Textile azo dyes discolouration using spent mushroom substrate: enzymatic degradation and adsorption mechanisms

ABSTRACT This study evaluated the adsorption and enzymatic degradation of azo dyes when using SMS. The laccase present in the SMS was characterised, and the maximum activity was obtained at pH 2, a temperature of 45°C, a Michaelis–Menten constant (Km) of 0.264 mM, and a maximum reaction rate (Vmax) of 117.95 µmol L−1 min−1. The presence of NaCl at 5 mM inhibited enzyme activity while no inhibition was observed by Na2SO4, typically found in textile wastewater. The maximum dye adsorption (57.22%) was achieved at pH 8.0, 25°C, and 100 g L−1 of SMS while the maximum enzymatic degradation (14.18%) was obtained under the same conditions, except at pH 4.0. The enzymes laccase, lignin peroxidase, and manganese peroxidase trapped in the SMS resulted in higher dye discolouration when compared to that extracted with aqueous solution, meaning that SMS has strong adsorption capacity and is a natural immobilisation matrix, which improves the enzymatic degradation of the dyes. Thus, SMS can be used in the treatment of textile effluents for dye removal by simultaneous mechanisms of adsorption and enzymatic degradation, with reduction of environmental impacts for SMS disposal and reduction of the costs associated with commercial enzymes and adsorbents. GRAPHICAL ABSTRACT


Introduction
Due to the use of large volumes of water and chemicals used during processing, textile industries are responsible for the generation of effluents with high polluting potential, mainly associated with the high concentration of dyes. On average, 20%-50% of the dyes applied in the dyeing process are eliminated in the effluents [1]. Textile dyes are highly perceptible and affect the aesthetics of the environment, reducing solar light penetration into the water column, disturbing biological cycles, and damaging the aquatic ecological community. These compounds also have carcinogenic and/or mutagenic characteristics and can cause disturbances in organisms, resulting in risks to human health [2].
Textile dyes have complex chemical structures and are stable to light, temperature, and microbial attack, configuring themselves as highly recalcitrant compounds. Thus, conventional effluent treatment processes have a limited capacity to remove dyes, requiring an excessive use of chemicals to achieve the levels of discolouration required by environmental legislation, generating large amounts of contaminated sludge, which results in high costs [3]. In addition, anaerobic treatment results in the formation of aromatic amines, which are more toxic substances than the dye itself [4].
The biodegradation of textile dyes by ligninolytic enzymes produced by white rot fungi has been highlighted due to the enzymes' ability to completely mineralise these compounds. However, the industrial production and purification of enzymes is a time-consuming and costly process, resulting in expensive enzymatic formulations. The cost of enzymes is one of their main bottlenecks for environmental application, requiring the search for readily available and cheap sources of enzymes. In this respect, the residual substrate from mushroom production is highlighted [5].
Mushrooms are grown on lignocellulosic substrates such as straw, wheat bran, rice bran, sugarcane bagasse, coffee straw, banana leaves, and sawdust [6]. After harvesting, the residual substrate is known as spent mushroom substrate (SMS), which consists of the remaining biomass, mycelium, and extracellular enzymes released by the fungi during their growth [7]. The white rot basidiomycetes class includes several species of edible mushrooms, such as those belonging to the Pleurotus genus, which occupy second position in world production [8].
The lack of adequate and profitable destination for SMS contributes to uncontrolled disposal, which can cause negative impacts on ecosystems [9]. Considering that 1 kg of cultivated mushrooms generate approximately 5 kg of SMS [10] and that mushroom production is growing, applications of SMS with reduced environmental impact must be studied and developed.
The ligninolytic enzymes present in the SMS mainly laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) have the ability to degrade phenolic compounds which have a structure similar to lignin, such as textile dyes [11]. Laccase is the key enzyme involved in degradation processes. It has low substrate specificity, and directly catalyses the oxidation of phenolic compounds with the simultaneous reduction of molecular oxygen in water, without the need for H 2 O 2 [12]. SMS has been reported as an adsorbent due to the abundant hydroxyl, carbonyl, and carboxyl groups present on its surface [13]. The cultivation of the fungus results in increased surface area and active sites, therefore, it has a greater adsorption capacity compared to the substrate before cultivation [14].
Regarding the application of the SMS in the removal of textile dyes, studies were carried out with crude enzyme broth extracted from SMS [15][16][17][18] under fungus growing conditions [19,20], and as an adsorbent [13,14,21]. The application of SMS in the treatment of textile wastewater proves to be a sustainable and advantageous alternative as it provides that both the adsorption and enzymatic degradation processes contribute to the removal of dyes, in addition to avoiding additional costs with enzymatic extraction and adsorbent preparation. Di Gregorio et al. [22] and Papinutti and Forchiassin [23] carried out preliminary assessments in this perspective, but thorough analyses of the simultaneous performance of these processes in the presence of a mixture of dyes and auxiliary products, and the characterisation of enzymes present in the SMS are still not reported in the literature.
The feasibility of wastewater treatment processes considers cost-effective and sustainable technologies, and enzymatic biodegradation has been claimed to meet these conditions. Since wastewater treatment does not require purified enzymes as is the case in food or pharmaceutical applications, the use of natural enzyme sources such as SMS is a promising alternative. In addition, natural enzyme sources could contain mediators required for enzyme hydrolysis and oxidation processes, such as syringaldazine for laccase [24].
The objective of the present study was to analyze the removal of dyes present in textile wastewater using SMS, and to evaluate the contribution of the adsorption and enzymatic degradation processes. The laccase enzyme from the SMS was characterised, and a factorial experiment was carried out to preliminarily check the best conditions for the discolouration of textile dyes by each process involved.

Spent mushroom substrate
The SMS used in the study was obtained from the production of Pleurotus ostreatus, and consisted of sawdust and wheat bran, supplemented with calcium carbonate. Post-harvest mushroom cultivation blocks were collected after 114 days of the fungi inoculation and 75 days after the second mushroom production cycle, when the SMS is considered a waste and no longer useful for the producing company. The SMS was kept refrigerated at 5°C to preserve the enzymes and the material structure until being used in the experiments. Prior to the experiments, the SMS blocks were broken down and homogenised manually so that the samples used were representative.

