*2.8. Extraction and Identification of Secondary Metabolites*

The secondary metabolites were extracted by a solvent extraction method from filtrates, where ethyl acetate and filtrate in a ratio of 1:1 (*v*/*v*) was used to extract exhaustively. The upper layer of the solvent contains the compounds which were collected separately from the aqueous layer (PDB medium) using a separation funnel. The solvent ethyl acetate was evaporated from the filtrate using a vacuum rotary evaporator at 40 ◦C, 70 rpm until the extract got reduced to 4 mL; it was maintained at −20 ◦C in the deep freezer until further use. Ethyl acetate extracts were analyzed by Gas Chromatography Mass Spectroscopy (GC-MS) analysis. The GC-MS analysis was performed using Thermo Scientific, Ceres 800, MS DSQ II (Waltham, MA, USA) and the silica column was packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250 µm df). For the separation of components, helium

gas was used as a carrier gas, which maintained the constant flow of 1 mL/min. The temperature of the injector was maintained at 260 ◦C, which was set for the chromatographic run. The sample of 1 µL of extract was injected into the instrument where the temperature of the oven was 60 ◦C (2 min), followed by 300 ◦C at the rate of 10 ◦C·min−<sup>1</sup> and 300 ◦C for 6 min. The conditions for the mass detector were a temperature of 230 ◦C for the transfer line, a temperature of 230 ◦C for the ion source, an ionization mode electron impact at 70 eV, a scan time of 0.2 s and a scan interval of 0.1 s. The comparison of spectrum of components were carried out with the database of spectrum of known components stored in the GC-MS NIST (2008) library [43].
