# Adsorption Potential ofSchizophyllum commune White Rot Fungus for Degradation of Reactive Dye and Condition Optimization: A Thermodynamic and Kinetic Study

Authors: Alia Raees; Haq Nawaz Bhatti; Sameer Alshehri; Farheen Aslam; Foziah F. Al-Fawzan; Siham A. Alissa; Munawar Iqbal; Arif Nazir
Journal: Adsorption Science & Technology
 (2023)

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## Abstract

The pollution due to dyes from textile sector is one of the major issues faced worldwide. This study was focused on the removal of the reactive dye, Drimaren Turquoise CL-B usingSchizophyllum commune, a white rot fungus (WRF) keeping in mind the current environmental conditions. Different parameters like pH, sources of carbon & nitrogen, temperature, concentration of dye and C/N ratio were used to investigate their effect on the process. Maximum dye removal of 95.45% was obtained at pH 4.5, temperature 35°C, inoculum size 3 mL, veratryl alcohol (mediator), glucose (carbon source) and ammonium nitrate (nitrogen source). The enzyme activity was determined by employing enzyme assay. Laccase and Lignin peroxidase (LiP) activity was low while Manganese peroxidase (MnP) activity was highest. Maximum bio-sorption was achieved at pH 1 and 313 K. The pseudo-2nd-order kinetic model & Freundlich isotherm was best suited for the process of removal of dye. From these data, it is concluded that white rot fungus could possibly be the excellent biomaterial for elimination of synthetic dyes from wastewater.


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## Body

## 1. Introduction

Many industries including paper, plastic, tannery, and textile broadly use dyes. Reactive dyes, being extensively used in textile, pose the greatest problem due to nonbiodegradable nature and needs an extensive treatment for complete removal of color from the effluents [1–6]. Nowadays, the industrial usage of dyes and other chemicals has become a great challenge with reference to environment [7, 8].Dyes are difficult to degrade in aquatic environment, since these are resilient to light and oxidizing agents [9–14]. The dyes are characterized as cationic, nonionic and anionic type depending on the structural features. The heavy metals in industrial effluents also pose environmental problems along with the dyes. Discarding industrial effluents without treatment into the environment makes the water bodies colored. This will limit the oxygenation capacity of the water body at receiving end and ultimately interrupts the photosynthetic process in the marine environment and responsible for severe toxicity. Prior to discharging the effluents in water bodies, it is highly appreciated to treat for removal of impurities [7, 15–18].Conventional methods of wastewater treatment are not efficient for the treatment of dye wastewater, since the dyes are nonbiodegradable and chemically stable. Some of the already employed methods including oxidation, adsorption, filtration, coagulation, and ionization are effective. These physical and chemical methods have limitations, e.g., excess chemical usage, concentrated sludge, operational costs, and ineffective color reduction. So, the researchers are thinking about some cost effective, ecofriendly methods that produce less sludge. In this view, the microbial biodegradation of dyes is considered one of the better alternatives.Previous studies suggested that the microbes can reduce dyes comprehensively. These microbes have the ability to degrade very complex natured pollutants [19–25]. White rot fungi are considered the best tool for biodegradation of lignin in nature. The fungi have an advantage over bacterial organisms that they can solubilize substrates using extracellular enzymes [26–31].Biosorption is sequestration of complex natured pollutants using biomass or their derivatives. The dead biomass is preferred over the live one because the sorption process is unaffected by toxicity of pollutants. Through the process of biodegradation, the dyes are broken down using enzymes; e.g., white rot fungi secrete ligninolytic enzymes to degrade variety of pollutants [32]. The working of white-rot fungi is primarily dependent on nutrient limitation. This is not actually affected by the concentration of pollutants [33–39].The key aim and objective of this research are to explore the ability of locally sequestered white rot fungus,S. commune, for the process of removal of dyes and, furthermore, to study the enzyme profile, to examine the influence of biodegradable carbon source, and to evaluate the sorption capability of S. commune biomass for the elimination of reactive dye under numerous experimental circumstances.


## 2. Material and Methods

Reactive textile dyes including Drimaren Orange CL-3R, Drimaren Red CL-58, Drimaren Yellow CL-2R, Drimaren Black CL-CX, Drimaren Navy CL-B110, and Drimaren Turquoise CL-B were collected from Clariant (Pvt.) Limited, Faisalabad, Pakistan. WRFS. commune was sequestered from decomposing wood located in Murree Hills and nearby vicinity. The strain was isolated and identified from the Department of Microbiology, UAF. Decaying wood samples were aseptically positioned onto potato dextrose agar (PDA) plates for the isolation of fungal mycelia, which were subcultured until pure mycelia were obtained and kept at 4°C [40]. The isolate was identified based on the morphology, hypha, and spores [41].


### 2.1. Sporulation Media and Inoculum Preparation

The potato dextrose agar (PDA) is a general-purpose medium used for the culture of fungi, and to avoid contamination from bacterial growth, some antibiotics were also added in PDA medium. In the current study, for the preparation of WRF slant, PDA medium is used. For slant preparation, 100 mL distilled water was taken to dissolve 3.9 g PDA and autoclaved at 121°C for 15 min. After autoclave, the warm 10 mL medium was aseptically transferred into a sterilized culture tube, put on a slanting position on a slanting rack, and allowed to cool. In the culture medium, due to the presence of agar-agar, it is solidified on cooling. Using aseptic loop, few spores ofS. commune were transferred into slant using all possible sterilized conditions in laminar air flow [42].For the preparation of fresh culture of WRF, 2.4 g potato dextrose broth was dissolved into water and later on was sterilized. After sterilization, the flask was maintained on room temperature, the sterile tartaric acid (10%) was used to lower the pH of the medium up to 4.5 to inhibit the bacterial growth. Using sterilized condition in laminar air flow, few spores of white rot fungi from slant culture were inoculated into a medium flask and placed at 120 rpm; 30°C; 72 h for the required number of spore’s inoculum (1×108 spores/mL) at 100 μL sample. This was transferred to a UV spectrophotometer, and optical density of sample was measured at 600 nm.


### 2.2. Decolorization Procedure and Time Course Study

Six duplicate containers were placed for the reactive dyes. Each flask contained 0.01% of the respective dye along with 95 mL sterilized Kirk’s basal nutrient medium [43]. The pH of the media used for removal of dyes was kept at 4.5. Each flask was inoculated with 2 mL suspension of the strain of WRF after being autoclaved and cooled. These dyestuff sets were kept in aseptic laminar air flow. The incubation parameters for the flasks in shaking incubator were optimum temperature (30°C) and centrifugation speed (120 rpm) for 10 days. The dye with most removal affect was picked up to optimize the removal process [44]. After 24 h, the removed sample (1 mL) from flask was centrifuged for 10 min. The absorbance was taken at λmax to determine the removal (%) by S. commune. Maximum removal was perceived for the dye Drimaren Turquoise CL-B by the culture of S. commune. This dye was subjected to optimization of different parameters [45].


### 2.3. Process of Optimization

The pH of 0.01% Drimaren Turquoise CL-B was adjusted using different initial pH values starting from 3 and up to 5.5 before inoculation. 2 mL of the spore suspension of the fungal strain was inoculated after being autoclaved followed by incubation in orbital shaker (120 rpm) at 30°C. Samples were centrifuged using a Sigma centrifuge machine (Germany) at7379×g for 10 min at 25°C. The effect of temperature on dye removal was studied using 25, 30, 35, 40, and 45°C. Nutrients with dye solution was autoclaved and then 2 mL of inoculum was added. Samples were removed after 24 h from each flask, centrifuged at 7379×g for 10 min, and absorbance was read on a spectrophotometer [46].Different materials including wheat bran, fructose, rice bran, sucrose, glucose, and maltose were used for the removal of the Drimaren Turquoise CL-B dye by the culture ofS. commune in a shake flask experiment. The experiment was directed at optimal pH and temperature for about 7 days. Glucose was evidenced to be the finest carbon source for Drimaren Turquoise CL-B removal by the culture of S. commune. The ammonium salts (tartrates and chlorides) are the most widely used nitrogen sources for removal of dyes using fungus biomass. These nitrogen sources are costly, so there was a need to find out relatively optimized source and ammonium nitrate which are considered optimum source. Five duplicate flasks were maintained for different inoculum size such as 2, 3, 4, 5, and 6 mL to study the effect on dye removal. Similarly, the mediator effect on the removal of dye was also monitored.


