Detecting carotenoids in salt crystals: insights into biosignature detection under Mars-like proton irradiation using in situ and ex situ Raman spectroscopy

Abstract The search for biosignatures of past microbial life has promoted the interest in halophilic archaea trapped inside fluid inclusions of salt crystals. These hypersaline environments are promising targets for the preservation of microbial cell envelope biomolecules. In this study, we focused on the preservation of bacterioruberin, a carotenoid pigment found in the cell envelope of Halobacterium salinarum , within fluid inclusions of salt crystals mimicking early Mars environments and modern Earth. Halite (NaCl) and sylvite (KCl) crystals were subjected to Mars-like proton irradiation, and the preservation of carotenoids was assessed using in situ and ex situ Raman spectroscopy. Our findings demonstrate that Raman spectroscopy efficiently detected carotenoids within fluid inclusions in non-irradiated crystals. However, post-irradiation analyses posed great challenges due to fluorescence induced by the formation of colour centres in the crystal lattice, which suppressed the carotenoid signal. Cleavage of irradiated crystals revealed preserved carotenoid pigments beyond the radiation penetration depth, suggesting potential preservation of biomolecules in deeper inclusions within larger crystals. Furthermore, in some cases, carotenoids were detected even within fluorescent zones, suggesting extensive preservation. This study underscores the potential of Raman spectroscopy for the detection of carotenoids as biosignatures in planetary exploration contexts, particularly as a preliminary screening tool. However, it also highlights the need for optimized protocols to overcome fluorescence-related limitations. These findings contribute to the methodologies for detecting and interpreting biosignatures in salt deposits, advancing the search for possible traces of past microbial life beyond Earth.