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].