2.6. Melanin Quantification

The concentration of melanin in shake flask supernatants was determined by spectrophotometric analysis by measuring the UV absorbance of the samples at 220 nm (Spectrophotometer V-530, Jasco, Tokio, Japan). A calibration curve was established in the range of 0.0005 to 0.01 g/L with the synthetic melanin standard after its dissolution in 0.1 M NaOH. To determine the melanin concentration in the supernatant samples, the initial measured absorbance of the growth medium was subtracted from the absorbance of each sample. The purity of the dried precipitated and purified melanin samples, obtained during the purification process, was calculated by determining the concentration of the melanin by measuring its absorbance at 220 nm after dissolving the samples in a defined volume of MilliQ water. Full UV-visible spectra, between 200 and 400 nm of the synthetic melanin standard of the 96 h supernatant cultures of the precipitated and purified melanin were also recorded during the purification process. In all cases, a 0.1 M NaOH solution was used as a blank.