for the efficient treatment of environmental pollutants and the reusability and stability of laccase serve as pivotal factors in cost reduction for industrial applications.In this work, we systematically investigated the repeated-batch decolorization of single and mixed dyes with crude laccase from Pleurotus eryngii.After several batches, the crude laccase preparation retained its decolorization activity for both single and mixed dyes.Our results showed that the crude laccase from Pleurotus eryngii was stable and reusable in repeated-batch degradation of dyes.These characteristics significantly reduced the cost of crude laccase from Pleurotus eryngii for practical application, markedly enhancing its value in real-world applications.This remarkable attribute distinguished the use of crude laccase from Pleurotus eryngii as a cost-effective and highly feasible solution for the efficient treatment of dye pollutants.

Strain and Culture Media
Pleurotus eryngii ACCC 50757 was obtained from the Agricultural Culture Collection of China (ACCC) and a GYP medium was used for laccase production (20 g/L of glu-cose, 5 g/L of yeast extract, 5 g/L of peptone, 1 g/L of MgSO 4 •7H 2 O, and 0.002 g/L of CuSO 4 •5H 2 O).

Induction of Laccase Production and Preparation of Crude Laccase
Pleurotus eryngii was inoculated onto a PDA medium and incubated at 28 • C for 7 days.Ten mycelial plugs were then collected using a puncher and transferred with an inoculation needle into the PDB medium.The inoculated medium was allowed to stand for 12 h and then incubated at 28 • C and 180 r/min for 7 days to obtain uniform mycelial pellets.
The mycelial pellets (5 mL) were transferred from PDB to GYP medium (100 mL) and incubated at 28 • C and 180 r/min for 3 days.Then, CuSO 4 at a final concentration of 2 mM was added to induce laccase production, and incubation was continued at 28 • C and 180 r/min.Samples were collected every day to determine the laccase activity.The culture broth was collected under maximum laccase activity and filtered through eight layers of gauze to remove the mycelia, with the filtrate stored at −20 • C overnight.After thawing at room temperature, the polysaccharides were removed with precipitation and filtration to obtain the crude laccase preparation.The polysaccharides precipitated included glucan, mannan, etc. Laccase activity was determined with 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per min.

Decolorization of the Dyes with Crude Laccase from Pleurotus eryngii and Calculation of the Decolorization Efficiency
Table 3 presents some information on the dyes used in this study, including azo dyes, anthraquinone dyes, triphenylmethane dyes, and indigo dye.

Induction of Laccase Production and Preparation of Crude Laccase
Pleurotus eryngii was inoculated onto a PDA medium and incubated at 28 °C for 7 days.Ten mycelial plugs were then collected using a puncher and transferred with an inoculation needle into the PDB medium.The inoculated medium was allowed to stand for 12 h and then incubated at 28 °C and 180 r/min for 7 days to obtain uniform mycelial pellets.
The mycelial pellets (5 mL) were transferred from PDB to GYP medium (100 mL) and incubated at 28 °C and 180 r/min for 3 days.Then, CuSO4 at a final concentration of 2 mM was added to induce laccase production, and incubation was continued at 28 °C and 180 r/min.Samples were collected every day to determine the laccase activity.The culture broth was collected under maximum laccase activity and filtered through eight layers of gauze to remove the mycelia, with the filtrate stored at −20 °C overnight.After thawing at room temperature, the polysaccharides were removed with precipitation and filtration to obtain the crude laccase preparation.The polysaccharides precipitated included glucan, mannan, etc. Laccase activity was determined as described in the literature [46].Laccase activity was determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min.

Decolorization of the Dyes with Crude Laccase from Pleurotus eryngii and Calculation of the Decolorization Efficiency
Table 3 presents some information on the dyes used in this study, including azo dyes, anthraquinone dyes, triphenylmethane dyes, and indigo dye.