SMS characterisation
The morphological analysis of the SMS prior to the dye removal experiments was carried out using scanning electron microscopy analysis (SEM, JEOL JSM-6390LV, USA), operating at 10 kV with magnifications ranging from 200 up to 1000x at a resolution of 1μm and the qualitative composition by X-ray diffraction analysis (EDS) with a NORAN Six X-ray microanalysis system.
The SMS surface chemistry was analyzed using Fourier transform infrared spectroscopy (FTIR) using an Agilent Technologies-Cary 660 spectrometer in the wavelength range of 400-4000 cm −1 , and a resolution of 4 cm −1 .
To assess changes in the functional groups of the synthetic textile effluent after treatment with SMS, FTIR analysis was performed on raw effluent (50 mg L −1 ), under the conditions determined to be most favourable. These liquid samples were oven dried for three days at 50°C and the resulting solid was analyzed on an Agilent Technologies-Cary 660 spectrometer in the wavelength range of 400-4000 cm −1 , and at a resolution of 4 cm −1 .

Enzyme assays
The enzymes present in the SMS were extracted with 0.1 M sodium acetate buffer (pH 5.0) and 155 g L −1 of SMS (wet weight), then stirred at 100 rpm with a magnetic stirrer for 1 h at room temperature. Subsequently, the mixture was centrifuged at 11000 rpm for 5 min, and the supernatant was used in the enzyme characterisation.
The laccase activity was colorimetrically determined by the oxidation of 2,2 -azinobis (3-ethylbenzathiazoline-6-sulfonic acid) (ABTS) substrate. The reaction mixture contained 4 mL of ABTS (1.8 mM), 0.5 mL of sodium acetate buffer (0.1 M; pH 5), and 0.5 mL of the sample. The reaction mixture was kept at 40°C for 5 min in a water bath and the resulting absorbance was spectrophotometrically measured at 420 nm (Hach, DR 3600) [26]. Two control samples were prepared, one replacing the sample with the buffer and the other replacing ABTS with the buffer.
LiP activity was determined by the oxidation of veratryl alcohol to veratraldehyde by the enzyme. The reaction mixture contained 0.5 mL of veratryl alcohol (10 mM), 0.5 mL of H 2 O 2 (0.4 mM), 1 mL of sodium tartrate buffer (100 mM; pH 3), and 0.5 mL of the sample. The reaction mixture was kept in a water bath at 30°C for 5 min and the resulting absorbance was spectrophotometrically measured at 310 nm (Hach, DR 5000) [27,28]. Two control samples were prepared, one replacing the sample with the buffer and the other replacing veratryl alcohol with the buffer.
MnP activity was obtained by the formation of the Mn (III)-malonate complex. The reaction mixture contained 1 mL of MnSO 4 (4 mM), 0.5 mL of H 2 O 2 (0.4 mM), 1 mL of malonate buffer (20 mM; pH 5), and 1 mL of the sample. The reaction mixture was kept in a water bath at 30°C for 5 min and the resulting absorbance was spectrophotometrically measured at 270 nm (Hach, DR 5000) [29]. Two control samples were prepared, one replacing the sample with the buffer and the other replacing MnSO 4 with the buffer.
The enzyme activity was expressed in international units (U), where one unit of enzyme activity is sufficient to oxidise 1 μmol of the substrate per minute [30].

Enzyme characterisation
Determination of the optimum pH was done so using buffer solutions at pH 2, 4, 6, and 8 using Mcllvaine buffer (0.1 M citric acid and 0.2 M disodium phosphate) and at pH 10 using borate buffer (3.092 g of boric acid, 3.728 g of potassium chloride, and 2.34 mL of sodium hydroxide 50%). The optimum enzyme temperature was determined by incubating the enzyme-substrate preparations in their optimum pH at 25, 35, 45, 55 and 65°C using controlled water baths.
The enzyme kinetics was analyzed using the Michalis-Menten model with different ABTS concentrations (0.1, 0.3, 0.5, 0.75, 1, 1.5, and 2 mM) under optimal conditions of pH and temperature previously obtained. The inhibition effect of NaCl and Na 2 SO 4 on laccase activity was evaluated using concentrations of 1, 2.5, 5, 7.5, and 10 mM of each salt. These salts were analyzed since they are in the synthetic effluent used in the tests and are usually found in real textile effluents, which can result in the inhibition of laccase activity.
The kinetic parameters K m (Michaelis Menten coefficient) and V max (maximum reaction velocity) were determined by non-linear adjustment of the Michaelis-Menten model (Equation 1), using the Origin® 2017 programme. The inhibition constant (K i ) was determined by Equation 2. To determine the type of inhibition, the Lineweaver-Burk double reciprocal linearisation was employed [31].
Note: K i inhibition constant (mM); [I] inhibitor concentration (mM); α obtained by dividing K m in the presence of inhibitor and K m in the absence of inhibitor.

Batch dye removal experiments
The synthetic textile wastewater composition was based on Mo et al. [32], consisting of two azo dyes and auxiliary chemicals in order to simulate real textile wastewater, as shown in Table 1.
The discolouration tests were performed in a thermostatic bath (Dubnoff, model 252) using Erlenmeyer flasks (250 mL) containing 150 mL of synthetic textile wastewater under reciprocal agitation at 200 rpm. For each tested condition, Erlenmeyer flasks some containing only synthetic wastewater and some containing only SMS with distilled water were considered as control experiments. The textile wastewater pH values were adjusted with 0.5 M HCl and 0.5 M NaOH. The samples were centrifuged at 11,000 rpm for 5 min to later determine the concentration of dyes and the activity of the laccase enzyme.
The concentration of dyes in the textile effluent was evaluated by spectrophotometry at 549 nm using the Hach spectrophotometer (DR 3600). This wavelength was obtained after the spectral scanning of the effluent ( Figure S1), and corresponds to the highest absorbance in the visible range. The absorbance value verified in each analysis was interpolated on the calibration curve ( Figure S2) inserted in the spectrophotometer to obtain the concentration of the dyes.

