2.4 Biochemical characterization
Biochemical analysis of all the samples were done for analyzing the changes in composition, i.e., total proteins, carbohydrates, and lipids content subjected to various growth conditions. The sulfo-phospho-vanillin test was used to estimate the total lipid content[44]. Briefly, 0.5 mg cells were pelleted and resuspended into 100 µL of distilled water. The cells were treated with 2 mL of 98 % H2SO4 and incubated at 100 °C for 10 minutes. After cooling for 5 minutes on ice, 5 mL of phosphovanillin reagent (0.6 g vanillin in 10 mL ethanol and 90 mL distilled water and 400 mL of 85 % phosphoric acid) was added, and the samples incubated for 10 minutes at 37 °C, absorbance was measured at 530 nm. Total carbohydrate was estimated using a modified phenol-sulfuric acid technique [45]. Around 0.25 mg cells were hydrolyzed; with 0.2 mL of 98 % H2SO4 at RT for 1 hour; following that, 5 % of phenol was added along with 1 mL of H2SO4 and incubated at RT for 20 minutes and the absorbance was measured at 490 nm. Protein content was quantified using a modified biuret technique. After pelleting 0.5 mg cells, 1 mL of extraction buffer (25 % NaOH in 1N methanol) was added. The reaction mixture was incubated at 80 °C for 15 minutes. To eliminate debris, the sample was cooled to RT and centrifuged. The supernatant was then treated with CuSO4 solution (0.21 % CuSO4 in 30 % NaOH) and kept at RT for 10 minutes before being measured at 310 nm[46].