*3.2. Detection of Pigments by Confocal Raman Spectroscopy*

Colonies of *C. antarcticus* grown on different analogues were analyzed by Confocal Raman spectroscopy after exposure to simulated space and Mars-like conditions. The results of Raman analyses identified the melanin spectra with two main peaks, according to [37]: an intense and broad peak at 1590–1605 cm−<sup>1</sup> and a second peak at lower wavenumber at 1340 cm−<sup>1</sup> (Figure 2). The presence of these peaks is probably due to the aromatic C-N bonds for the first peak [38] and to the stretching of the C-C bonds within the rings of the aromatic melanin monomers for the second peak [39]. The presence of other peaks and shoulders in all spectra, with the most prominent one around 1425 cm<sup>−</sup>1, served as a proof that the samples did not undergo thermal degradation and that the signal acquired is of melanin and not burnt organic matter (or amorphous carbon). Figure S1 shows the signal coverage for the image scan analyses, calculated from the application of a SNR superior to

5; results were normalized to the corresponding SNR of the non-irradiated sample (Ctr) for each analogue.

**Figure 2.** Raman spectra of melanin pigments of *C. antarcticus* grown on (**A**) OS exposed to space simulated conditions; (**B**) P-MRS and (**C**) S-MRS exposed 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 Control (sample kept in the lab, in the dark at room temperature) = green spectrum. OS = Original Substrate, P-MRS = Phyllosilicatic Mars Regolith Simulant, S-MRS = Sulfatic Mars Regolith Simulant.

No significant melanin changes are reported in all the collected spectra; similar peak positions (1602–1604 cm<sup>−</sup>1) were detected in samples exposed to simulated space conditions (OS) and in samples exposed to Mars-like conditions (P-MRS and S-MRS), both in Bottom and Top conditions, in comparison with the respective Control samples (Figure S2A,B).