Induction of Laccase Production and Preparation of Crude Laccase
Pleurotus eryngii was inoculated onto a PDA medium and incubated at 28 °C for 7 days.Ten mycelial plugs were then collected using a puncher and transferred with an inoculation needle into the PDB medium.The inoculated medium was allowed to stand for 12 h and then incubated at 28 °C and 180 r/min for 7 days to obtain uniform mycelial pellets.
The mycelial pellets (5 mL) were transferred from PDB to GYP medium (100 mL) and incubated at 28 °C and 180 r/min for 3 days.Then, CuSO4 at a final concentration of 2 mM was added to induce laccase production, and incubation was continued at 28 °C and 180 r/min.Samples were collected every day to determine the laccase activity.The culture broth was collected under maximum laccase activity and filtered through eight layers of gauze to remove the mycelia, with the filtrate stored at −20 °C overnight.After thawing at room temperature, the polysaccharides were removed with precipitation and filtration to obtain the crude laccase preparation.The polysaccharides precipitated included glucan, mannan, etc. Laccase activity was determined as described in the literature [46].Laccase activity was determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min.

Decolorization of the Dyes with Crude Laccase from Pleurotus eryngii and Calculation of the Decolorization Efficiency
Table 3 presents some information on the dyes used in this study, including azo dyes, anthraquinone dyes, triphenylmethane dyes, and indigo dye.

Induction of Laccase Production and Preparation of Crude Laccase
Pleurotus eryngii was inoculated onto a PDA medium and incubated at 28 °C for 7 days.Ten mycelial plugs were then collected using a puncher and transferred with an inoculation needle into the PDB medium.The inoculated medium was allowed to stand for 12 h and then incubated at 28 °C and 180 r/min for 7 days to obtain uniform mycelial pellets.
The mycelial pellets (5 mL) were transferred from PDB to GYP medium (100 mL) and incubated at 28 °C and 180 r/min for 3 days.Then, CuSO4 at a final concentration of 2 mM was added to induce laccase production, and incubation was continued at 28 °C and 180 r/min.Samples were collected every day to determine the laccase activity.The culture broth was collected under maximum laccase activity and filtered through eight layers of gauze to remove the mycelia, with the filtrate stored at −20 °C overnight.After thawing at room temperature, the polysaccharides were removed with precipitation and filtration to obtain the crude laccase preparation.The polysaccharides precipitated included glucan, mannan, etc. Laccase activity was determined as described in the literature [46].Laccase activity was determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min.

Decolorization of the Dyes with Crude Laccase from Pleurotus eryngii and Calculation of the Decolorization Efficiency
Table 3 presents some information on the dyes used in this study, including azo dyes, anthraquinone dyes, triphenylmethane dyes, and indigo dye.

Induction of Laccase Production and Preparation of Crude Laccase
Pleurotus eryngii was inoculated onto a PDA medium and incubated at 28 °C for 7 days.Ten mycelial plugs were then collected using a puncher and transferred with an inoculation needle into the PDB medium.The inoculated medium was allowed to stand for 12 h and then incubated at 28 °C and 180 r/min for 7 days to obtain uniform mycelial pellets.
The mycelial pellets (5 mL) were transferred from PDB to GYP medium (100 mL) and incubated at 28 °C and 180 r/min for 3 days.Then, CuSO4 at a final concentration of 2 mM was added to induce laccase production, and incubation was continued at 28 °C and 180 r/min.Samples were collected every day to determine the laccase activity.The culture broth was collected under maximum laccase activity and filtered through eight layers of gauze to remove the mycelia, with the filtrate stored at −20 °C overnight.After thawing at room temperature, the polysaccharides were removed with precipitation and filtration to obtain the crude laccase preparation.The polysaccharides precipitated included glucan, mannan, etc. Laccase activity was determined as described in the literature [46].Laccase activity was determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per min.