### 2.4. Ligninolytic Enzyme Profile

The fermented samples were removed, at the end of each experiment, from removal media and centrifuged. This was done to find out the patterns related to working of enzymes involved in removal process of different dyestuffs by different fungi. Supernatant was withdrawn, kept at -4°C, and studied for determination of enzyme effects. Laccase action was performed by 1 mL of ABTS added in 1 mL buffer and 100μL enzyme solution. The absorbance of samples was observed at 420 nm. Controlled cuvette contained 100 μL of purified water. The MnP activity was estimated by adding 1 mL MnSO4 in 1 mL malonate buffer with 0.5 μL H2O2 and 100 μL enzyme solution. Mn+3 forms a composite with malonate that is measured at the absorbance of 270 nm. Absorbance of sample will be taken after 10 min up to 30 min. Lignin peroxidase (LiP) test was done using 2.7 mL mixture along with 1 mL of buffer (pH 3) and veratryl alcohol (4 mM), 0.5 mL of H2O2, and 100 μL solution of enzyme and noted at 310 nm.


### 2.5. Biosorption Study

The spore suspension ofS. commune was obtained after autoclaving using filtration of inoculum of fungi. The dried biomass of S. commune was obtained after drying spore suspensions in oven at 80°C for 24 h. The dried biomass was finally grinded (Molineux, France) and sieved through (OCT-DIGITAL 4527-01) sieve. The 0.25 mm size of sample was selected for further use. The sample was now placed in sealed vessels.


### 2.6. Immobilization of Biomass and Batch Studies

One gram ofS. commune biomass was dissolved in 2% Na-alginate (100 mL). The mixture was reacted with 0.1 M CaCl2 after being homogenized to get beads of uniform size. This immobilized mass was used for further experimentations. Different conditions of pH ranging from 1 to 9, temperature from 30 to 70°C, biosorbent dose in the range of 0.05, 0.1, 0.15, 0.20, and 0.30 g, concentration of dye (25, 50, 75, and 100 ppm) and contact time of 24 h were studied to evaluate the removal of Drimaren Turquoise CL-B [47–49].The following equation was used to find out dye uptakeq (mg/g);
(1)q=Ci−CeV1000w,where Ci (mg/g) is the initial dye concentration, Ce (mg/g) is dye concentrations at any time, V (mL) represents volume of the solution, and w (g) is the mass of the sorbent. The collected data was analyzed statistically [39, 50].


## 2.1. Sporulation Media and Inoculum Preparation

The potato dextrose agar (PDA) is a general-purpose medium used for the culture of fungi, and to avoid contamination from bacterial growth, some antibiotics were also added in PDA medium. In the current study, for the preparation of WRF slant, PDA medium is used. For slant preparation, 100 mL distilled water was taken to dissolve 3.9 g PDA and autoclaved at 121°C for 15 min. After autoclave, the warm 10 mL medium was aseptically transferred into a sterilized culture tube, put on a slanting position on a slanting rack, and allowed to cool. In the culture medium, due to the presence of agar-agar, it is solidified on cooling. Using aseptic loop, few spores ofS. commune were transferred into slant using all possible sterilized conditions in laminar air flow [42].For the preparation of fresh culture of WRF, 2.4 g potato dextrose broth was dissolved into water and later on was sterilized. After sterilization, the flask was maintained on room temperature, the sterile tartaric acid (10%) was used to lower the pH of the medium up to 4.5 to inhibit the bacterial growth. Using sterilized condition in laminar air flow, few spores of white rot fungi from slant culture were inoculated into a medium flask and placed at 120 rpm; 30°C; 72 h for the required number of spore’s inoculum (1×108 spores/mL) at 100 μL sample. This was transferred to a UV spectrophotometer, and optical density of sample was measured at 600 nm.


## 2.2. Decolorization Procedure and Time Course Study

Six duplicate containers were placed for the reactive dyes. Each flask contained 0.01% of the respective dye along with 95 mL sterilized Kirk’s basal nutrient medium [43]. The pH of the media used for removal of dyes was kept at 4.5. Each flask was inoculated with 2 mL suspension of the strain of WRF after being autoclaved and cooled. These dyestuff sets were kept in aseptic laminar air flow. The incubation parameters for the flasks in shaking incubator were optimum temperature (30°C) and centrifugation speed (120 rpm) for 10 days. The dye with most removal affect was picked up to optimize the removal process [44]. After 24 h, the removed sample (1 mL) from flask was centrifuged for 10 min. The absorbance was taken at λmax to determine the removal (%) by S. commune. Maximum removal was perceived for the dye Drimaren Turquoise CL-B by the culture of S. commune. This dye was subjected to optimization of different parameters [45].


## 2.3. Process of Optimization

The pH of 0.01% Drimaren Turquoise CL-B was adjusted using different initial pH values starting from 3 and up to 5.5 before inoculation. 2 mL of the spore suspension of the fungal strain was inoculated after being autoclaved followed by incubation in orbital shaker (120 rpm) at 30°C. Samples were centrifuged using a Sigma centrifuge machine (Germany) at7379×g for 10 min at 25°C. The effect of temperature on dye removal was studied using 25, 30, 35, 40, and 45°C. Nutrients with dye solution was autoclaved and then 2 mL of inoculum was added. Samples were removed after 24 h from each flask, centrifuged at 7379×g for 10 min, and absorbance was read on a spectrophotometer [46].Different materials including wheat bran, fructose, rice bran, sucrose, glucose, and maltose were used for the removal of the Drimaren Turquoise CL-B dye by the culture ofS. commune in a shake flask experiment. The experiment was directed at optimal pH and temperature for about 7 days. Glucose was evidenced to be the finest carbon source for Drimaren Turquoise CL-B removal by the culture of S. commune. The ammonium salts (tartrates and chlorides) are the most widely used nitrogen sources for removal of dyes using fungus biomass. These nitrogen sources are costly, so there was a need to find out relatively optimized source and ammonium nitrate which are considered optimum source. Five duplicate flasks were maintained for different inoculum size such as 2, 3, 4, 5, and 6 mL to study the effect on dye removal. Similarly, the mediator effect on the removal of dye was also monitored.


## 2.4. Ligninolytic Enzyme Profile

The fermented samples were removed, at the end of each experiment, from removal media and centrifuged. This was done to find out the patterns related to working of enzymes involved in removal process of different dyestuffs by different fungi. Supernatant was withdrawn, kept at -4°C, and studied for determination of enzyme effects. Laccase action was performed by 1 mL of ABTS added in 1 mL buffer and 100μL enzyme solution. The absorbance of samples was observed at 420 nm. Controlled cuvette contained 100 μL of purified water. The MnP activity was estimated by adding 1 mL MnSO4 in 1 mL malonate buffer with 0.5 μL H2O2 and 100 μL enzyme solution. Mn+3 forms a composite with malonate that is measured at the absorbance of 270 nm. Absorbance of sample will be taken after 10 min up to 30 min. Lignin peroxidase (LiP) test was done using 2.7 mL mixture along with 1 mL of buffer (pH 3) and veratryl alcohol (4 mM), 0.5 mL of H2O2, and 100 μL solution of enzyme and noted at 310 nm.


## 2.5. Biosorption Study

The spore suspension ofS. commune was obtained after autoclaving using filtration of inoculum of fungi. The dried biomass of S. commune was obtained after drying spore suspensions in oven at 80°C for 24 h. The dried biomass was finally grinded (Molineux, France) and sieved through (OCT-DIGITAL 4527-01) sieve. The 0.25 mm size of sample was selected for further use. The sample was now placed in sealed vessels.


## 2.6. Immobilization of Biomass and Batch Studies

One gram ofS. commune biomass was dissolved in 2% Na-alginate (100 mL). The mixture was reacted with 0.1 M CaCl2 after being homogenized to get beads of uniform size. This immobilized mass was used for further experimentations. Different conditions of pH ranging from 1 to 9, temperature from 30 to 70°C, biosorbent dose in the range of 0.05, 0.1, 0.15, 0.20, and 0.30 g, concentration of dye (25, 50, 75, and 100 ppm) and contact time of 24 h were studied to evaluate the removal of Drimaren Turquoise CL-B [47–49].The following equation was used to find out dye uptakeq (mg/g);
(1)q=Ci−CeV1000w,where Ci (mg/g) is the initial dye concentration, Ce (mg/g) is dye concentrations at any time, V (mL) represents volume of the solution, and w (g) is the mass of the sorbent. The collected data was analyzed statistically [39, 50].


