## 3 Results and Discussion
Figure 6: _Cont._  
**Study of ligninolytic enzymes during biodegradation:** Fungi have a non-specific ligninolytic enzyme system; they produce these enzymes in response to nutrients in the surrounding. The dyes have aromatic structures and induce the production of ligninolytic enzymes, which lead to dye oxidation and degradation [2]. The ligninolytic enzymes including lignin peroxidase (LiP), manganese peroxidase (MnP) and increase were studied parallel to the biodegradation during each optimization step. It was observed that the activities of all three ligninolytic enzymes were increased with their increase in the biodegradation process. These findings confirmed the involvement of these enzymes in the biodegradation of textile effluents. After the optimization of physical parameters, maximum activities were 831.65 \(\pm\) 5.43 UmL\({}^{-1}\)min\({}^{-1}\), 609.86 \(\pm\) 2.98 UmL\({}^{-1}\)min\({}^{-1}\) and 325.27 \(\pm\) 4.12 UmL\({}^{-1}\)min\({}^{-1}\) for LiP, MnP and lactose, respectively. The increase in the activities were observed after the addition of carbon and nitrogen sources that cause an increase in the biodegradation of the effluents. The enzymatic activities were 925.54 \(\pm\) 4.98 UmL\({}^{-1}\)min\({}^{-1}\), 687 \(\pm\) 3.21 UmL\({}^{-1}\)min\({}^{-1}\) and 398.20 \(\pm\) 2.34 UmL\({}^{-1}\)min\({}^{-1}\) for LiP, MnP and lactose, respectively. These results suggest the role of ligninolytic enzymes of _P. betulinus_ IEBL-3 in the biodegradation of the textile industrial effluents. A higher enzymatic activity of LiP indicated that this enzyme has a more active role in the biodegradation of industrial effluents under the study compared to the other two enzymes [25,26]. The ligninolytic enzymes were involved in the oxidation of phenolic substrates, which are part of the dyes in the effluents. The oxidized products of phenolics dyes act as a substrate for other enzymes, which are produced in fewer amounts by the fungi [27]. [PERSON] et al. [27] also reported the biodegradation of dyes by ligninolytic enzymes from fungi and found that there was an enhancement in the biodegradation with an increase in enzymatic activity. The azo dyes are decolorized by ligninolytic enzymes produced by fungi when the mixture is supplemented with optimized conditions. This decolorization is achieved by the oxidative degradation of dyes contents in the mixture by ligninolytic enzymes including LiP, MnP and lactose [3,28]. **HPLC analysis of the effluents:** During the biodegradation process, fungi degrade the complex dyes by an oxidation process, absorbing some into its biomass or mineralizing them into resultant elements. To monitor this, HPLC was performed before and after the biological treatment of the effluents by _P. betulinus_ IEBL-3. There were three secondary amines available as standards, i.e., N-methylaniline, 3-methyldiphenylamine and phenylamin (Figure 7). Monitoring of the industrial effluents by HPLC showed a degradation of the dyes and the apparition of new peaks. Some of them may correspond to smaller  
Figure 6: Response surface 3D graphs showing effect of carbon and nitrogen sources on the biodegradation of Five Star Textile (MT) industrial effluent by _P. betulinus_ IEBL-3; graphs represent positive interaction between (**a**) ammonia and fructose, (**b**) ammonia and a. nitrate and (**c**) a. sulfate and a. nitrate. (**d**) Statistical analysis indicating the significant effect of parameters. molecules such as N-methylaniline, 3-methyldiphenylamine and phenylalanine, while others remain unknown (Figures 8-10) [29]. The current findings indicated that _P. betulinus_ IEBL-3 has the ability of biodegradation textile industrial effluent under optimized conditions and can be used for wastewater treatment at industrial scale. The degradation or mineralization of dyes in the effluents is due to the activity of ligninolytic enzymes including manganese peroxidase, lignin peroxidase and lacase.