Decolorization of the Dyes with Crude Laccase from Pleurotus eryngii and Calculation of the Decolorization Efficiency
Table 3 presents some information on the dyes used in this study, including azo dyes, anthraquinone dyes, triphenylmethane dyes, and indigo dye.Indigo dye Indigo Carmine IC 610 The 1 mL decolorization system included the crude laccase preparation (1 U/mL), dyes at final concentrations of 50, 100, 200, 400, and 800 mg/L (diluted from 5 g/L stock solutions), and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The acetic acid-so-The 1 mL decolorization system included the crude laccase preparation (1 U/mL), dyes at final concentrations of 50, 100, 200, 400, and 800 mg/L (diluted from 5 g/L stock solutions), and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The acetic acidsodium acetate buffer (pH 5.0) was used to dissolve the stock solution of dyes.The mixture was thoroughly mixed and then incubated in a water bath at 30 • C for 24 h in the dark.The change in the maximum absorbance value before and after 24 h of dye degradation was determined using a UV-visible spectrophotometer.The decolorization efficiency was calculated as follows: decolorization efficiency (%) = (A 0 − A t )/A 0 × 100%, where A 0 is the initial absorbance and A t denotes the absorbance after 24 h of decolorization.

Time Courses of Decolorization of Dyes at Different Concentrations
The 1 mL decolorization system consisted of the crude laccase preparation (1 U/mL), dyes at final concentrations of 50, 100, 200, 400, and 800 mg/L (diluted from 5 g/L stock solutions), and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The mixture was thoroughly mixed and incubated in a water bath at 30 • C in the dark, with samples collected at 0, 0.5, 1, 2, 3, 6, 9, 12, and 24 h.The absorption peaks were observed using a UV-visible spectrophotometer and the absorbance at the maximum absorption wavelength was measured.The decolorization efficiency was calculated as described in Section 4.3, and the time course of dye decolorization was plotted according to the decolorization efficiency at different time points.

Kinetic Equations of Single-Dye Degradation
The 5 g/L stock solution was diluted to 10, 20, 50, 80, 100, 120, 160, and 200 mg/L.The absorbance A at the maximum absorption wavelength of the standard dye solution was determined, and the A-c curve was plotted.The absorbance at the maximum absorption wavelength of each concentration of dye standards at 10, 20, 50, 80, 100, 120, 160, and 200 mg/L was measured using a UV-visible spectrophotometer.A straight line is plotted for the dye concentration c against absorbance A, and the slope K of the line is the molar absorption coefficient of each dye.The values of the coefficients are shown in Table S1.With a molar absorption coefficient K of the dye, the dye concentration at time t (C t ) of decolorization can be calculated from the time-course curve.For the first-order reaction, the integral form of the reaction rate equation can be expressed by ln C t C 0 = −kt where C 0 is the initial dye concentration, C t is the dye concentration at time t, and k denotes the reaction rate constant (min −1 ), which directly reflects the rate of reaction.The ln c-t curve was plotted with t on the horizontal axis and ln C t C 0 on the vertical axis, and k was subsequently calculated.

Decolorization of Mixed Dyes: Determination of Decolorization Efficiency of Each Dye in the Mixtures
The 1 mL dye mixture decolorization system included 1 U/mL of the crude laccase preparation, two-dye mixtures (each with final concentrations of 50, 100, 200, 400, and 800 mg/L), and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The reaction system was thoroughly mixed and incubated in a water bath at 30 • C for 24 h in the dark.The absorption peaks were observed using a UV-visible spectrophotometer and the absorbance at the maximum absorption wavelength was measured to calculate the decolorization efficiency of each dye, as described in Section 4.3.

Decolorization Kinetics of the Mixed Dyes
During the degradation of the dyes, samples were collected at 0, 10, 20, 30, 40, 50, 60, 90, 120, and 180 min.The absorbance at the maximum absorption wavelength was measured using a UV-visible spectrophotometer, and the ln c-t curve was plotted, with the k value calculated as described in Section 4.5.

Effects of Metal Salts at Different Concentrations on Decolorization
The decolorization system of the experimental group included 1 U/mL of the crude laccase preparation, 100 mg/L of dye, metal salts (MnSO 4 , MnCl 2 , MgSO 4 , MgCl 2 , ZnSO 4 , ZnCl 2 , K 2 SO 4 , KCl, CuSO 4 , CuCl 2 , Na 2 SO 4 , NaCl, Al 2 (SO 4 ) 3 , AlCl 3 , CdSO 4 , CdCl 2 , CaCl 2 , CoCl 2 , NiCl 2 , and LiCl at final concentrations of 50, 100, 200, 400, and 800 mM), and 100 mM of acetic acid-sodium acetate buffer (pH = 5.0).The control group consisted of a 1 mL decolorization system without metal salts.The reaction system was thoroughly mixed and incubated in a water bath at 30 • C for 24 h in the dark, and the absorbance of each dye at the maximum absorption wavelength was measured at 0 and 24 h.The decolorization efficiency was calculated as described in Section 4.3.