## 3. Results and Discussion

White rot fungi have the ability to degrade very complexed nature polymers, e.g., lignin which is characteristics of woody plants. Furthermore, WRF can also degrade dyes with the help of enzymes including manganese peroxidase (MnP) and lignin peroxidase (LiP). They can make use of some other enzymes for removal of dyes especially the ones producing H2O2. For removal of the largest class of dyes (azo dyes), WRF are better customer than other microorganisms [51]. Culture of WRF showed variable removal potential for different dyes (Table 1). The culture of S. commune removed Drimaren orange CL-3R up to 59.95%, Drimaren red CL-58 up to 63.95%, Drimaren black CL-CX up to 63.35%, Drimaren Navy CL-B 110 up to 89.50%, Drimaren Yellow CL-2R up to 91.94%, and the culture of S. commune showed maximum removal on Drimaren turquoise CL-B (97.17%) (Figure 1). During the dye removal, the aromatic ring cleavage occurs. This cleavage is related with the uniqueness of the ring substituents. With the presence of easily biodegradable functional groups, the removal process moved up to larger extent [52]. Drimaren Turquoise CL-B dye was selected for subsequent optimization studies.Table 1
Time course study for the removal of reactive dyes usingS. commune.
DyesRemoval (%)Days12345678910Drimaren Red CL-5839.444.5554.3558.859.8561.7562.7363.0163.9563.19Drimaren Orange CL-3R18.9530.5542.3555.8056.3558.1559.8559.9159.9559.83Drimaren Black CL-CX28.730.2048.2553.6559.0560.4562.0562.4563.3563.25Drimaren Navy CL-B 11059.3261.3085.889.5086.4087.2587.5388.2388.9188.75Drimaren Yellow CL-2R25.336.5538.8342.7742.8944.1244.4354.9567.3791.94Drimaren Turquoise CL-B44.0750.666.3572.6080.5194.6095.9596.6597.1796.20Figure 1
Removal of reactive dyes usingSchizophyllum commune culture.


### 3.1. Optimization of Removal Parameters

#### 3.1.1. Effect of Hydrogen Ion Concentration

Initial trial was run on pH 4.5. Different pH levels 3, 3.5, 4, 4.5, 5, and 5.5 were employed for the proper optimization of pH level for dye removal. The maximum dye removal after 7 days (95.45%) was observed at pH 4.5, followed by 90% at pH 4 and 89.85% at pH 3.5. 87.3% removal was observed at pH 5.5. At pH 3, the least dye removal (81.8%) was found. An inverse relationship between dye removal, and acidity level was observed. The minimum removal (81.8%) of dye was detected at pH 3. Enzymes ofS. commune did not work actively on highly acidic and basic pH (Tables 2 and 3; Figure 2). It means that for proper functioning of enzymes, pH is a very vital parameter. Mostly, acidic conditions are favorable for WRF Lignolytic enzymes. Previous studies reported the effect of pH on removal of Solar golden orange R using S. commune. The maximum removal efficiency (73%) was detected at pH 4.5 [42]. On the other hand, some enzymatic species show their efficiency in basic conditions. The pH range for white rot fungi is 4-8 [53].Table 2
Removal (%) of Drimaren Turquoise CL-B at different pH, temperature, carbon sources, nitrogen sources, inoculum size, and mediator effect.
DaysMean1234567pH364.578.179.0579.3579.7580.881.877.623.57184.1584.8586.1587.3188.6589.2586.6471.585.3185.9587.487.5589.659085.344.574.586.987.3586.4589.1591.295.4588.4457384.685.486.186.989.0589.8584.985.572.382.1582.384.6584.886.5587.382.86Temperature (°C)304882.884.487.3588.891.3591.882.07354982.590.190.390.693.0593.6584.174036.579.585.988.989.959292.6580.77452978.583.385.8587.890.6590.3577.92Carbon sourcesGlucose84.387.4590.9591.9593.193.3594.790.83Fructose79.2583.2588.9589.758787.5591.6586.77Maltose78.6581.587.1087.1587.5588.1588.7585.55Sucrose78.4581.4586.0587.0589.387.5387.8585.38Rice bran41.3451.2555.956.6556.7157.8557.9053.94Wheat bran69.1572.473.9572.8575.2574.178.9573.81Nitrogen sourcesAm. tartrate69.681.683.6583.57———79.6Am. nitrate68.291.5593.7595.15———87.16Urea35.1540.959.264.5———49.94Am. sulphate57.879.6585.390.45———78.3Peptone33.5543.4540.842.5———40.07Inoculum size (mL)259.3571.785.488.4589.690.5591.6482.38351.3576.3588.791.593.994.394.484.35452.377.689.791.392.0592.994.184.27554.1575.5587.991.490.592.993.183.64652.5578.18090.892.5593.1593.582.95MediatorABTS84.9891.193.17————89.75MnSO487.0390.7887.7————88.5Ver. alcohol89.0795.291.5————91.9Guaicol87.794.1992.15————91.3Table 3
Enzyme assay for dye screening experiment along with the effect of pH, temperature and carbon source.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)DyesDrimaren Red CL-580.05515.40.143122.980.166178.45Drimaren Orange CL-3R0.11532.20.137117.820.233250.475Drimaren Black CL-CX0.08222.960.142122.120.137147.275Drimaren Navy CL-B 1100.06718.760.344295.840.149160.175Drimaren Yellow CL-2R0.09125.480.134115.240.05660.2Drimaren Turquoise CL-B0.13337.240.05749.020.145155.875pH30.0174.760.02319.780.03133.3253.50.03710.360.02824.080.03739.77540.04913.720.05345.580.04952.6754.50.05415.120.06656.760.07984.92550.0267.280.05950.740.02223.655.50.0215.880.04135.260.01920.425Temperature (°C)300.09326.042.161857.60.06165.575350.14640.882.0517630.05660.2400.16847.042.131831.80.05559.125450.13437.522.011728.60.096103.2Carbon sourceGlucose0.12735.560.10388.580.04548.375Fructose0.10930.520.276237.360.04649.45Maltose0.11933.320.351301.860.023525.2625Sucrose0.1130.82.071780.20.026528.4875Rice bran0.20156.280.118101.480.01111.825Wheat bran0.17849.840.476409.360.053557.5125Figure 2
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) pH, (b) temperature, and (c) carbon source.
(a)(b)(c)


#### 3.1.2. Temperature

The medium was incubated on 30, 35, 40, and 45°C estimation of optimized level of temperature for highest dye removal by WRF. The maximum removal was observed in the medium incubated at 35°C. Kumar et al. [54] reported the same results for the removal of reactive azo dye. The optimum temperature found for WRF growth is 35°C, but some species (P. chrysosporium) even grows at higher temperature, i.e., 37°C [53]. Optimum temperature was found to vary between 25 and 37°C among isolates. Schizophyllum commune shows maximum activity on 35°C. The culture of S. commune showed 93.65% removal of dye at 35°C followed by 92.65% removal of dye on 40°C. On the other hand, the culture of white rot fungi showed less removal, i.e., 91.8% and 90.35% at 30 and 45°C, respectively. Results showed that at temperature 30°C and 40°C there is nonsignificant difference but at 45°C there is significance difference in the removal of dye.


#### 3.1.3. Carbon Sources

Different carbon sources were added to get the maximum removal of dye. The enzymes of WRF are not substrate specific, so they are produced to display their function on a wide range of substrates. Removal was done by all the substrates, but maximum removal was shown by glucose. Fructose is a monosaccharide with keto-group and shows furanose ring structure. Fructose showed 91.65% removal. Minimum removal was done by rice bran i.e. 57.90%. Wheat bran also showed less removal (78.95%). Maltose is a disaccharide of glucose and showed 88.75% removal.