Experimental factorial design
To evaluate the combined effect of variables on the discolouration of the textile wastewater, a 2³ factorial design was performed with one repetition at the central point, resulting in nine treatments performed in duplicate [33]. The independent variables considered were pH, temperature, and SMS concentration; the respective levels are shown in Table 2. These levels were defined based on the general characteristics of the textile wastewater and on preliminary tests. Color removal and laccase activity were established as the dependent variables.
In the same combinations of the analyzed variables (Table S1) and in parallel to the discolouration tests, experiments were carried out using the SMS without the presence of enzymes in order to determine color removal due solely to the adsorption mechanism. For this, the SMS was pre-treated at 90°C for 15 min for enzyme denaturation and loss of activity. Enzymatic degradation was estimated by the difference between total removal with the SMS and the adsorbed percentage.
The Statistica software (StatSoft, USA) was used for the experimental design and statistical analysis. A first order polynomial equation was determined to identify the possible interactions of the selected factors. The effects were obtained using the least squares method and statistically evaluated by analysis of variance (ANOVA). The statistical significance of the regression coefficients was 90%.
Under the best conditions of pH, temperature, and SMS concentration, a kinetic test was performed with sample collection at the following contact times: 0, 15, 30, 60, 120, 180 and 240 min. In addition to the removal of color and laccase activity, the activities of LiP and MnP were determined.
A comparison was made between the enzymatic degradation using aqueous enzymatic extract from SMS and the direct application of SMS. The enzymatic extraction was carried out in Erlenmeyers with 125 mg L −1 of SMS in distilled water, incubated in a thermostatic bath (Dubnoff brand, model 252) with agitation at 200 rpm for two hours. A concentrated solution of the synthetic textile effluent (2000 mg L −1 ) was added to the enzymatic extract, resulting in a final concentration of 50 mg L −1 of dyes. In the assay involving the simultaneous processes of enzymatic degradation and adsorption, the percentage of degradation of the dyes was obtained from the difference between the removal with SMS and with SMS without enzymes, in the SMS concentration of 125 g L −1 and dyes concentration of 50 mg L −1 . Both the decolourisation tests with the enzymatic extract and the SMS were conducted under the best conditions of pH, temperature, and SMS obtained in the previous experiments.

Adsorption studies
To evaluate the dye adsorption process to the SMS, the kinetic models, isotherms, and thermodynamic parameters were studied. The amounts of dyes adsorbed by the SMS at a given time (q t ) and at equilibrium (q e ) were calculated using the corresponding mass balance equations: where, q is the amount of solute adsorbed per gram of adsorbent (mg g −1 ); C 0 , C e, and C t are the concentration (mg L −1 ) at the beginning, at a given time, and at equilibrium, respectively; V is the volume (L) of effluent used and m is the mass (g) of the adsorbent. Isotherms and kinetic models were applied in a non-linear adjustment using Origin® 2017 software. The kinetic models of Pseudo-first order (PFO), Pseudo-second order (PSO), intraparticle diffusion (ID), and the Elovich equation were applied in order to determine the kinetic mechanism that governs the adsorption process. The non-linear form of the PFO model The non-linear form of the PSO model The non-linear form of the ID model The non-linear form of the Elovich model where, q e and q t are the adsorption capacity at time t and at equilibrium, respectively (mg g −1 ); t: reaction time (min); K 1 (min −1 ) and K 2 (g mg −1 min −1 ) are the rate constants of PFO and PSO adsorption, respectively; K d (mg g −1 min −1/2 ) is the intraparticle diffusion constant, and C (mg g −1 ) is the constant related to diffusion resistance; β (mg g −1 ) is the desorption constant, and α (mg g −1 min −1 ) is the initial adsorption rate. The initial adsorption velocity (h: mg g −1 min −1 ) was calculated based on the PSO model using Equation 7.
The tests for the determination of the adsorption isotherms were carried out with concentrations of dyes that varied from 5 to 60 mg L −1 at three different temperatures (25,35, and 45°C). The experiment was carried out with 150 mL of textile effluent and 125 g L −1 of SMS after enzyme inactivation, under agitation of 200 rpm in a thermostatic bath (Dubnoff brand, model 252) for two hours, under the most favourable temperature and pH conditions for adsorption. The control samples consisted of only textile effluent and only SMS in distilled water. The samples were subjected to centrifugation at 11,000 rpm for 5 min for later determination of the concentration of dyes. To adjust the experimental data and characterise the adsorption process, the nonlinear models of the Freundlich, Langmuir, Sips, Redlich-Peterson and Temkin were studied.
The non-linear form of the Freundlich model The non-linear form of the Langmuir model The non-linear form of the Sips model The non-linear form of the Redlich-Peterson model The non-linear form of the Temkin model where, C e : concentration of the adsorbate at equilibrium (mg L −1 ); q e : adsorption capacity at equilibrium (mg g −1 ); are the Freundlich, Langmuir, Sips, Redlich-Peterson, and Temkin constants, respectively; 1/n: constant related to the heterogeneity of the surface; q max : maximum adsorption capacity (mg g −1 ); β S : heterogeneity coefficient of the bio adsorber's surface; a S : affinity coefficient; g: exponent of the Redlich-Peterson isotherm; K R : Redlich-Peterson affinity constant (L g −1 ); R: gas constant (8.3144 J mol −1 K −1 ); T: temperature (K); b: constant related to biosorption heat (J mol −1 ). Langmuir's model was also explained by the separation factor (R L ) that was calculated using Equation 13 [34].
As enthalpy and entropy did not show a linear relationship with temperature, Equation 15 was used to calculate the ΔH°a ds , and ΔS°a ds was obtained by Equation 16 [35]. (17) where, ΔG°a ds : Gibbs free energy (kJ mol −1 ); R: universal gas constant (8.314 J mol −1 K −1 ); T: temperature (K); K D : dimensionless thermodynamic equilibrium constant obtained from adsorption isotherms; ΔH°a ds : enthalpy in adsorption (J mol −1 ); ΔS°a ds : adsorption entropy (J mol −1 ); a, b, c: empirical parameters obtained by the relationship between lnK D and T −1 .