Effects of Organic Solvents at Different Concentrations on Decolorization
The decolorization system of the experimental group included 1 U/mL of the crude laccase preparation, 100 mg/L of dye, organic solvents (glycerol, butanediol, propanediol, ethylene glycol, methanol, ethanol, isopropanol, acetonitrile, acetone, DMSO, and DMF at final concentrations of 10%, 20%, 40%, 50%, 70%, and 80%, v/v), and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The control group consisted of a 1 mL decolorization reaction system without organic solvents, and the reaction system was thoroughly mixed and incubated in a water bath at 30 • C for 24 h in the dark.The absorbance of each dye at the maximum absorption wavelength was measured at 0 and 24 h, and the decolorization efficiency was calculated as described in Section 4.3.The decolorization system of the experimental group included 100 mg/L of dye and 100 mM of acetic acid-sodium acetate buffer (pH = 5.0).The reaction system was thoroughly mixed and the initial absorbance A 01 was measured.Subsequently, 0.5 U/mL of the crude laccase preparation was added and thoroughly mixed.The reaction system was incubated in a water bath at 30 • C for 24 h in the dark, the absorbance A t1 was measured, and the 24 h decolorization efficiency of the first-cycle reaction was calculated as described in Section 4.3.The decolorization efficiency of the first batch was set to 100% for calculation of the relative decolorization efficiency of subsequent batches.After the first decolorization batch, 100 mg/L of the same dye was added to the decolorization system and the absorbance A 02 was measured.The reaction system was thoroughly mixed and incubated at 30 • C for 24 h.Then, the absorbance A t2 was measured to calculate the relative decolorization efficiency.Seven consecutive batches of decolorization were carried out in this study.

Repeated-Batch Decolorization of Mixed Dyes
The decolorization system of the experimental group included mixed dyes (each with a final concentration of 100 mg/L) and 100 mM acetic acid-sodium acetate buffer (pH = 5.0).The mixture was thoroughly mixed and the initial absorbance A 01 of each dye was measured.The remaining steps were the same as in Section 4.10.1.Twenty wheat or rice seeds of the same size were selected and placed in a Petri dish.Then, 5 mL reaction systems for the experimental group, which included single dyes (final concentrations of 200, 400, 600, and 800 mg/L), 0.5 U/mL of crude laccase preparation, and 100 mM of acetic acid-sodium acetate buffer (pH = 5.0), were obtained.Laccase was not added to the control group.The mixture was then incubated in a water bath at 30 • C for 24 h in the dark and then added to the Petri dish with the seeds.The seeds were incubated at 28 • C for 3-5 days and the lengths of the shoots and roots were measured.The control acetic acid-sodium acetate buffer (pH = 5.0) (not adding dye).Differences in root and shoot length between the experimental groups (dyes with decolorization) and control group (dyes without decolorization) were analyzed using GraphPad Prism 8 software, with p < 0.01 indicating a highly significant difference (**) and p < 0.05 a significant difference (*).

Detoxification of the Mixed Dyes
Twenty wheat or rice seeds of the same size were selected and placed in a petri dish.The 5 mL reaction system of the experimental group included mixed dyes (each at final concentrations of 200, 400, 600, and 800 mg/L), 0.5 U/mL of crude laccase preparation, and 100 mM of acetic acid-sodium acetate buffer (pH = 5.0).The remaining steps were the same as in Section 4.11.1.

Statistical Analysis
All measurements were repeated three times, with the data expressed as mean ± standard deviation.The significance of differences between the mean values was determined using a t test or analysis of variance.All data were analyzed using GraphPad Prism 8 software.Differences in the mean values between groups were assessed using the t test, with p < 0.01 indicating a highly significant difference (**) and p < 0.05 a significant difference (*).

