*3.1. Detection of Pigments by Spectrophotometric Analyses*

Spectrophotometric analyses were performed on fungal melanin extracted from *C. antarcticus* colonies after exposure to simulated space conditions (OS) and Mars-like conditions (P-MRS and S-MRS). The wavelength of maximum absorbance was scanned at a range of 200 to 800 nm. The UV-visible absorbance spectrum of the purified pigments showed a strong absorbance in the UV region, and a characteristic absorption peak was observed at 230 nm corresponding to typical melanin UV absorption. The strong absorption in the UV region with a progressive decrease at high wavelength is due to the presence of complex conjugated structures in the melanin molecule [34,35]. The decrease in the absorption with increasing wavelength is linear in the case of melanin.

Figure 1 shows the melanin spectra extracted from samples exposed to simulated space UV radiation (OS Top; red line), vacuum (OS Bottom; blue line), to Mars-like UV radiation (P-MRS and S-MRS Top; red line) and Mars-like atmosphere (P-MRS and S-MRS Bottom; blue line) compared with relative control (black line). No change in melanin absorbance at 230 nm has been reported in all the experimental conditions (Top, Bottom and Control), compared with those reported in [36] for extracted melanin pigments from *C. antarcticus*. In addition, UV–VIS analyses of melanin pigments extracted for P-MRS and S-MRS analogues revealed a bulge at ~300 nm, not shown in OS spectra, probably due to the presence of regolith during the detection process.

**Figure 1.** UV–VIS-spectra of melanin pigment extracted from *C. antarcticus* colonies grown on (**A**) OS exposed to simulated space conditions; (**B**) P-MRS and (**C**) S-MRS exposed to Mars-like conditions on different layers: Top (exposed to sun light with 0.1% Neutral Density filters) = red spectrum, Bottom (dark control in space, not exposed to space radiation) = blue spectrum and CTR (sample kept in the lab, in the dark at room temperature) = black spectrum. OS = Original Substrate, P-MRS = Phyllosilicatic Mars Regolith Simulant, S-MRS = Sulfatic Mars Regolith Simulant.
