*2.2.2 Preparation of mineral salt medium*

The mineral salt medium used in this study was composed of KH2PO4 (0.2 g/L), K2HPO4 (0.2 g/L), CaCl2.H2O (0.1 g/L), NaCl (0.8 g/L), MgSO4.7H2O (0.2 g/L), MnSO4.7H2O (0.01 g/L), FeSO4.7H2O (0.02 g/L), and yeast (2.0 g/L) with pH adjusted to 7.0. The basal mineral media was supplemented with 100 mg/L of BPA. These were sterilized in an autoclave at 15 psi and (121°C) for 20 minutes.

### *2.2.3 Actinobacterial growth during the biodegradation process*

Biomass concentration was estimated at every 24 h from the absorbance of appropriately diluted culture medium at 620nm according to the predetermined correlation between optical density and dry weight of biomass [34, 35]. The media were incubated at room temperature for 312 h at 160 r⋅min−1.

## **2.3 Effect of actinobacterial consortium on laccase production and BPA degradation**

The effect of actinobacterial consortium on laccase production and BPA degradation was investigated for a period of 312 h in sterilized mineral salt media supplemented with 100 mg/L of BPA at pH 7.0. The consortia were developed using equal volume of individual seed culture. BPA degradation efficiency and laccase production were monitored, as described below, at 24-h interval throughout the incubation period.

#### *2.3.1 Enzyme assays*

The activity of laccase was measured using the common substrate, ABTS (**E**420 = 36,000 M−1 cm−1) a modified method of Bourbonnais and Paice [36]. This was done by monitoring spectrophotometrically change in absorbance at 420 nm (A420) correlated with the rate of oxidation of 1-mM ABTS in 1-mM Tris–HCl buffer pH 7.0. The experiment was performed in 1-mL cuvettes at 30°C. Reaction mixture contained 750-μL ABTS and 250 μL of enzyme solution.

At an interval of 1 minute for 5 minutes, the absorbance of the mixture was measured. One unit of laccase activity was defined as the amount of enzyme that oxidized 1 mM of ABTS per minute under standard assay conditions. Laccase activity was expressed as U/mL. The enzyme activity was calculated using the Eq. (1).

× <sup>=</sup> × × *Absorbance Total volume of mixture minute Enzyme activity Total time Extinction coefficient volume of enzyme* (1)

#### *2.3.2 Determination of BPA degradation*

The percentage removal of BPA was determined using Folin–Ciocalteu reagent according to the method of Yordanova *et al.* [37]. The residual of BPA supplemented in the mineral salt media was determined at 24-h intervals throughout the degradation period. Aliquots of the culture media were withdrawn at intervals and centrifuged for 10 min at 3500 r⋅min−1. One milliliter of the supernatant was added to 10 mL of distilled H2O and 1 mL of Folin–Ciocalteu reagents. The mixture was left for 5 min, and 2 mL of 20% Na2CO3 (w/v) was added to the mixture. The solution was kept in the dark for 60 min, and therefore, absorbance at 750 nm was measured [37]. The degradation rate was expressed as the difference between the initial and final absorbance. This was estimated in percentage as follows (Eq. (2)):

$$\text{BPA Depradation} \left( \% \right) = \frac{\text{Initial } \left( \text{BPA} \right) \text{concentration} - \text{Final } \left( \text{BPA} \right)}{\text{Initial } \left( \text{BPA} \right) \text{concentration}} \times 100 \tag{2}$$

### **2.4 Gas chromatography: mass spectrometry (GC: MS) analysis of BPA degradation metabolites**

From the quantitative confirmation analysis employing a Shimadzu gas chromatograph GC-2010 series connected to a Shimadzu spectrometer GCMS-QP2010 PLUS (Japan), the separation of the compounds was achieved by employing a DB5MS capillary column (60 m) (Supelco), the carrier gas was helium and maintained at constant flow of (0.9 mL⋅min−1). A sample volume of 1 μL was injected in the splitless mode at an inlet temperature of 280°C. The MS transfer line temperature was maintained at 280°C, whereas the ion source temperature was 180°C.