SMS characterisation
The SMS presented a highly porous structure due to the sawdust integrated into the fungal hyphae, as observed in Figure 1. The sawdust porosity is increased by the action of lignocellulolytic enzymes that break down the cellulose structure of the biomass. Porous materials are preferred for adsorption purposes. SMS surface area also indicates adsorption capacity. As reported in other studies, similar SMS had a surface area of 2.417 m 2 g −1 [36], 2.46 m 2 g −1 [37] and 4.11 m 2 g −1 [38].
Additionally, fungal hyphae act as a matrix that keeps the extracellular enzymes attached to the material structure. Adsorbed dye molecules are much closer to the immobilised enzymes in the SMS structure, probably increasing their chance to bind the laccase active sites, in comparison to enzymes and dye molecules diluted in the liquid fraction. During fungal mycelia growth in a solid substrate such as SMS, the extracellular polymeric substances (EPS) are naturally produced, with the functions of avoiding dehydration, nutrient storage, and enzyme adherence to the external support. The immobilisation of fungal enzymes was already extensively proposed as an effective technique for dye degradation.
The quantitative analysis of chemical elements of the SMS with and without enzymes by the EDS technique is shown in Table S10. The SMS is basically composed of carbon (C), oxygen (O), and calcium (Ca). Only a small percentage of magnesium (Mg) was detected in the SMS in one of the analyzed points. The presence of Ca in the SMS can be attributed to the calcium carbonate added to the substrate to neutralise the acidity. Alhujaily et al. [39] also found that C, O, and Ca are the elements present in highest quantities in the SMS. Figure S3 shows the FTIR of the SMS with and without enzymes, before and after the treatment of the synthetic textile effluent. The SMS consists mainly of lignin, cellulose, hemicelluloses, and simple sugars, chitin, and minerals [40]. These components have several functional groups that can enable the adsorption of textile dyes.
The bands from 3442.27 to 3432.63 cm −1 are assigned to the functional group OHof the cellulose and lignin phenol group. The peak at 2917.73 cm −1 corresponds to the elongation of the aliphatic CH 2 group. The bands at 1648.81 and 1646.89 cm −1 are referring to the aldehyde group C = O [41]. Peak 1322.91 cm −1 corresponds to CH 2 vibration in cellulose [42]. The syringyl ring and C-O stretch in lignin and xylan were identified in the band 1241.92 cm −1 [43]. Peaks at 1054.85 and 1056.79 cm −1 correspond to the C-O stretch in polysaccharides [44]. The C-H deformation outside the plane of the aromatic ring is detected at wave numbers 611.32 and 613.24 cm −1 [45].
The SMS, both with and without enzymes, showed higher intensity peaks in the wave numbers corresponding to the functional groups C = O (1646.89 cm −1 ), OH -(3442.77 and 3433.62 cm −1 ), C-O (1054.85 and 1056.79 cm −1 ), and CH 2 (1322.91 cm −1 ), in descending order. The SMS without enzymes showed greater intensity of the bands compared to the SMS with enzymes, but no new peak was added. It was also found that in the SMS without enzymes, there was a small displacement for lower wavenumbers 3442.27 to 3432.63 cm −1 and from 1506.11 to 1504.18 cm −1 and for larger ones from 1054.85 to 1056.78 cm −1, when compared to SMS. Considering that these differences are not accentuated and that the adsorption capacity may not be significantly affected by these differences, the SMS studied can be used to identify the exclusive performance of the enzymes.
Both with and without enzymes, the SMS after treatment showed peaks of lesser intensity in comparison with the respective SMS without contact with the effluent. The reduction in the intensity of the SMS peaks after treatment can be attributed to the release of compounds present in the SMS into the aqueous solution. No new peak was added to the SMS after contact with the textile effluent, however shifts in the wavenumbers were observed, which may indicate the adsorption of the dye.
As shown in Figure S5, the SMS zero charge point (pH PZC ) is 5.5, which indicates that at a pH below 5.5, SMS will have a positive charge and anion adsorption will be favoured, while at a pH above 5.5, SMS will have a negative charge and positive species will be better adsorbed. In the studies by Alhujaily et al. [39] and Yan and Wang [36], the SMS showed approximate pH PZC values of 6.5 and 7.2, respectively.
A significant increase in peak intensity around 1627.61 cm −1 was observed in the textile effluent after treatment ( Figure S4). Wavenumbers from 1500 to 1700 cm −1 are associated with proteins, specifically the stretching of the C = O bond of the peptide, therefore, this increase in intensity is probably associated with the proteins released by the SMS [46]. Peaks of 1421.26 and 1419.33 cm −1 appeared after the treatment of the textile effluent, which refer to section C = C-H in the C-H curvature of the plane [47]. The peaks at 1141.64 cm −1 (sulfonate group) and 997.01 cm −1 (symmetrical elongation of the sulfonic groups) identified in the raw textile effluent disappeared after treatment with SMS [48]. In the treated effluents, peaks of 1047.14 cm −1 appeared, attributed to the S = O stretching mode of the SO 3 group [49]. The 620.79 cm −1 wavenumber present in the raw effluent, which corresponds to C-H, decreased the intensity and was shifted to smaller wavenumbers after treatment [50]. Figure 2 shows the effect of pH 2.0-10 on laccase activity from the SMS. The results indicated that the laccase has activity at acidic pH levels (2)(3)(4)(5)(6) and is inactivated at basic pH (8-10) levels. Laccase activity decreased from 78.14 U L −1 at pH 2 to 19.58 U L −1 at pH 6. It is already established that laccase enzymes need an acidic environment for their ideal catalytic activity, as has been verified in the present study.

Effect of pH and temperature
The effect of pH on laccase activity is the result of changes in the reaction, caused by the substrate, oxygen, or by the enzyme itself [51]. The absence of laccase activity at basic pH levels can be attributed to the hydroxide anions (OH -) present in greater quantity, which bind to the T2/T3 centre of the laccase structure and interrupt the internal electron transfer between T1 and T2/T3 [52]. Both the excess of OHand H + can cause the disruption of hydrogen bonds and disulfide bonds at charged laccase sites, which reduces the functional capacity of the enzyme. These opposite effects induce a laccase pH profile in a typical bell shape, with low activity at extreme pH values [53]. However, this study showed that at pH 2.0 laccase mantains its activity at the highest level.
The ideal pH of the laccase is also highly dependent on the substrate and for ABTS, the optimal pHs are generally between 3 and 5 [54]. However, other laccases showed an optimum pH below 3 with ABTS, corroborating the findings for the laccase from the SMS. One of the recombinant isoenzymes of Pleurotus ostreatus showed an optimum pH equal to 2.5 [52], similar to the isoenzyme produced by Pleurotus sajor-caju that showed greater activity at pH 2.4 [55]. The laccase of Cerrena unicolor exhibited an optimum pH of 2.6 and was totally inactive at a pH greater than 6.6. Although at pH 2.0 the highest laccase activity was seen, extreme pH levels can reduce the enzyme stability and are not characteristic for real textile wastewater. Therefore, pH 4.0 was chosen as the optimum pH value for further experiments.
Regarding the temperature effect, the highest laccase activity range (57.45 U L −1 -55.50 U L −1 ) was obtained with temperatures ranging from 45°C to 55°C. These values are in agreement with the optimum temperatures of 40°C -70°C for fungal ligninolytic enzymes [52]. The temperature of textile wastewater is highly variable, but the typical temperature is in the range of 35°C-45°C , including the temperature of the highest enzymatic activity of the SMS laccase [56].
The reduction in laccase activity at a temperature above 55°C can be associated with the interruption of the tertiary structure and conformational change of the active site. Das et al. [57] observed that laccase activity of Pleurotus ostreatus also decreased at temperatures above 55°C, but showed high activity in the range of 35°C-55°C. In addition, El-batal et al. [51] observed a rapid loss of activity at temperatures above 60°C and the optimum temperature was in the range of 30°C-50°C for Pleurotus ostreatus laccase. Similar to the laccase from the SMS, laccase activity of Agaricus bisporus gradually increased with increasing temperature until it reached maximum activity at 55°C. This behaviour was associated with the increase in the kinetic energy of the molecules by the elevation of the temperature, which resulted in greater interaction of the ABTS substrate with the enzyme's active sites [58].

