2.1.2. Fungal Melanin Extraction

Melanin was extracted from dried colonies of *C. antarcticus* grown on different substrata, optimizing the protocol reported by [26]. Fungal cells were collected by centrifugation at 16,100 rcf for 20 min, washed with phosphate-buffered saline (PBS) (pH 7.4). Cells were suspended in 20 μL of a buffer composed by 0.25 μL of enzyme from *Trichoderma harzarium* (Sigma #L1412, St. Louis, MO, USA), 50 μL of 0.2 M sodium citrate (pH 5.5), 15 μL of 0.2 M citric acid and 18.2 g of sorbitol, up to the final volume of 500 μL.

Samples were incubated overnight at 30 ◦C, shaken at 50 rpm, washed with PBS two times and collected by centrifugation (10 min at 16,100 rcf). The supernatant was removed by adding 1 mL of 4 M guanidine thiocyanate (VWR International srl, Radnor, PA, USA), and samples were incubated overnight on stirrer. Samples were washed with PBS and centrifuged for 15 min at 16,100 rcf for two times. Then, 1 mg/mL of Proteinase K, previously dissolved in the reaction buffer (4 μL of TRIS HCl, 1 μL of CaCl2 and 1 mL of SDS, up the final volume of 20 mL) was added to the samples; they were incubated at 37 ◦C for 4 h and after centrifuged for 5 min at 16,100 rcf. Samples were washed two times by adding 1 mL of PBS 4×, and three times by adding 1 mL of chloroform.

A volume of 2 mL of a 6 M HCl solution was added to the samples, and they were boiled for 1 h. Samples were transferred in dialysis membrane (SnakeSkin Dialysis Tubing, 3.5 K MWCO-Thermo Scientific, Waltham, MA, USA) in sterile water for 3 days, changing the water every day. Finally, samples were lyophilized overnight with the Lyophilizer FreeZone 2.5 L (Freeze Dry Systems, LabConco, Kansas city, MO, USA) and then used for the analyses.