Conclusions
In this study, we prepared a crude laccase from a fungal strain Pleurotus eryngii with high laccase production.On this basis, we systematically investigated the decolorization efficacy and reaction rates of single dyes in four structural classes.The crude laccase from Pleurotus eryngii showed efficient decolorization of single dyes (azo, anthraquinone, triphenylmethane, and indigo dyes).We also investigated the tolerance of the crude laccase from P. eryngii to metal salts and organic solvents at different concentrations during the degradation of single dyes.Our results showed that the crude laccase from P. eryngii was highly tolerant to SO 4 2− salts and organic solvents, such as glycerol and ethylene glycol, during the decolorization of various types of dyes.Based on the degradation of single dyes, we further investigated the decolorization of mixed dyes, and the crude laccase from P. eryngii was found to be efficient in the decolorization of mixed dyes (mixtures of various types of dyes).The crude laccase from P. eryngii was also efficient in the repeated-batch decolorization of single dyes as well as two-and four-dye mixtures.The crude laccase preparation showed a high stability and reusability in repeated-batch decolorization.The crude laccase from P. eryngii was efficient in the detoxification of different types of single dyes and mixed dyes containing different types of dyes, and the phytotoxicity of decolorized dyes (single and mixed dyes) was significantly reduced.In conclusion, our results indicated that the crude laccase from Pleurotus eryngii had unique advantages and properties.These included efficient decolorization of a broad spectrum of dyes, efficient decolorization of mixed dyes (mixtures of various types of dyes), strong tolerance to different metal salts and organic solvents during dye degradation, repeated-batch decolorization of single and mixed dyes with high enzyme stability, and efficient detoxification of the phytotoxicity of single and mixed dyes.The crude laccase from Pleurotus eryngii offers great potential for the bioremediation and efficient treatment of pollutants containing single and mixed dyes with various molecular structures.Our results offer valuable insight and practical guidance for using crude laccase from Pleurotus eryngii in the efficient treatment of industrial wastewater containing dyes with diverse molecular structures.

Figure 1 .
Figure 1.The production of laccase by Pleurotus eryngii after the addition of 2 mM CuSO4 as the inducer.A 2 mM solution of CuSO4 was added to the fungal culture on the third day.

Figure 1 .
Figure 1.The production of laccase by Pleurotus eryngii after the addition of 2 mM CuSO 4 as the inducer.A 2 mM solution of CuSO 4 was added to the fungal culture on the third day.

Figure 4 .
Figure 4. Effects of metal salts at different concentrations on the degradation of the azo dye NC (New Coccine) with the crude laccase from P. eryngii.The metal salts used in this study were MnSO4, MnCl2, MgSO4, MgCl2, ZnSO4, ZnCl2, K2SO4, KCl, CuSO4, CuCl2, Na2SO4, NaCl, Al2(SO4)3, AlCl3, CdSO4, and CdCl2.The control group consisted of a 1 mL decolorization system without metal salts.(A) The final concentration of metal salt was 50 mM.(B) The final concentration of metal salt was 100 mM.(C) The final concentration of metal salt was 200 mM.(D) The final concentration of metal salt was 400 mM.(E) The final concentration of metal salt was 600 mM.(F) The final concentration of metal salt was 800 mM.Red color: control, no metal salts added.Grey color: SO4 2− salts.Black color: Cl − salts.

Figure 4 .
Figure 4. Effects of metal salts at different concentrations on the degradation of the azo dye NC (New Coccine) with the crude laccase from P. eryngii.The metal salts used in this study were MnSO 4 , MnCl 2 , MgSO 4 , MgCl 2 , ZnSO 4 , ZnCl 2 , K 2 SO 4 , KCl, CuSO 4 , CuCl 2 , Na 2 SO 4 , NaCl, Al 2 (SO 4 ) 3 , AlCl 3 , CdSO 4 , and CdCl 2 .The control group consisted of a 1 mL decolorization system without metal salts.(A) The final concentration of metal salt was 50 mM.(B) The final concentration of metal salt was 100 mM.(C) The final concentration of metal salt was 200 mM.(D) The final concentration of metal salt was 400 mM.(E) The final concentration of metal salt was 600 mM.(F) The final concentration of metal salt was 800 mM.Red color: control, no metal salts added.Grey color: SO 4 2− salts.Black color: Cl − salts.