Kinetic parameters
The Michaelis-Menten model was evaluated using the ABTS substrate (0.1-2 mM) at pH 4 and 45°C in the absence and presence of NaCl and Na 2 SO 4 salts, respectively. The kinetic parameters are shown in Table  3. The laccase from SMS showed a K m of 0.264 mM and a Vmax of 117.95 μmol L −1 min −1 . The low K m value implies high affinity of the laccase to the ABTS substrate and is in the range of 0.004-0.77 mM mentioned in the literature [59].
According to Das et al. [57] the reaction between ABTS and the Pleurotus ostreatus laccase resulted in a higher K m value (0.52 mM). A lower Km value (0.157 mM) was obtained for the laccase of Pleurotus pulmonarius [60]. Values close to the K m 0.264 mM obtained for laccase from the SMS were verified for basidiomycete fungal laccase from Steccherinum murashkinskyi (0.275 mM) [61] and Trametes versicolor (0.290 mM) [62].
The high V max (117.95 μmol L −1 min −1 ) of the SMS laccase indicates that the enzymes are active in the process of converting the substrate to product. Lower Vmax values were obtained for the laccase of Pleurotus ostreatus (22 μmol min −1 ) [63] and Pleurotus florida (0.07 μmol min −1 ) [64]. High Vmax values were obtained for the laccase of Pleurotus ostreatus   Figure 3 shows the inhibition effect of NaCl and Na 2 SO 4 at concentrations from 0 to 10 mM. The initial reaction rate increased rapidly with low substrate concentrations, tending to saturate with high ABTS concentrations.
Regarding the salts evaluated as inhibitors, NaCl promoted an inhibition of laccase activity, with greater intensity as the NaCl concentration was increased. Laccase inhibition by NaCl may be associated with the fact that chloride prevents electron transfer from the substrate to type 1 copper or from this to type 3 copper in the laccase structure [67]. Meanwhile, the reaction rate was the same in the absence and presence of different concentrations of Na 2 SO 4 , which implies that this salt is not characterised as a laccase inhibitor ( Figure 2C).
As in the present study, Zilly et al. [68] also found that NaCl inhibited the laccase of the fungus Ganoderma lucidum even at low concentrations, and that Na 2 SO 4 promoted mild stimulation. The authors attributed this stimulation to the increase in the enzyme's affinity to the substrate due to a small decrease in the K m value, but it did not interfere in the acceleration of the catalytic reaction because V max was not affected. Unlike this study, for laccase from SMS, Na 2 SO 4 promoted both a brief decrease in K m and an increase in V max (Table 3).
Zilly et al. [68] also stated that the inhibition caused by NaCl was of the mixed type that involves competitive and non-competitive inhibition, because at the same time that it reduced the affinity of the laccase to ABTS (increase in K m ) it decreased the reaction rate (decrease in V max ). As shown in Table 3, it can be seen that this behaviour can also be observed in the present study. Similarly, Yang et al. [69] found that NaCl was a mixedtype inhibitor for both free and immobilised laccase.
In contrast, in the Linewaever-Burk linearisation ( Figure 2B), the lines referring to NaCl concentrations and the absence of inhibitor intersect in the same area of the graph's abscissa, however they intersect different areas of the ordinate, inferring a non-competitive inhibition. In this type of inhibition, the inhibitor binds to the enzyme at a different site from the active site, allowing normal binding of the substrate with the enzyme, however, complete inactivation of the enzyme occurs which prevents the conversion of the substrate into product. With the decrease in the amount of active enzymes in the medium as the inhibitor binds to the enzyme, the V max is reduced, while the K m is not affected as the inhibitor does not block the active enzyme site [70]. However, it was found that K m increased in the presence of NaCl. In contrast, Enaud et al. [71] found a competitive inhibition between ABTS and NaCl by representing Lineweaver-Burk, albeit a mixed inhibition according to the Cornish-Bowden model.
The laccase inhibition was 4.12% with 1 mM, and 21.61% with 10 mM NaCl (Table 3). In comparison with these results, higher percentages of inhibition, approximately 40% and 60%, were verified for the commercial laccase of Trametes versicolor at concentrations of 5 and 10 mM NaCl, respectively [72]. Two laccase isoenzymes produced by the white rot fungus Pycnoporus sp. were also inhibited to a greater extent (19.16% and 8.48%) at a concentration of 1 mM NaCl.
K i values ranged from 4.44 to 10.169 mM for concentrations of 1 and 5 mM NaCl, respectively (Table 3). While the percentage of inhibition increased as the concentration of NaCl was increased, K i did not follow a trend and showed a higher value in the mean concentration of NaCl tested (5 mM). Higher K i values indicate a lower affinity of the laccase to NaCl.
The concentration of chlorides in textile effluents is highly variable and most of them are associated with the use of NaCl in the production process. As reported by Valh et al. [73] the concentration of chlorides in textile effluents is in the range of 200-6000 mg L −1 . The synthetic textile effluent used in this study had a concentration of 250 mg L −1 which corresponds to 4.3 mM NaCl, so it is assumed that an inhibition between 7.67 and 11.81% may have occurred. Considering the highest concentration of NaCl studied for the inhibition of laccase 10 mM equivalent to 584.4 mg L −1 an inhibition of only 21.61% of the laccase activity would be achieved, considering only the NaCl effect. Higher percentages of inhibition can be avoided by carrying out the dye degradation by SMS enzymes stage after the removal of salts in the treatment plant.

