*2.5. Characterization of Melanin Production in the Culture Filtrate*

The dry weights of the fungal biomass, melanin in the culture filtrate, and residual concentrations of glucose and total nitrogen (TN) were monitored throughout the cultivation process. Spore suspensions (106 spores/mL) of the strain were prepared using the above-described methods. Afterward, 1 mL of the suspension was inoculated into individual flasks containing 100 mL of sterilized peptone yeast glucose (PYG; peptone: 10 g/L; yeast: 5 g/L; glucose: 20 g/L) media and PYG media supplemented with 1 mM CuSO4. The glucose concentration of the PYG media was adjusted from 5 g/L to 20 g/L. The inoculated culture media were maintained at a constant 150 rpm agitation on a rotary shaker at 27 ◦C. At each measurement point, the biomass and cell-free culture media were separated by centrifugation. The cells were weighed after filtering the sample through Whatman 1.2 μm glass fiber filters (Clifton, NJ, USA). The obtained supernatant was then acidified (pH 2) with 1 M HCl and incubated for 24 h at 21 ◦C to enable melanin formation. The newly formed melanin pellets were weighed after filtering the sample as described above. The filtered supernatant excluding the melanin pellets was used to measure the residual glucose and TN concentrations. The residual glucose concentrations were monitored using a high-performance liquid chromatography (HPLC) system (Shimadzu, Tokyo, Japan) equipped with a refractive index detector (RID-20A, Shimadzu, Tokyo, Japan) and an Aminex HPX-87H ion exchange column (300 mm × 7.8 mm) (Bio-Rad, Hercules, CA, USA) at a 0.5 mL/min flow rate. The mobile phase was a 5 mM H2SO4 solution prepared in deionized water. TN concentrations were determined using the HS-TN(CA)-L kit and an HS-1000PLUS water analyzer (Humas, Daejeon, Korea).