#### 3.1.4. Evaluation of Nitrogen Sources

Various sources of nitrogen were added to the medium to evaluate their effects on removal process (Table4; Figure 3) and have shown variable effects. Another study reported that nitrogen addition had no considerable effect on dye removal using B. adusta along with P. tremellosa. Moreover, nitrogen source have made great contribution in reduction of removal time [55]. The maximum removal was observed using ammonium nitrate as a nitrogen source. All the other sources have shown less removal than nitrates, and the minimum was observed by peptone 42.5%.Table 4
Enzyme assay for different nitrogen sources, inoculum size, and mediator.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)Nitrogen sourceAmmonium tartrate0.12635.280.175150.50.0886Ammonium nitrate0.18150.680.312268.320.118126.85Urea0.2261.60.173148.780.173185.975Ammonium Sulphate0.11933.320.09480.840.094101.05Peptone0.21961.320.07665.360.07681.7Inoculum size (mL)20.17348.442.0761785.360.21225.7530.0164.480.229196.940.224240.840.0267.280.281241.660.186199.9550.08323.240.452388.720.247265.52560.0287.840.297255.420.07176.325MediatorABTS0.10429.122.01030.282.02144MnSO40.13136.680.676581.362.02144Veratryl alcohol0.06618.482.017200.392421.4Guaiacol0.12133.880.161138.462.02144Figure 3
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) nitrogen source, (b) inoculum size, and (c) mediator.
(a)(b)(c)


#### 3.1.5. Inoculum Size

To find out the outcome of inoculum size on the removal process of dye, 2 to 6 mL of inoculum were added. It has been observed that first there is an increase in the removal of Drimaren Turquoise CL-B by an increase in the inoculum size, but further increase in inoculum size caused less removal. Maximum removal was shown by 3 mL inoculum size, i.e., 94.4%.


#### 3.1.6. Mediators

The inducers or mediators have a great contribution for the production of ligninolytic enzymes when used along with WRF. For this reason, various inducers/mediators including veratryl alcohol, xyloidine, veratraldehyde, 3,4,5-trimethoxy, and cinnamic acid have been employed to evaluate the induction of ligninolytic enzymes of WRF. Veratryl alcohol is nonphenolic compound and is better substrate for LiP. On the other hand, it is not relatively good substrate for MnP [56].


### 3.2. Biosorption Study

#### 3.2.1. Effect of pH

The acidity and basicity of medium play a pivotal role with reference to removal process. In this study, pH value ranges from 1 to 9 were applied for evaluation of effect of pH on removal of Drimaren Turquoise CL-B usingS. commune (Table 5; Figure 4). We found decrease in equilibrium sorption capacity with rise in pH. The highest (15.74 mg/g) and lowest (0.325 mg/g) dye removal was noted at highly acidic and highly basic conditions, respectively. This shows that acidic medium favors the removal of dye and also helps the binding of dye with cationic fungal biomass. A similar result was described in previous study [57] for the removal of Reactive Navy-4 dye. In another study, maximum biosorption of Remazol Navy reactive dye was monitored at pH 2 [58].Table 5
Effect of pH, temperature, dye concentration, biosorbent dose, and time on the removal of Drimaren Turquoise CL-B usingS. commune biomass.
pHCe (mg/L)Ci−Ce (mg/L)q (mg/g)% removal118.5131.4915.74562.98227.2422.7611.3845.52342.917.093.54514.18445.524.482.248.96547.252.751.3755.5648.531.470.7352.94748.911.090.5452.18849.250.750.3751.5949.350.650.3251.3Temperature (K)30318.7331.2715.63562.5431316.7233.2816.6466.5632319.0630.9415.4761.8833321.01528.98514.492557.9734323.9826.0213.0152.04Dye concentration (mg/L)253.7321.2710.6385.085019.3330.6715.3361.347530.22544.7722.3859.6910044.5555.4527.7255.4520093.96106.0453.0253.02300149.26150.7475.3750.24Biosorbent dose (g)0.0517.0932.9132.9165.820.114.9335.07417.5370.1480.1513.8136.1912.0672.380.211.1938.819.7077.620.311.1938.819.7077.62Time (min)048.281.720.863.443024.2525.7512.87551.56022.3927.6113.80555.2212021.5728.4314.21556.8624020.5229.4814.74058.9636020.5229.4814.74058.9696020.6729.3314.6658.66144020.6729.3314.6658.66Figure 4
Effect of (a) pH, (b) temperature, and (c) dye concentration on the removal of Drimaren Turquoise CL-B bySchizophyllum commune biomass.
(a)(b)(c)


#### 3.2.2. Influence of Temperature

Temperature is very vital for the biosorption process. Since the textile dye effluents are discharged around (50–60°C), so temperature is vital design factor disturbing the sorption capacity using biomass. The biosorption of Drimaren Turquoise CL-B onto WRF performed at different temperatures exhibited that the sorption is an exothermic process. The sorption potential is decreased with increase in temperature. The maximum (16.64 mg/g) and minimum (13.01 mg/g) dye removal was recorded at 313 K and 343 K, respectively. Aksu and Tezer [59] explored the effect of heat change on the sorption of dye Remazol Black-B with the assistance from R. arrhizus. They have demonstrated that optimum temperature was 35°C for removal of dye and sorption capacity decreased rise in temperature. This may be attributed to the reduced surface activity.


#### 3.2.3. Effect of Initial Dye Concentration

Color removal efficiency is affected by dye initial concentration. Predominantly, the concentration brings a vital force to overwhelm the mass transfer phases of dye. The higher the initial concentration is, the higher the removal process will be. The removal of dye was analyzed using different initial dye concentrations (25, 50, 75, 100, 200, and 300 mg/L) of fungal biomass. With the rise in dye concentration, the increase in the amount of dye adsorbed was observed. Almeida et al. [60] reported the removal of MB with different concentration and concluded that MB was highly retained by the adsorbent as the initial dye concentration increased. Chu and Chen [61] inspected the consequence of concentration on removal of dye with the help of sludge. In their observation, they reported that sorption of dye increased with rise in dye concentration.


#### 3.2.4. Modeling for Equilibrium Studies

Demonstrating the equilibrium statistics provides detailed evidence for comparison among different materials under diverse working circumstances. This kind of data modelling is ultimate for dye removal process. To understand the removal process, a number of theories have been presented. Langmuir and Freundlich equations are the best tool to examine the association between sorbed (qe) and aqueous concentrations (Ce) at equilibrium. The Langmuir isotherm model defines the monolayer formation of solute molecules on the biosorbent surface. The following linearized form of equation relates the qmax (mg/g) and other parameters:
(2)Ceqe=1qmaxKL+CeXmwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution, and KL is Langmuir constants. The heterogeneous capacity, qe (mg/g) of S. commune biomass for Drimaren Turquoise CL-B, was also determined using Freundlich isotherm:
(3)logqe=1nlogCe+logKwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution (mg/L), and Kandn are Freundlich constants. The constants Kand1/n were determined by linear regression from the plot of logqe against logCe. K is a measure of the degree or strength of removal. The magnitude of the experimental qmax for S. commune biomass was found to be 75.37 mg/g (Table 6). The data suggest that Freundlich (Figure 5) model better represented the sorption process.Table 6
Comparison of Langmuir, Freundlich isotherms, pseudo-1st-order, and pseudo-2nd-order kinetics model parameters for removal of Drimaren turquoise CL-B.
DyeLangmuir isotherms parametersFreundlich isotherm parametersExperimental valuesDrimaren Turquoise CL-Bqmax (mg/g)KL (L/mg)R2qmax (mg/g)K (mg/g)1/nR2qmax (mg/g)117.650.00940.656975.036.880.17040.985275.37Pseudo-first-order kinetic modelPseudo-second-order kinetic modelExperimental valuesqe (mg/g)k1,ads(min-1)R2qe (mg/g)k2,ads (mg/g)R2qe (mg/g)2.110.002760.693814.680.135114.74Figure 5
Adsorption isotherms. (a) Freundlich and (b) Langmuir for the removal of Drimaren Turquoise CL-B usingS. commune biomass.
(a)(b)


#### 3.2.5. Effect of Biosorbent Dose and Contact Time

Biosorbent dose is considered a significant contributor in biosorption process. Figure6 represents the biosorbent dose effects on dye removal using fungal biomass. When the biosorbent dose was initially increased, the removal of dye increased but it was up to a certain limit (0.2 g). Following on, there was no effect of increase in biosorbent dose on the dye removal. The maxima (77.62%) were found at 0.2 g dose. Almeida et al. [60] have also elaborated similar effects for the MnP biosorption. The effect of contact time on dye removal process has been shown in Figure 6. The removal efficiency of dye was fast. Equilibrium time for the biosorption of Turquoise Reactive dye was found to be 240 min (qe 14.74 mg/g).Figure 6
Effect of (a) biosorbent dose and (b) time on the removal of Drimaren Turquoise CL-B.
(a)(b)