Discolouration assays
A factorial experimental design was carried out to evaluate the effect of the pH, temperature, and SMS concentration and their interactions on the adsorption and enzymatic degradation of textile dyes as well as on laccase activity, after a contact time of 2 h. Table 4 presents the conditions of each test and the respective results.
Using the SMS as an adsorbent and source of enzymes, a maximum removal percentage of 57.22% was obtained at pH 8, 25°C, and 100 g L −1 (Run 6). A similar removal rate of 56.41% was found under the same conditions except at a temperature of 45°C (Run 8). However, under these conditions, adsorption was the only mechanism responsible for dye removal and no percentage of enzymatic degradation was detected. Therefore, these were also the best conditions for the isolated adsorption process using the SMS.
The highest contribution of enzymatic degradation in the removal of textile dyes was 14.18% at pH 4, 25°C, and 100 g L −1 (Run 5) followed by 6.79% under the same pH and temperature conditions but 30 g L −1 of SMS (Run 1). The highest laccase activity was 58.09 U L −1 in Run 6, precisely in which enzymatic degradation was not identified, while 52.15 U L −1 corresponded to the highest enzymatic degradation obtained in Run 5. There is no direct correlation between laccase activity and dye   degradation due to the complex interactions that the studied variables have on the reaction. Therefore, high enzyme activities may have resulted in low dye degradation because the experimental conditions prevented the enzymes from using the dyes as a substrate at pH 8.0. However, they do not affect the enzyme activity at pH 4.5 and with ABTS as substrate, which are the optimised conditions to be used in laccase assays. This aspect shows the importance to evaluate the enzyme activity and the enzyme catalysis using real substrates such as industrial dyes. A model equation was developed based on the regression coefficients (Table S2 to S5). Total dye removal (adsorption + enzymatic degradation) is represented by Equation 3, adsorption by Equation 4, enzymatic degradation by Equation 5, and the laccase activity by Equation 6. The temperature and SMS concentration interactions (T x SMS) were rejected for the laccase activity and the isolated mechanisms of adsorption and enzymatic degradation since they were not statistically significant and caused a decrease in the model's adjusted coefficient of determination (R 2 ). For the same reasons, the effect of pH and pH x SMS interaction were also removed for the laccase activity. Other effects, even if not significant, were maintained as they caused a reduction in the adjusted R 2 when removed.
The R 2 and R 2 adjusted values were 0.996 and 0.986 for total removal, 0.993 and 0.982 for adsorption, 0.930 and 0.814 for enzymatic degradation, and 0.921 and 0.873 for laccase activity, respectively. The R 2 values for both models were higher than 0.9, which indicates a high correlation and a good adjustment between the observed and predicted responses. Therefore, the model is reliable in obtaining removal percentages for both mechanisms and laccase activity. The high values of adjusted R 2 also confirm the model's significance. The ANOVA results for the experimental models are shown in Tables S6 to S8.  Figure 4 shows the Pareto graph that allows the identification of significant effects at a 90% significance level (p = 0.1). For the total removal (adsorption + enzymatic degradation), the separate mechanisms of adsorption and degradation, and for the laccase activity, the interactions of the variables were not significant; only the isolated variables caused significant changes in the responses. For total removal with the SMS, the variables that had a significant and positive effect were the SMS concentration followed by pH. Temperature caused a negative effect. In the same way for adsorption, however, the temperature was not significant and the interaction of pH with SMS was close to causing a significant effect.

Removal (%) Adsorption
For enzymatic degradation only pH was significant, and for laccase activity, only SMS concentration. Although the effects are not significant, a correlation between laccase activity and enzymatic degradation can be seen, as the variables caused the same effect both in laccase and in the degradation of dyes by these enzymes, with the exception of pH x T where enzymatic degradation caused a positive effect, and in laccase activity a negative effect. The isolated variables pH and T and the interactions pH x SMS and SMS x T promoted a negative effect, whereas the concentration of SMS promoted a positive effect.
In comparison, the SMS concentration and temperature caused a positive effect for both adsorption and enzymatic degradation. However, pH caused a positive effect for adsorption, or rather, high pH values result in higher adsorption, while enzymatic degradation is favoured by low pH values. Thus, the use of SMS may provide dye removal in a wider pH range due to the complementarity of adsorption and enzymatic degradation. Textile wastewater may suffer pH fluctuations due to the different processes and dyes involved in the industrial process, therefore, adsorption and enzymatic degradation may compensate the lesser contribution of the other according to the pH.
Corroborating the results obtained in this study, enzymes extracted from the SMS also resulted in higher degradation of textile dyes in an acidic medium with an optimum pH of between 4 and 4.5 [17] and higher adsorption to SMS at low temperature [13,74]. In nonconformity, higher percentages of adsorption of anionic dyes by the SMS were obtained at acidic pH values [21,75] and better degradation by enzymes extracted from SMS at higher temperatures [17].
The plotted response surfaces show the effects of the interaction of two factors with the third maintained at the zero level. Figure 5 shows the effect of the interaction of pH and temperature factors for each response evaluated with 65 g L −1 of SMS. A higher rate of dye removal is achieved with high pH values and low temperature. For isolated adsorption, higher removal was observed with high pH values but regardless of temperature. Enzymatic degradation is favoured at low temperatures with acidic pH. Laccase activity in the presence of the dye is high over a wide range of pH and temperature, however the maximum activity is achieved at low temperature and pH. Figure 6 shows the response surfaces for the interaction of the SMS concentration and pH at 35°C. Both the total removal and the adsorption were higher with the increase of SMS concentration and pH. The increase in SMS concentration and the reduction in pH favoured enzymatic degradation. Higher SMS concentrations also contributed to a high level of laccase activity at low pH, despite the lesser influence of this factor.
The response surfaces shown in Figure 7 illustrate the effects of the interaction of SMS concentration and temperature at pH 6.0. Maximum dye removal occurred at higher SMS concentrations and lower temperature. The highest adsorption was also obtained in high SMS concentrations, but with no effect of temperature. With the increase in SMS concentration and decrease in temperature, enzymatic degradation is favoured, as well as laccase activity. Figure 8 shows the dye removals from 5 to 240 min at the optimised pH (4.0), temperature (25°C), and SMS concentration (125 g SMS L −1 ). In these tests, in addition to the laccase activity, the lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were determined. The global dye removal considering adsorption and enzymatic degradation ranged from 45.33% in 5 min to 75.85% in 240 min. The enzymatic degradation contributed to 10.53% whereas the adsorption accounted for 65.32% in 240 h.

