## 2 Methodology
**} & \multirow{2}{*}{**pH**} & \multirow{2}{*}{**Fungal**} & \multirow{2}{*}{**Days**} & \multirow{2}{*}{**Temp. **} & \multirow{2}{*}{**Effluent**} \\  & & & & (\({}^{\circ}\)C) & & & & & **Size (mL)** & & \\ \hline
8.0 & 6.0 & 02 & 32.5 & 0.030 & 6.0 & 6.0 & 05 & 32.5  
**Study of ligninolytic enzymes:** Three ligninolytic enzymes, i.e., LiP, MnP and Laccase, were studied during the biodegradation optimization, to check their role in the process. Lignin peroxidase activity assay was performed by the method of [17] by using veratryl alcohol (3,4-dimethoxybenzylalcohol) as a substrate. The reaction mixture contained 1 mL of tartarate buffer (100 mM, pH = 4), 1 mL veratryl alcohol and 500 \(\mu\)L H\({}_{2}\)O\({}_{2}\) (0.2 M). The reaction started after the addition of 100 \(\mu\)L of biodegradation mixture as an enzyme sample. The blank contained distilled H\({}_{2}\)O instead of enzyme sample. The activity was measured by taking the absorbance after the interval of 10 min at 310 nm and was calculated by using following formula. \[\textbf{A}=\textbf{E310}\times\textbf{c}\times\textbf{L}\]  
\[\textbf{C}310=9300\text{ M}^{-1}\text{cm}^{-1}\]  
where:  
\(\textbf{E}\)= molar extinction coefficient,  
\(\textbf{c}=\text{concentration}\),  
\(\textbf{A}=\text{absorbance}\),  
\(\textbf{L}=\text{path length}\). Manganese peroxidase activity assay was performed by the method described by [18]. Manganese sulfate (MnSO\({}_{4}\)) was used as a substrate and H\({}_{2}\)O\({}_{2}\) as an oxidizing ag/ ent. The reaction mixture consisted of 1 mL of MnSO\({}_{4}\), 1 mL sodium melonate buffer (50 mM, pH = 4.5) and 500 \(\mu\)L H\({}_{2}\)O\({}_{2}\). The addition of 100 \(\mu\)L of enzyme solution started the reaction. The complex products were monitored spectrophotometrically at 270 nm initially and after 10 min. The activity of MnP was calculated by using following formula. \[\textbf{A}=\textbf{C270}\times\textbf{c}\times\textbf{L}\]  
\[\textbf{C}\text{Z70}=\text{11},\text{590 M}^{-1}\text{cm}^{-1}\]  
Laccase activity assay was performed by the method reported by [19] by using ABTS as substrate. Laccase activity assay was performed by mixing 1 mL ABTS solution in 1 mL of sodium melonate buffer (50 mM, pH = 4.5) and a 100 \(\mu\)L enzyme sample. The absorbance  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**No. **} & \multirow{2}{*}{**Glucose (\%)**} & \multirow{2}{*}{**Fructose (\%)**} & \multicolumn{2}{c}{**Amm. **} & \multirow{2}{*}{**Amm. **} & \multirow{2}{*}{**Ammonia (\%)**} & \multirow{2}{*}{**No. **} & \multirow{2}{*}{**Glucose (\%)**} & \multirow{2}{*}{**Fructose (\%)**} & \multirow{2}{*}{**Amm. **} & \multirow{2}{*}{**Amm. **} & \multirow{2}{*}{**Amm. **} \\  & & & **Nitrate** & & & & & & & \\  & & **(\%)** & & & & & & & \\ \hline  & 1.00 & 1.00 & 2.00 & 2.00 & 2.00 & 1.00 & 2.00 & 1.00 & 2.00 & 1.00 \\  & 1.00 & 2.00 & 2.00 & 2.00 & 2.00 & 2.00 & 1.00 & 2.00 & 2.00 & 1.00 \\  & 2.00 & 1.00 & 1.  
was noted at 410 nm initially and after 10 min. The difference in absorbance was used to calculate the lactose activity by using following formula. \[\textbf{A}=\textbf{E436}\times\textbf{c}\times\textbf{L}\]  
\[\textbf{C436}=\textbf{36}\textbf{,}000\text{ M}^{-1}\text{cm}^{-1}\]  
**Determination of efficiency of biodegradation process:** To monitor the efficiency of _P. betulinus_ IEBL-3 during biodegradation and to check the efficacy of the process, BOD and COD values were measured before and after the treatment. **Determination of BOD**: The Biological Oxygen Demand was measured for 5 days by following the method reported by [20] and described earlier in [16]. The effluent under observation was packed in an airtight bottle for 5 days, and dissolved oxygen was measured initially as well as at the last day. The oxygen taken during these days was oxygen used by microorganisms for the biodegradation of the material.