#### 3.2.6. Kinetic Modeling

To describe the kinetics of dye biosorption, several reaction-based and diffusion-based models were tested for the simulation of the obtained experimental data [62]. In this study, the biomass was employed as a free cell suspension where binding sites were readily available for dye uptake. So, the biosorption rate can be safely assumed. The pseudo-1st-order Lagergren model is generally expressed as
(4)logqe−q=logqe−k1ads2.303twhere qe (mg/g) and q are the amounts of adsorbed dye on the biosorbent at the equilibrium and at any time t, respectively; and k1ads is the Lagergren rate constant of the first-order biosorption. The pseudo-2nd-order model (Figure 7) is based on the assumption that the rate of occupation of removal sites is proportional to the square of the number of unoccupied sites:
(5)1q=1k2ads.qe2+1qetwhere k2ads is the rate constant of the 2nd-order biosorption (g/mg.min). The Lagergren model failed to estimate qe since the experimental values of qe were different from estimated. The biosorption process followed 2nd-order mechanism as indicated by a close agreement between experimental and estimated qe values along with higher R2. Other researchers reported almost similar kinds of results for removal of dyes [16, 63–69].Figure 7
Reaction kinetics plots. (a) Pseudo-1st-order and (b) pseudo-2nd-order for the removal of Drimaren Turquoise CL-B.
(a)(b)


## 3.1. Optimization of Removal Parameters

### 3.1.1. Effect of Hydrogen Ion Concentration

Initial trial was run on pH 4.5. Different pH levels 3, 3.5, 4, 4.5, 5, and 5.5 were employed for the proper optimization of pH level for dye removal. The maximum dye removal after 7 days (95.45%) was observed at pH 4.5, followed by 90% at pH 4 and 89.85% at pH 3.5. 87.3% removal was observed at pH 5.5. At pH 3, the least dye removal (81.8%) was found. An inverse relationship between dye removal, and acidity level was observed. The minimum removal (81.8%) of dye was detected at pH 3. Enzymes ofS. commune did not work actively on highly acidic and basic pH (Tables 2 and 3; Figure 2). It means that for proper functioning of enzymes, pH is a very vital parameter. Mostly, acidic conditions are favorable for WRF Lignolytic enzymes. Previous studies reported the effect of pH on removal of Solar golden orange R using S. commune. The maximum removal efficiency (73%) was detected at pH 4.5 [42]. On the other hand, some enzymatic species show their efficiency in basic conditions. The pH range for white rot fungi is 4-8 [53].Table 2
Removal (%) of Drimaren Turquoise CL-B at different pH, temperature, carbon sources, nitrogen sources, inoculum size, and mediator effect.
DaysMean1234567pH364.578.179.0579.3579.7580.881.877.623.57184.1584.8586.1587.3188.6589.2586.6471.585.3185.9587.487.5589.659085.344.574.586.987.3586.4589.1591.295.4588.4457384.685.486.186.989.0589.8584.985.572.382.1582.384.6584.886.5587.382.86Temperature (°C)304882.884.487.3588.891.3591.882.07354982.590.190.390.693.0593.6584.174036.579.585.988.989.959292.6580.77452978.583.385.8587.890.6590.3577.92Carbon sourcesGlucose84.387.4590.9591.9593.193.3594.790.83Fructose79.2583.2588.9589.758787.5591.6586.77Maltose78.6581.587.1087.1587.5588.1588.7585.55Sucrose78.4581.4586.0587.0589.387.5387.8585.38Rice bran41.3451.2555.956.6556.7157.8557.9053.94Wheat bran69.1572.473.9572.8575.2574.178.9573.81Nitrogen sourcesAm. tartrate69.681.683.6583.57———79.6Am. nitrate68.291.5593.7595.15———87.16Urea35.1540.959.264.5———49.94Am. sulphate57.879.6585.390.45———78.3Peptone33.5543.4540.842.5———40.07Inoculum size (mL)259.3571.785.488.4589.690.5591.6482.38351.3576.3588.791.593.994.394.484.35452.377.689.791.392.0592.994.184.27554.1575.5587.991.490.592.993.183.64652.5578.18090.892.5593.1593.582.95MediatorABTS84.9891.193.17————89.75MnSO487.0390.7887.7————88.5Ver. alcohol89.0795.291.5————91.9Guaicol87.794.1992.15————91.3Table 3
Enzyme assay for dye screening experiment along with the effect of pH, temperature and carbon source.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)DyesDrimaren Red CL-580.05515.40.143122.980.166178.45Drimaren Orange CL-3R0.11532.20.137117.820.233250.475Drimaren Black CL-CX0.08222.960.142122.120.137147.275Drimaren Navy CL-B 1100.06718.760.344295.840.149160.175Drimaren Yellow CL-2R0.09125.480.134115.240.05660.2Drimaren Turquoise CL-B0.13337.240.05749.020.145155.875pH30.0174.760.02319.780.03133.3253.50.03710.360.02824.080.03739.77540.04913.720.05345.580.04952.6754.50.05415.120.06656.760.07984.92550.0267.280.05950.740.02223.655.50.0215.880.04135.260.01920.425Temperature (°C)300.09326.042.161857.60.06165.575350.14640.882.0517630.05660.2400.16847.042.131831.80.05559.125450.13437.522.011728.60.096103.2Carbon sourceGlucose0.12735.560.10388.580.04548.375Fructose0.10930.520.276237.360.04649.45Maltose0.11933.320.351301.860.023525.2625Sucrose0.1130.82.071780.20.026528.4875Rice bran0.20156.280.118101.480.01111.825Wheat bran0.17849.840.476409.360.053557.5125Figure 2
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) pH, (b) temperature, and (c) carbon source.
(a)(b)(c)


### 3.1.2. Temperature

The medium was incubated on 30, 35, 40, and 45°C estimation of optimized level of temperature for highest dye removal by WRF. The maximum removal was observed in the medium incubated at 35°C. Kumar et al. [54] reported the same results for the removal of reactive azo dye. The optimum temperature found for WRF growth is 35°C, but some species (P. chrysosporium) even grows at higher temperature, i.e., 37°C [53]. Optimum temperature was found to vary between 25 and 37°C among isolates. Schizophyllum commune shows maximum activity on 35°C. The culture of S. commune showed 93.65% removal of dye at 35°C followed by 92.65% removal of dye on 40°C. On the other hand, the culture of white rot fungi showed less removal, i.e., 91.8% and 90.35% at 30 and 45°C, respectively. Results showed that at temperature 30°C and 40°C there is nonsignificant difference but at 45°C there is significance difference in the removal of dye.


### 3.1.3. Carbon Sources

Different carbon sources were added to get the maximum removal of dye. The enzymes of WRF are not substrate specific, so they are produced to display their function on a wide range of substrates. Removal was done by all the substrates, but maximum removal was shown by glucose. Fructose is a monosaccharide with keto-group and shows furanose ring structure. Fructose showed 91.65% removal. Minimum removal was done by rice bran i.e. 57.90%. Wheat bran also showed less removal (78.95%). Maltose is a disaccharide of glucose and showed 88.75% removal.


### 3.1.4. Evaluation of Nitrogen Sources

Various sources of nitrogen were added to the medium to evaluate their effects on removal process (Table4; Figure 3) and have shown variable effects. Another study reported that nitrogen addition had no considerable effect on dye removal using B. adusta along with P. tremellosa. Moreover, nitrogen source have made great contribution in reduction of removal time [55]. The maximum removal was observed using ammonium nitrate as a nitrogen source. All the other sources have shown less removal than nitrates, and the minimum was observed by peptone 42.5%.Table 4
Enzyme assay for different nitrogen sources, inoculum size, and mediator.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)Nitrogen sourceAmmonium tartrate0.12635.280.175150.50.0886Ammonium nitrate0.18150.680.312268.320.118126.85Urea0.2261.60.173148.780.173185.975Ammonium Sulphate0.11933.320.09480.840.094101.05Peptone0.21961.320.07665.360.07681.7Inoculum size (mL)20.17348.442.0761785.360.21225.7530.0164.480.229196.940.224240.840.0267.280.281241.660.186199.9550.08323.240.452388.720.247265.52560.0287.840.297255.420.07176.325MediatorABTS0.10429.122.01030.282.02144MnSO40.13136.680.676581.362.02144Veratryl alcohol0.06618.482.017200.392421.4Guaiacol0.12133.880.161138.462.02144Figure 3
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) nitrogen source, (b) inoculum size, and (c) mediator.
(a)(b)(c)


### 3.1.5. Inoculum Size

To find out the outcome of inoculum size on the removal process of dye, 2 to 6 mL of inoculum were added. It has been observed that first there is an increase in the removal of Drimaren Turquoise CL-B by an increase in the inoculum size, but further increase in inoculum size caused less removal. Maximum removal was shown by 3 mL inoculum size, i.e., 94.4%.