Kinetic study
The discolouration due to enzymatic degradation increased with the time, accompanied by the increasing laccase activity, ranging from 33 U L −1 in 5 min to 90.25 U L −1 in 240 min. In addition, the highest MnP activity (371.04 U L −1 ) was obtained in 240 min while LiP activity was not detected at all.
According to Figure 8, adsorption increased in the first 60 min and stabilised, probably due to the saturation of the adsorbent pores. However, the laccase activity and enzymatic degradation increased linearly until 240 min. This behaviour suggests that the adsorption is a fast process, followed by the enzymatic degradation that is constant and slower.
Likewise, Alhujaily et al. [21] also found that the SMS adsorption of direct dyes black 22, red 5b, blue 71 and the reactive black 5 increased rapidly in the first 60 min and gradually increased up to 240 min, when maximum adsorption was reached. For the adsorption of the methylene blue dye by SMS, Yan and Wang [36]  also found that the highest adsorption occurred in the initial 60 min. Even faster adsorption to SMS was observed for malachite green, safranin T, and methylene blue, in which 78% of the dyes were adsorbed in the first 5 min and slowly increased until 30 min [74]. This behaviour is associated with the large number of active sites available at the beginning of the reaction. Subsequently, few sites remain accessible, which results in slow adsorption, in addition to the effect of repulsive forces between the adsorbed dye and free molecules that hinder contact with the sites.
According to Figure 9, the combination of adsorption and enzymatic degradation resulted in increased discolouration, probably due to the enzymatic oxidation of the azo groups.
The color of azo dyes are related to the chromophore azo groups (-N = N-) contained in their chemical structure, and these chromophore azo groups are commonly associated with -OH or -NH 2 auxochromic groups that intensify the performance of these chromophores [76]. The azo dyes' degradation mediated by laccase starts by cleavage of the azo bond followed by oxidative cycling, desulfonation, deamination, demethylation, and dihydroxylation, depending on the structure of the dye and the laccase enzyme [77].

Comparison of SMS and the enzymatic extract
In order to verify whether the enzymatic degradation of textile dyes was enhanced when using the enzymes together with the SMS, a comparative analysis was carried out with the enzymatic extract from the SMS. This assay also included a comparison of the optimum adsorption pH (8.0) and the optimum enzymatic activity pH (4.0). The results are shown in Figure 10.
At pH 8, the enzymatic degradation (0.44%) and laccase activity (44.58 U L −1 ) due to the enzyme extract were lower than that obtained at pH 4.0. At pH 4, the extract was able to degrade 2.61%, whereas in the presence of the SMS the removal percentage reached 15.43%, in agreement with the laccase activity, which increased from 39.72 to 48.98 U L −1 . These results indicate that when the enzymes are applied together with the SMS they are more efficient in the degradation of textile dyes, mainly at pH 4.
This increase in the degradation of dyes by enzymes contained in the SMS can be attributed to the direct contact with phenolic compounds that are generated during the degradation of lignin by white rot fungi. Examples of these are acetosiringone, syringaldehyde, p-cumaric acid, vanillin, acetovanillone, and methylsyringe, which can act as natural mediators of laccase in the degradation of dyes [78].   The presence of salts, chelating agents, by-products, and surfactants in real textile wastewater can affect the application of enzymes for wastewater treatment purposes. However, natural immobilised enzymes such as those found in SMS combined with the physical-chemical mechanism of adsorption can overcome these limitations, as presented in this work.

Kinetic adsorption models
The adjustments of the models and the kinetic parameters obtained are shown in Figure 11 and Table  S11. The experimental data were the result of the discolouration tests carried out at pH 8, 125 g L −1 of SMS, and 25°C. All the coefficients of determination (R 2 ) of the applied models were higher than 0.90, which indicates that both mechanisms govern the adsorption kinetics, the same was observed by Toptas et al. [75] in the adsorption of dyes by SMS.
However, the Pseudo-second order model (PSO) presented the highest determination coefficient (0.998) and the equilibrium adsorption capacity provided by the model (q e mod ) is the same obtained experimentally (qe exp ) (Table S11) suggesting that this is the predominant kinetic mechanism in the adsorption process. The adequacy of the PSO model suggests the chemical nature of adsorption, which can be attributed to the covalent bond between the anionic dyes and the adsorbent through the sharing of electrons. Other studies have found that the main kinetic mechanism involved in the adsorption of dyes by SMS is the PSO [14,21,36,79].  The PFO adsorption rate constant (K 1 ) and the PSO rate constant (K 2 ) obtained were 0.110 min −1 and 0.302 g mg −1 min −1 , respectively (Table S11). The K 1 value is close to that found for the adsorption of methylene blue by the SMS (0.095 min −1 ) [13] and the reactive black 5 by the modified SMS (0.094 min −1 ) [39]. The K 2 value is lower than that obtained for the adsorption of reactive black 5 (0.082 g mg −1 min −1 ) [21] and higher for malachite green (0.595 g mg −1 min −1 ) and methylene blue (0.503 g mg −1 min −1 ) by the SMS [13]. Higher values of K 1 and K 2 indicate that a shorter time is needed to reach equilibrium.
The adjustment of the experimental data to the Elovich model (R 2 of 0.982) confirms that the adsorption is due to the chemical bonds between the dyes and the adsorbent (Table S11). The initial adsorption rate (α) provided by this model was 1.09 mg g −1 min −1 , much lower than 27.69 mg g −1 min −1 obtained in the adsorption of reactive black dye 5 by SMS [21]. Corroborating with the low adsorption rate, a lower initial velocity (h: 0.098 mg g −1 min −1 ) was obtained because of the PSO model. The Elovich model provided a high desorption constant value (β) (13.85 mg g −1 ), which indicates a greater extent of surface coverage.
According to the intraparticle diffusion graph shown in Figure 11B, the first segment of the line does not cut the origin, and the linear coefficient (C) is different from zero (Table S11), so intraparticle diffusion is not the only limiting step and the process is also controlled by other mechanisms [80]. From the experimental data, the presence of two adsorption stages was verified ( Figure 5B), where the first stage corresponds to a fast adsorption in which the intraparticle diffusion has a greater effect, since the value of K d (0.242 mg g −1 min −0.5 ) is greater and C is less than zero (−0.08 mg g −1 ). In the second stage, adsorption occurs gradually, C becomes positive (0.406 mg g −1 ), and K d decreases (0.04 mg g −1 min −0.5 ), indicating the greatest effect of the boundary layer and the lowest contribution of intraparticle diffusion. The higher R 2 value (0.999) for the first stage compared to the second stage (0.9003) reveals that adsorption is mainly regulated by intraparticle diffusion (Table S4).
Applying the intraparticle diffusion model for the adsorption of textile dyes by SMS, Alhujaily et al. [21] identified the presence of three stages: intra-film diffusion, intraparticle diffusion, and steady state, while Yan and Wang [36] verified only two stages intraparticle diffusion and steady state. For sawdust as an adsorbent, a product that makes up the SMS, there were also two stages intra film diffusion followed by intraparticle diffusion [81].

