## 2 Methodology  
**Chemicals and Reagent:** 2,2'-azino-bis3-ethylbenzothazoline-6-sulfonic acid (ABTS), Manganese sulfate and veratryl alcohol were of Sigma-Aldrich and purchased from the local supplier. Ligninolytic enzymes (Laccase, lignin peroxidase and manganese peroxidase) were produced from _P. betulinus_ IEBL-3. Untreated industrial effluents (Mujahid textiles (MT), Sitara Textiles (ST) and Five Star Textile (FST) were collected from the local industrial units. All other chemicals used for the enzymatic assay, media preparation were of analytical grade, for HPLC analysis chemicals were of HPLC grade and purchased from local chemical supplier. **Fungal collection, isolation and maintenance:** _P. betulinus_ IEBL-3 was obtained from the IEBL Laboratory, Department of Biochemistry Arid Agriculture University Rawalpindi. It was isolated and maintained on the malt extract agar media having pH 5.5. The culture plates were streaked aseptically with a loop full of fungal spores from the pure culture obtained from IEBL. The plates were rapped and put in an incubator at 30 \({}^{\circ}\)C for 4 days. The culture obtained after day 4 was used to prepare the broth culture. Agar-less malt extract broth media ( malt extract 20 g/L, glucose 20 g/L, peptone 3 g/L and distilled water) was prepared in a flask, inoculated aseptically and placed in a shaking incubator for 3-4 days. The fungal spores in the broth were maintained at 1 \(\times\) 10\({}^{7}\) to 10\({}^{9}\) spores/mL and monitored with a Biomass Monitor (ABER-220). It was used for the biodegradation of effluents [11]. **Biodegradation of textile effluents:** Considering the dyes biodegradation potential of _P. betulinus_ IEBL-3 [12] the biodegradation of industrial effluents was optimized in the current study for expanding its industrial applications. For this, 90 mL of effluent was taken in a 250 mL flask, mixed with 10 mL broth media and fungal inoculum, and the effluent concentration was maintained as mentioned in Table 1. The experimental mixture was put on a shaking incubator at 120 rmp for a specified time period (Table 1). The absorbance was checked before and after the experiment at \(\lambda\)max of each effluent by withdrawing 2 mL of the sample from the mixture. The biodegradation process was optimized at two stages with a variation of physical and nutritional parameters by using the Response Surface Methodology (RSM) under the Box Bhenken Design (BBD) [5]. At each stage five parameters were optimized simultaneously, and experiments were designed using the JMP software [13]. The MT effluent has a burgundy color, \(\lambda\)max = 513 nm and initial pH 10.92. The FST effluent has dark blue color, \(\lambda\)max = 642 nm and initial pH 10.45. Finally, the ST effluent has dark red color, \(\lambda\)max = 518 nm and initial pH 13.32. For the biodegradation process, the pH was adjusted in an acidic range by adding acid. **Optimization of physical parameters**: The fungal growth and its biodegradation potential is associated with certain factors such as pH, temperature, time period, dye conc. and fungal inoculum size [14]. These parameters were optimized simultaneously by using RSM under BBD. The experimental design results in 46 experimental combinations with six central points (Table 1). **Optimization of nutritional parameters:** The presence of nutrients increases the growth of the strain, and, as a consequence, the biodegradation (co-metabolic biodegradation) process may be enhanced. To check this, two carbon sources (i.e., glucose and fructose) and three nitrogen sources (i.e., ammonium sulfate, ammonium nitrate and ammonia) were added simultaneously as a mixture in the biodegradation medium. There were 46 with 6 central points, under BBD and all were run in triplicate (Table 2) [15; 16]. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**No. **} & \multirow{2}{*}{**pH**} & \multirow{2}{*}{**Fungal**} & \multirow{2}{*}{**Days**} & \multirow{2}{*}{**Temp. **} & \multirow{2}{*}{**Effluent**} & \multirow{2}{*}{**No.