### 3.1.6. Mediators

The inducers or mediators have a great contribution for the production of ligninolytic enzymes when used along with WRF. For this reason, various inducers/mediators including veratryl alcohol, xyloidine, veratraldehyde, 3,4,5-trimethoxy, and cinnamic acid have been employed to evaluate the induction of ligninolytic enzymes of WRF. Veratryl alcohol is nonphenolic compound and is better substrate for LiP. On the other hand, it is not relatively good substrate for MnP [56].


## 3.1.1. Effect of Hydrogen Ion Concentration

Initial trial was run on pH 4.5. Different pH levels 3, 3.5, 4, 4.5, 5, and 5.5 were employed for the proper optimization of pH level for dye removal. The maximum dye removal after 7 days (95.45%) was observed at pH 4.5, followed by 90% at pH 4 and 89.85% at pH 3.5. 87.3% removal was observed at pH 5.5. At pH 3, the least dye removal (81.8%) was found. An inverse relationship between dye removal, and acidity level was observed. The minimum removal (81.8%) of dye was detected at pH 3. Enzymes ofS. commune did not work actively on highly acidic and basic pH (Tables 2 and 3; Figure 2). It means that for proper functioning of enzymes, pH is a very vital parameter. Mostly, acidic conditions are favorable for WRF Lignolytic enzymes. Previous studies reported the effect of pH on removal of Solar golden orange R using S. commune. The maximum removal efficiency (73%) was detected at pH 4.5 [42]. On the other hand, some enzymatic species show their efficiency in basic conditions. The pH range for white rot fungi is 4-8 [53].Table 2
Removal (%) of Drimaren Turquoise CL-B at different pH, temperature, carbon sources, nitrogen sources, inoculum size, and mediator effect.
DaysMean1234567pH364.578.179.0579.3579.7580.881.877.623.57184.1584.8586.1587.3188.6589.2586.6471.585.3185.9587.487.5589.659085.344.574.586.987.3586.4589.1591.295.4588.4457384.685.486.186.989.0589.8584.985.572.382.1582.384.6584.886.5587.382.86Temperature (°C)304882.884.487.3588.891.3591.882.07354982.590.190.390.693.0593.6584.174036.579.585.988.989.959292.6580.77452978.583.385.8587.890.6590.3577.92Carbon sourcesGlucose84.387.4590.9591.9593.193.3594.790.83Fructose79.2583.2588.9589.758787.5591.6586.77Maltose78.6581.587.1087.1587.5588.1588.7585.55Sucrose78.4581.4586.0587.0589.387.5387.8585.38Rice bran41.3451.2555.956.6556.7157.8557.9053.94Wheat bran69.1572.473.9572.8575.2574.178.9573.81Nitrogen sourcesAm. tartrate69.681.683.6583.57———79.6Am. nitrate68.291.5593.7595.15———87.16Urea35.1540.959.264.5———49.94Am. sulphate57.879.6585.390.45———78.3Peptone33.5543.4540.842.5———40.07Inoculum size (mL)259.3571.785.488.4589.690.5591.6482.38351.3576.3588.791.593.994.394.484.35452.377.689.791.392.0592.994.184.27554.1575.5587.991.490.592.993.183.64652.5578.18090.892.5593.1593.582.95MediatorABTS84.9891.193.17————89.75MnSO487.0390.7887.7————88.5Ver. alcohol89.0795.291.5————91.9Guaicol87.794.1992.15————91.3Table 3
Enzyme assay for dye screening experiment along with the effect of pH, temperature and carbon source.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)DyesDrimaren Red CL-580.05515.40.143122.980.166178.45Drimaren Orange CL-3R0.11532.20.137117.820.233250.475Drimaren Black CL-CX0.08222.960.142122.120.137147.275Drimaren Navy CL-B 1100.06718.760.344295.840.149160.175Drimaren Yellow CL-2R0.09125.480.134115.240.05660.2Drimaren Turquoise CL-B0.13337.240.05749.020.145155.875pH30.0174.760.02319.780.03133.3253.50.03710.360.02824.080.03739.77540.04913.720.05345.580.04952.6754.50.05415.120.06656.760.07984.92550.0267.280.05950.740.02223.655.50.0215.880.04135.260.01920.425Temperature (°C)300.09326.042.161857.60.06165.575350.14640.882.0517630.05660.2400.16847.042.131831.80.05559.125450.13437.522.011728.60.096103.2Carbon sourceGlucose0.12735.560.10388.580.04548.375Fructose0.10930.520.276237.360.04649.45Maltose0.11933.320.351301.860.023525.2625Sucrose0.1130.82.071780.20.026528.4875Rice bran0.20156.280.118101.480.01111.825Wheat bran0.17849.840.476409.360.053557.5125Figure 2
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) pH, (b) temperature, and (c) carbon source.
(a)(b)(c)


## 3.1.2. Temperature

The medium was incubated on 30, 35, 40, and 45°C estimation of optimized level of temperature for highest dye removal by WRF. The maximum removal was observed in the medium incubated at 35°C. Kumar et al. [54] reported the same results for the removal of reactive azo dye. The optimum temperature found for WRF growth is 35°C, but some species (P. chrysosporium) even grows at higher temperature, i.e., 37°C [53]. Optimum temperature was found to vary between 25 and 37°C among isolates. Schizophyllum commune shows maximum activity on 35°C. The culture of S. commune showed 93.65% removal of dye at 35°C followed by 92.65% removal of dye on 40°C. On the other hand, the culture of white rot fungi showed less removal, i.e., 91.8% and 90.35% at 30 and 45°C, respectively. Results showed that at temperature 30°C and 40°C there is nonsignificant difference but at 45°C there is significance difference in the removal of dye.


## 3.1.3. Carbon Sources

Different carbon sources were added to get the maximum removal of dye. The enzymes of WRF are not substrate specific, so they are produced to display their function on a wide range of substrates. Removal was done by all the substrates, but maximum removal was shown by glucose. Fructose is a monosaccharide with keto-group and shows furanose ring structure. Fructose showed 91.65% removal. Minimum removal was done by rice bran i.e. 57.90%. Wheat bran also showed less removal (78.95%). Maltose is a disaccharide of glucose and showed 88.75% removal.


## 3.1.4. Evaluation of Nitrogen Sources

Various sources of nitrogen were added to the medium to evaluate their effects on removal process (Table4; Figure 3) and have shown variable effects. Another study reported that nitrogen addition had no considerable effect on dye removal using B. adusta along with P. tremellosa. Moreover, nitrogen source have made great contribution in reduction of removal time [55]. The maximum removal was observed using ammonium nitrate as a nitrogen source. All the other sources have shown less removal than nitrates, and the minimum was observed by peptone 42.5%.Table 4
Enzyme assay for different nitrogen sources, inoculum size, and mediator.
LaccaseMnPLiPAbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)AbsEnzyme activity(U/mL/min)Nitrogen sourceAmmonium tartrate0.12635.280.175150.50.0886Ammonium nitrate0.18150.680.312268.320.118126.85Urea0.2261.60.173148.780.173185.975Ammonium Sulphate0.11933.320.09480.840.094101.05Peptone0.21961.320.07665.360.07681.7Inoculum size (mL)20.17348.442.0761785.360.21225.7530.0164.480.229196.940.224240.840.0267.280.281241.660.186199.9550.08323.240.452388.720.247265.52560.0287.840.297255.420.07176.325MediatorABTS0.10429.122.01030.282.02144MnSO40.13136.680.676581.362.02144Veratryl alcohol0.06618.482.017200.392421.4Guaiacol0.12133.880.161138.462.02144Figure 3
Removal of Drimaren Turquoise CL-B and enzyme activity ofSchizophyllum commune at different (a) nitrogen source, (b) inoculum size, and (c) mediator.
(a)(b)(c)


## 3.1.5. Inoculum Size

To find out the outcome of inoculum size on the removal process of dye, 2 to 6 mL of inoculum were added. It has been observed that first there is an increase in the removal of Drimaren Turquoise CL-B by an increase in the inoculum size, but further increase in inoculum size caused less removal. Maximum removal was shown by 3 mL inoculum size, i.e., 94.4%.


## 3.1.6. Mediators

The inducers or mediators have a great contribution for the production of ligninolytic enzymes when used along with WRF. For this reason, various inducers/mediators including veratryl alcohol, xyloidine, veratraldehyde, 3,4,5-trimethoxy, and cinnamic acid have been employed to evaluate the induction of ligninolytic enzymes of WRF. Veratryl alcohol is nonphenolic compound and is better substrate for LiP. On the other hand, it is not relatively good substrate for MnP [56].