Adsorption isotherms
The tests for the determination of the adsorption isotherms were carried out with 125 g L −1 of SMS, pH 8, and two hours of contact. The data obtained experimentally were adjusted by the models and the results are shown in Figure 12 and (Table S12). R 2 values close to 1 were found for both models studied, however higher R 2 values were obtained for the Sips isotherm, indicating that this model best represents the experimental data.
The Sips isotherm is a combination of the Langmuir and Freundlich models, which defines the dimensionless heterogeneity coefficient (β S ). The β S values for the adsorption of dyes by the SMS were 1.05, 1.03, and 0.97 for temperatures of 25, 35, and 45°C, respectively (Table S12). This tendency to reduce the β S value may infer that the surface becomes more heterogeneous at high temperatures. When β S values are equal to 1, the Sips isotherm is reduced to the Langmuir model and reveals that the adsorbent surface is homogeneous, which can be observed in the present study, since the β S values are very close to 1. Corroborating this result, the R 2 values of the Langmuir model are higher than those of Freundlich.
The Sips affinity coefficient (a S ) was higher at 25°C (0.028 L mg −1 ) compared to the values obtained at 35°C (0.015 L mg −1 ) and 45°C (0.019 L mg −1 ), which indicates that the interaction between the dyes and the adsorbent is greater at low temperatures. When increasing the temperature from 25°C to 35°C, the Sips constant (K S ) decreased from 0.053 to 0.042 L g −1 , but increased again by 45°C (0.057 L g −1 ). This behaviour was also observed in the constants of the other models (K L , K F , K RP , and K T ), which after decreasing by 35°C to a temperature of 45°C reach values higher or very close to those obtained at 25°C (Table S12).
The suitability of the Langmuir model indicates that there is a monolayer coverage of dyes on the homogeneous surface of the SMS. The highest q max value was 3.303 mg g −1 at 35°C, while at this temperature a lower affinity of the dye for the adsorbent sites was found due to the lower K L value (0.013 L mg −1 ) ( Table  S12). The value of q max found in the present study is lower than that found by other authors in the adsorption of dyes by SMS [13,14,21,75], as well as for other adsorbents such as sawdust [41]. This result was expected since the SMS was not subjected to any procedure that could increase the adsorption capacity, such as cleaning, drying, grinding, or modification. This is due to the focus of the research, which is the direct application of SMS, with the objective of using enzymes and eliminating costly steps that make the use and manipulation of this waste more expensive due the treatment plants themselves.
Generally, a favourable adsorption has a value of n of the Freundlich isotherm between 1 and 10, which can be seen in the adsorption of dyes by the SMS, since the values of n were from 1.144 to 1.269 [82]. In relation to the Redlich-Peterson model, the values of exponent g are close to 1 (Table S12), indicating that there is a condition of low adsorbate concentration and that this isotherm is reduced to the Langmuir equation, as observed in the Sips isotherm [83]. The decrease in parameter b of the Temkin isotherm with an increase in temperature from 25°C to 35°C indicates that the adsorption is exothermic, and the increase in b from 35°C to 45°C reveals that the process is endothermic (Table S12).
The separation factors (R L ) shown in Figure S6 are the result of the Langmuir model and correspond to the degree of development of the adsorption process. At both temperatures, the R L values are adjusted to the following ratio 0<R L <1, which implies that the adsorbate prefers the solid to the liquid phase, and therefore, the adsorption is favourable [84]. In comparison with other temperatures, it is possible to verify that at 35°C the R L values are higher, indicating that the adsorption is less favourable at that temperature.
Table S13, shows the thermodynamic parameters related to the adsorption of textile dyes by the SMS. Negative ΔG°values indicate that the reaction occurs spontaneously, not requiring external energy to convert reagents into products. At temperatures of 298.15 and 308.15 K, ΔH°is negative, indicating that heat is released, so the reaction is exothermic; whereas at 318.15 K, heat absorption and the process take place and it is given as endothermic. This behaviour corroborates the results verified by the variation of parameter b of the Temkin isotherm. The values of ΔH°for 298.15 and 308.15 K are between the range of 80-200 kJ mol −1 , which indicates that the adsorption is chemical in nature; whereas for 318.15 K, ΔH°is between 2.1 and 20.9 kJ mol −1 inferring the occurrence of physisorption. Negative ΔS°v alues at 298.15 and 308.15 K suggest a reduction in randomness at the solid-liquid interface [85].

Conclusions
This study showed that SMS has high laccase and MnP activity, making it able to degrade textile dyes. In addition, the salts typically used in industrial textile processes did not show a significant inhibition effect towards laccase activity. Considering that the azo dyes used in this study are less susceptible to enzymatic degradation due to their complex chemical structures, the degradation percentages obtained are promising, since they were subjected to a mixture of two azo dyes without any type of enzyme purification or concentration.
In addition to the enzymatic degradation, SMS showed high dye removal effect through the adsorption mechanism, without any pre-treatment or biochar production. In addition, enzymatic degradation was enhanced when using SMS compared to the enzymatic extract due to the presence of laccase mediating compounds and the role of SMS as an immobilisation matrix. Thus, a combined effect of adsorption and enzymatic degradation was observed for dye removal. Adsorption and enzymatic degradation were favoured for higher SMS concentrations and lower temperatures. While higher adsorption occurs at basic pH levels, enzymatic degradation is benefited by an acidic pH.
Based on the results obtained in this study, SMS can be used in the treatment of textile wastewater with the objective of removing dyes through the simultaneous mechanisms of adsorption and enzymatic degradation. The proposed use of SMS for dye discolouration contributes to the recovery and reuse of residual biomass and their enzymes, coupled with the removal of recalcitrant compounds contained in textile wastewater, through adsorption and enzymatic degradation mechanisms.