2. 10 .
Effects of Organic Solvents at Different Concentrations on the Degradation of Dyes: Organic Solvent Tolerance 2.10.1.Effects of Organic Solvents at Different Concentrations on the Degradation of the Azo Dye NC

Figure 5 .
Figure 5. Effects of organic solvents at different concentrations on the degradation of the azo dye NC (New Coccine) with the crude laccase from P. eryngii.The organic solvents used in this study were glycerol, butanediol, propylene glycol, ethylene glycol, methanol, ethanol, isopropanol, acetonitrile, acetone, DMSO, and DMF.The control group consisted of a 1 mL decolorization system without metal salts.(A) The final concentration of organic solvent was 10% (v/v).(B) The final concentration of organic solvent was 20% (v/v).(C) The final concentration of organic solvent was 40% (v/v).(D) The final concentration of organic solvent was 50% (v/v).(E) The final concentration of organic solvent was 70% (v/v).(F) The final concentration of organic solvent was 80% (v/v).Red color: control, no organic solvents added.Black color: different organic solvents added.

Figure 5 .
Figure 5. Effects of organic solvents at different concentrations on the degradation of the azo dye NC (New Coccine) with the crude laccase from P. eryngii.The organic solvents used in this study were glycerol, butanediol, propylene glycol, ethylene glycol, methanol, ethanol, isopropanol, acetonitrile, acetone, DMSO, and DMF.The control group consisted of a 1 mL decolorization system without metal salts.(A) The final concentration of organic solvent was 10% (v/v).(B) The final concentration of organic solvent was 20% (v/v).(C) The final concentration of organic solvent was 40% (v/v).(D) The final concentration of organic solvent was 50% (v/v).(E) The final concentration of organic solvent was 70% (v/v).(F) The final concentration of organic solvent was 80% (v/v).Red color: control, no organic solvents added.Black color: different organic solvents added.

45 Figure 6 .
Figure 6.Repeated-batch decolorization of single dyes with the crude laccase from P. eryngii.The decolorization efficiency of the first batch was set to 100% for calculation of the relative decolorization efficiency of subsequent batches.Seven consecutive batches of decolorization were carried out in this study.(A) Repeated-batch decolorization of the azo dyes NC and RB5.(B) Repeated-batch decolorization of the anthraquinone dyes RBBR and RB4.(C) Repeated-batch decolorization of the triphenylmethane dyes MG and BB.(D) Repeated-batch decolorization of the indigo dye IC.

Figure 6 .
Figure 6.Repeated-batch decolorization of single dyes with the crude laccase from P. eryngii.The decolorization efficiency of the first batch was set to 100% for calculation of the relative decolorization efficiency of subsequent batches.Seven consecutive batches of decolorization were carried out in this study.(A) Repeated-batch decolorization of the azo dyes NC and RB5.(B) Repeated-batch decolorization of the anthraquinone dyes RBBR and RB4.(C) Repeated-batch decolorization of the triphenylmethane dyes MG and BB.(D) Repeated-batch decolorization of the indigo dye IC.

Table 1 .
The reaction rate constants and R 2 values for different single dyes.

Table 3 .
Full names, abbreviations, structures, and maximum absorption wavelengths of different dyes used in this study.

Table 3 .
Full names, abbreviations, structures, and maximum absorption wavelengths of different dyes used in this study.

Table 3 .
Full names, abbreviations, structures, and maximum absorption wavelengths of different dyes used in this study.

Table 3 .
Full names, abbreviations, structures, and maximum absorption wavelengths of different dyes used in this study.

Table 3 .
Full names, abbreviations, structures, and maximum absorption wavelengths of different dyes used in this study.