## 3.2. Biosorption Study

### 3.2.1. Effect of pH

The acidity and basicity of medium play a pivotal role with reference to removal process. In this study, pH value ranges from 1 to 9 were applied for evaluation of effect of pH on removal of Drimaren Turquoise CL-B usingS. commune (Table 5; Figure 4). We found decrease in equilibrium sorption capacity with rise in pH. The highest (15.74 mg/g) and lowest (0.325 mg/g) dye removal was noted at highly acidic and highly basic conditions, respectively. This shows that acidic medium favors the removal of dye and also helps the binding of dye with cationic fungal biomass. A similar result was described in previous study [57] for the removal of Reactive Navy-4 dye. In another study, maximum biosorption of Remazol Navy reactive dye was monitored at pH 2 [58].Table 5
Effect of pH, temperature, dye concentration, biosorbent dose, and time on the removal of Drimaren Turquoise CL-B usingS. commune biomass.
pHCe (mg/L)Ci−Ce (mg/L)q (mg/g)% removal118.5131.4915.74562.98227.2422.7611.3845.52342.917.093.54514.18445.524.482.248.96547.252.751.3755.5648.531.470.7352.94748.911.090.5452.18849.250.750.3751.5949.350.650.3251.3Temperature (K)30318.7331.2715.63562.5431316.7233.2816.6466.5632319.0630.9415.4761.8833321.01528.98514.492557.9734323.9826.0213.0152.04Dye concentration (mg/L)253.7321.2710.6385.085019.3330.6715.3361.347530.22544.7722.3859.6910044.5555.4527.7255.4520093.96106.0453.0253.02300149.26150.7475.3750.24Biosorbent dose (g)0.0517.0932.9132.9165.820.114.9335.07417.5370.1480.1513.8136.1912.0672.380.211.1938.819.7077.620.311.1938.819.7077.62Time (min)048.281.720.863.443024.2525.7512.87551.56022.3927.6113.80555.2212021.5728.4314.21556.8624020.5229.4814.74058.9636020.5229.4814.74058.9696020.6729.3314.6658.66144020.6729.3314.6658.66Figure 4
Effect of (a) pH, (b) temperature, and (c) dye concentration on the removal of Drimaren Turquoise CL-B bySchizophyllum commune biomass.
(a)(b)(c)


### 3.2.2. Influence of Temperature

Temperature is very vital for the biosorption process. Since the textile dye effluents are discharged around (50–60°C), so temperature is vital design factor disturbing the sorption capacity using biomass. The biosorption of Drimaren Turquoise CL-B onto WRF performed at different temperatures exhibited that the sorption is an exothermic process. The sorption potential is decreased with increase in temperature. The maximum (16.64 mg/g) and minimum (13.01 mg/g) dye removal was recorded at 313 K and 343 K, respectively. Aksu and Tezer [59] explored the effect of heat change on the sorption of dye Remazol Black-B with the assistance from R. arrhizus. They have demonstrated that optimum temperature was 35°C for removal of dye and sorption capacity decreased rise in temperature. This may be attributed to the reduced surface activity.


### 3.2.3. Effect of Initial Dye Concentration

Color removal efficiency is affected by dye initial concentration. Predominantly, the concentration brings a vital force to overwhelm the mass transfer phases of dye. The higher the initial concentration is, the higher the removal process will be. The removal of dye was analyzed using different initial dye concentrations (25, 50, 75, 100, 200, and 300 mg/L) of fungal biomass. With the rise in dye concentration, the increase in the amount of dye adsorbed was observed. Almeida et al. [60] reported the removal of MB with different concentration and concluded that MB was highly retained by the adsorbent as the initial dye concentration increased. Chu and Chen [61] inspected the consequence of concentration on removal of dye with the help of sludge. In their observation, they reported that sorption of dye increased with rise in dye concentration.


### 3.2.4. Modeling for Equilibrium Studies

Demonstrating the equilibrium statistics provides detailed evidence for comparison among different materials under diverse working circumstances. This kind of data modelling is ultimate for dye removal process. To understand the removal process, a number of theories have been presented. Langmuir and Freundlich equations are the best tool to examine the association between sorbed (qe) and aqueous concentrations (Ce) at equilibrium. The Langmuir isotherm model defines the monolayer formation of solute molecules on the biosorbent surface. The following linearized form of equation relates the qmax (mg/g) and other parameters:
(2)Ceqe=1qmaxKL+CeXmwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution, and KL is Langmuir constants. The heterogeneous capacity, qe (mg/g) of S. commune biomass for Drimaren Turquoise CL-B, was also determined using Freundlich isotherm:
(3)logqe=1nlogCe+logKwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution (mg/L), and Kandn are Freundlich constants. The constants Kand1/n were determined by linear regression from the plot of logqe against logCe. K is a measure of the degree or strength of removal. The magnitude of the experimental qmax for S. commune biomass was found to be 75.37 mg/g (Table 6). The data suggest that Freundlich (Figure 5) model better represented the sorption process.Table 6
Comparison of Langmuir, Freundlich isotherms, pseudo-1st-order, and pseudo-2nd-order kinetics model parameters for removal of Drimaren turquoise CL-B.
DyeLangmuir isotherms parametersFreundlich isotherm parametersExperimental valuesDrimaren Turquoise CL-Bqmax (mg/g)KL (L/mg)R2qmax (mg/g)K (mg/g)1/nR2qmax (mg/g)117.650.00940.656975.036.880.17040.985275.37Pseudo-first-order kinetic modelPseudo-second-order kinetic modelExperimental valuesqe (mg/g)k1,ads(min-1)R2qe (mg/g)k2,ads (mg/g)R2qe (mg/g)2.110.002760.693814.680.135114.74Figure 5
Adsorption isotherms. (a) Freundlich and (b) Langmuir for the removal of Drimaren Turquoise CL-B usingS. commune biomass.
(a)(b)


### 3.2.5. Effect of Biosorbent Dose and Contact Time

Biosorbent dose is considered a significant contributor in biosorption process. Figure6 represents the biosorbent dose effects on dye removal using fungal biomass. When the biosorbent dose was initially increased, the removal of dye increased but it was up to a certain limit (0.2 g). Following on, there was no effect of increase in biosorbent dose on the dye removal. The maxima (77.62%) were found at 0.2 g dose. Almeida et al. [60] have also elaborated similar effects for the MnP biosorption. The effect of contact time on dye removal process has been shown in Figure 6. The removal efficiency of dye was fast. Equilibrium time for the biosorption of Turquoise Reactive dye was found to be 240 min (qe 14.74 mg/g).Figure 6
Effect of (a) biosorbent dose and (b) time on the removal of Drimaren Turquoise CL-B.
(a)(b)


### 3.2.6. Kinetic Modeling

To describe the kinetics of dye biosorption, several reaction-based and diffusion-based models were tested for the simulation of the obtained experimental data [62]. In this study, the biomass was employed as a free cell suspension where binding sites were readily available for dye uptake. So, the biosorption rate can be safely assumed. The pseudo-1st-order Lagergren model is generally expressed as
(4)logqe−q=logqe−k1ads2.303twhere qe (mg/g) and q are the amounts of adsorbed dye on the biosorbent at the equilibrium and at any time t, respectively; and k1ads is the Lagergren rate constant of the first-order biosorption. The pseudo-2nd-order model (Figure 7) is based on the assumption that the rate of occupation of removal sites is proportional to the square of the number of unoccupied sites:
(5)1q=1k2ads.qe2+1qetwhere k2ads is the rate constant of the 2nd-order biosorption (g/mg.min). The Lagergren model failed to estimate qe since the experimental values of qe were different from estimated. The biosorption process followed 2nd-order mechanism as indicated by a close agreement between experimental and estimated qe values along with higher R2. Other researchers reported almost similar kinds of results for removal of dyes [16, 63–69].Figure 7
Reaction kinetics plots. (a) Pseudo-1st-order and (b) pseudo-2nd-order for the removal of Drimaren Turquoise CL-B.
(a)(b)


