2.5. Pigment extraction, purification and yield analysis

The pigment extraction process was initiated by dissolving the whole of the harvested biomass in 50 mL of 100 % methanol (v/v) assisted by vortexing to confirm a homogenous solution. The pigment appeared to be inside the cell as the centrifuged broth exhibited no extraction of the produced pigment. Hence the pigment was concluded to be intracellular. The dissolved cells of isolated bacterial strains were then lysed by ultrasonication treatment in a bath sonicator (Digital Ultrasonic Cleaner LCMU-2, Labman Scientific Instruments, Chennai, India) for 30 min. After sonication, the solution mixture was centrifuged at 7000 rpm at room temperature for 15 min to obtain a colorless pellet and colored supernatant [26,58]. This methanol solution along with the extracted pigment was then air-dried for 3 days in a dust-free environment. After drying, the extracted crude pigment was weighed.

The purification of the crude pigment was performed by Column Chromatography assisted with Thin Layer Chromatography (TLC). Initially, a homogenous mixture of dried crude pigment and silica were prepared and column chromatography was performed using dissolved Silica Gel G (GRM7480, Himedia, New Delhi, India) in chloroform in 1:4 (w/v) ratio. The fractions were eluted using a solvent mixture of ethanol:chloroform in 1:4 (v/v) ratio [59,60]. Each eluted fractions were examined by TLC analysis in pre-coated TLC plates (TLC Silica Gel 60 F₂₅₄, Merck, India) using the same solvent as the mobile phase for examining the purity of the fractions. For each TLC run, the retention factor (R_f) value was calculated by Eq. (1) [27].

Elute showing single mobile front having identical R_f value was considered as a single component. Such fractions were cumulated and the solvent was evaporated to obtain the purified dried component. From the obtained data, stoichiometric equations were produced to represent each of the steps and net yield was calculated.