## 3.2.1. Effect of pH

The acidity and basicity of medium play a pivotal role with reference to removal process. In this study, pH value ranges from 1 to 9 were applied for evaluation of effect of pH on removal of Drimaren Turquoise CL-B usingS. commune (Table 5; Figure 4). We found decrease in equilibrium sorption capacity with rise in pH. The highest (15.74 mg/g) and lowest (0.325 mg/g) dye removal was noted at highly acidic and highly basic conditions, respectively. This shows that acidic medium favors the removal of dye and also helps the binding of dye with cationic fungal biomass. A similar result was described in previous study [57] for the removal of Reactive Navy-4 dye. In another study, maximum biosorption of Remazol Navy reactive dye was monitored at pH 2 [58].Table 5
Effect of pH, temperature, dye concentration, biosorbent dose, and time on the removal of Drimaren Turquoise CL-B usingS. commune biomass.
pHCe (mg/L)Ci−Ce (mg/L)q (mg/g)% removal118.5131.4915.74562.98227.2422.7611.3845.52342.917.093.54514.18445.524.482.248.96547.252.751.3755.5648.531.470.7352.94748.911.090.5452.18849.250.750.3751.5949.350.650.3251.3Temperature (K)30318.7331.2715.63562.5431316.7233.2816.6466.5632319.0630.9415.4761.8833321.01528.98514.492557.9734323.9826.0213.0152.04Dye concentration (mg/L)253.7321.2710.6385.085019.3330.6715.3361.347530.22544.7722.3859.6910044.5555.4527.7255.4520093.96106.0453.0253.02300149.26150.7475.3750.24Biosorbent dose (g)0.0517.0932.9132.9165.820.114.9335.07417.5370.1480.1513.8136.1912.0672.380.211.1938.819.7077.620.311.1938.819.7077.62Time (min)048.281.720.863.443024.2525.7512.87551.56022.3927.6113.80555.2212021.5728.4314.21556.8624020.5229.4814.74058.9636020.5229.4814.74058.9696020.6729.3314.6658.66144020.6729.3314.6658.66Figure 4
Effect of (a) pH, (b) temperature, and (c) dye concentration on the removal of Drimaren Turquoise CL-B bySchizophyllum commune biomass.
(a)(b)(c)


## 3.2.2. Influence of Temperature

Temperature is very vital for the biosorption process. Since the textile dye effluents are discharged around (50–60°C), so temperature is vital design factor disturbing the sorption capacity using biomass. The biosorption of Drimaren Turquoise CL-B onto WRF performed at different temperatures exhibited that the sorption is an exothermic process. The sorption potential is decreased with increase in temperature. The maximum (16.64 mg/g) and minimum (13.01 mg/g) dye removal was recorded at 313 K and 343 K, respectively. Aksu and Tezer [59] explored the effect of heat change on the sorption of dye Remazol Black-B with the assistance from R. arrhizus. They have demonstrated that optimum temperature was 35°C for removal of dye and sorption capacity decreased rise in temperature. This may be attributed to the reduced surface activity.


## 3.2.3. Effect of Initial Dye Concentration

Color removal efficiency is affected by dye initial concentration. Predominantly, the concentration brings a vital force to overwhelm the mass transfer phases of dye. The higher the initial concentration is, the higher the removal process will be. The removal of dye was analyzed using different initial dye concentrations (25, 50, 75, 100, 200, and 300 mg/L) of fungal biomass. With the rise in dye concentration, the increase in the amount of dye adsorbed was observed. Almeida et al. [60] reported the removal of MB with different concentration and concluded that MB was highly retained by the adsorbent as the initial dye concentration increased. Chu and Chen [61] inspected the consequence of concentration on removal of dye with the help of sludge. In their observation, they reported that sorption of dye increased with rise in dye concentration.


## 3.2.4. Modeling for Equilibrium Studies

Demonstrating the equilibrium statistics provides detailed evidence for comparison among different materials under diverse working circumstances. This kind of data modelling is ultimate for dye removal process. To understand the removal process, a number of theories have been presented. Langmuir and Freundlich equations are the best tool to examine the association between sorbed (qe) and aqueous concentrations (Ce) at equilibrium. The Langmuir isotherm model defines the monolayer formation of solute molecules on the biosorbent surface. The following linearized form of equation relates the qmax (mg/g) and other parameters:
(2)Ceqe=1qmaxKL+CeXmwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution, and KL is Langmuir constants. The heterogeneous capacity, qe (mg/g) of S. commune biomass for Drimaren Turquoise CL-B, was also determined using Freundlich isotherm:
(3)logqe=1nlogCe+logKwhere qe is the dye sorbed (mg/g), Ce the equilibrium concentration of dye solution (mg/L), and Kandn are Freundlich constants. The constants Kand1/n were determined by linear regression from the plot of logqe against logCe. K is a measure of the degree or strength of removal. The magnitude of the experimental qmax for S. commune biomass was found to be 75.37 mg/g (Table 6). The data suggest that Freundlich (Figure 5) model better represented the sorption process.Table 6
Comparison of Langmuir, Freundlich isotherms, pseudo-1st-order, and pseudo-2nd-order kinetics model parameters for removal of Drimaren turquoise CL-B.
DyeLangmuir isotherms parametersFreundlich isotherm parametersExperimental valuesDrimaren Turquoise CL-Bqmax (mg/g)KL (L/mg)R2qmax (mg/g)K (mg/g)1/nR2qmax (mg/g)117.650.00940.656975.036.880.17040.985275.37Pseudo-first-order kinetic modelPseudo-second-order kinetic modelExperimental valuesqe (mg/g)k1,ads(min-1)R2qe (mg/g)k2,ads (mg/g)R2qe (mg/g)2.110.002760.693814.680.135114.74Figure 5
Adsorption isotherms. (a) Freundlich and (b) Langmuir for the removal of Drimaren Turquoise CL-B usingS. commune biomass.
(a)(b)


## 3.2.5. Effect of Biosorbent Dose and Contact Time

Biosorbent dose is considered a significant contributor in biosorption process. Figure6 represents the biosorbent dose effects on dye removal using fungal biomass. When the biosorbent dose was initially increased, the removal of dye increased but it was up to a certain limit (0.2 g). Following on, there was no effect of increase in biosorbent dose on the dye removal. The maxima (77.62%) were found at 0.2 g dose. Almeida et al. [60] have also elaborated similar effects for the MnP biosorption. The effect of contact time on dye removal process has been shown in Figure 6. The removal efficiency of dye was fast. Equilibrium time for the biosorption of Turquoise Reactive dye was found to be 240 min (qe 14.74 mg/g).Figure 6
Effect of (a) biosorbent dose and (b) time on the removal of Drimaren Turquoise CL-B.
(a)(b)


## 3.2.6. Kinetic Modeling

To describe the kinetics of dye biosorption, several reaction-based and diffusion-based models were tested for the simulation of the obtained experimental data [62]. In this study, the biomass was employed as a free cell suspension where binding sites were readily available for dye uptake. So, the biosorption rate can be safely assumed. The pseudo-1st-order Lagergren model is generally expressed as
(4)logqe−q=logqe−k1ads2.303twhere qe (mg/g) and q are the amounts of adsorbed dye on the biosorbent at the equilibrium and at any time t, respectively; and k1ads is the Lagergren rate constant of the first-order biosorption. The pseudo-2nd-order model (Figure 7) is based on the assumption that the rate of occupation of removal sites is proportional to the square of the number of unoccupied sites:
(5)1q=1k2ads.qe2+1qetwhere k2ads is the rate constant of the 2nd-order biosorption (g/mg.min). The Lagergren model failed to estimate qe since the experimental values of qe were different from estimated. The biosorption process followed 2nd-order mechanism as indicated by a close agreement between experimental and estimated qe values along with higher R2. Other researchers reported almost similar kinds of results for removal of dyes [16, 63–69].Figure 7
Reaction kinetics plots. (a) Pseudo-1st-order and (b) pseudo-2nd-order for the removal of Drimaren Turquoise CL-B.
(a)(b)


## 4. Conclusions

The following conclusions are drawn.A large amount (10-15%) of the dyes are discharged in the textile effluents during the dyeing process. The textile industries are not following the chemical and physical treatments for dye wastewater due to high cost and disposal problems. Nowadays, the industries are looking towards some green, cost-effective biosorbents for the removal of pollutants. WRF are efficient for the removal of dyes owing to their extra cellular ligninolytic enzyme system. Reactive dye, Drimaren turquoise CL-B, was decolorized usingS. commune. Different parameters effecting the removal process were optimized, and maximum removal (95.45%) was obtained at pH 4.5 and 35°C. As far as enzymatic activity is concerned, laccase and LiP activity was totally absent while MnP activity was dominant. Maximum biosorption was achieved at pH 1 and 313 K. There is a need to be more focused on such microorganisms and the conditions for their optimized working, which are able to biodegrade synthetic dyes in wastewater.



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*Source: 4725710-2023-02-02.xml*