Abstract EANA2026-26

The search for life beyond Earth strongly relies on terrestrial analogue environments capable of preserving subtle molecular and isotopic traces of biological activity. Volcanic caves are particularly relevant in this context because their basaltic substrates, protected interiors and steep physicochemical gradients resemble potential subsurface habitats on Mars. Among them, Capitão Kidd Cave, located on Selvagem Grande Island (Madeira, Portugal), represents an exceptional but still unexplored oceanic subterranean system where mineral deposits may act as long-term archives of microbial and environmental processes.

This study presents a multiproxy biogeochemical characterisation of organic matter preserved in nine speleothem samples from Capitão Kidd Cave (CK), combining bulk carbon isotope analysis (EA/IRMS), Pyrolysis-Compound-Specific Isotope Analysis (Py-CSIA), direct-infusion Orbitrap ultra-high-resolution mass spectrometry with 3D van Krevelen visualisation, and GDGT lipid biomarker analysis. Bulk δ¹³C values showed a strong agreement with compound-specific δ¹³C signatures obtained from linear n-alkanes by Py-CSIA, supporting the robustness of the pyrolysis-based isotopic approach for microgram-scale cave mineral matrices. Compound-specific δ¹³C values of individual n-alkanes revealed strongly depleted signatures in several speleothem samples, with values below −35‰, pointing to a possible contribution of methane-related microbial processes. Conversely, sediment sample CK03 and speleothem sample CK09 exhibited δ¹³C values consistent with C3 photosynthetic sources, including photosynthetic microorganisms. A particular case is represented by sample CK03, where the simultaneous occurrence of long- and short-chain n-alkanes, with gradual change of isotope values, suggests a mixed organic matter origin, potentially involving plant-derived material, seabird-associated inputs and decomposer microbial biomass.

Ultra-high-resolution molecular fingerprints further revealed a chemically diverse organic pool, including lipids, protein-like compounds, carbohydrates, oxygenated molecules, aromatic structures, unsaturated hydrocarbons and CRAM-like components. These molecular domains indicate the coexistence of relatively labile biological signatures and more transformed organic matter within the cave deposits. In parallel, GDGT distributions were dominated by archaeal isoprenoid lipids, including isoGDGT-0, isoGDGT-1, isoGDGT-2, isoGDGT-3 and crenarchaeol, suggesting a relevant archaeal contribution to the preserved organic signal. The existence of these glycerol lipids is in line with the very light isotope signature of speleothem samples, which may support the existence of specific archaea communities that produce these particular lipidic compounds.

The preservation of molecular, isotopic and lipid biosignatures in the speleothems of Selvagem Grande reinforces the value of basaltic cave systems as terrestrial analogues for the Martian subsurface. The integration of ultra-high-resolution molecular profiling with compound-specific isotope analysis offers a sensitive and minimally destructive strategy for detecting faint organic signals in complex mineral matrices. In the framework of the ExoMars Rosalind Franklin Mission, these results provide relevant analytical and interpretative criteria for future life-detection strategies on Mars.

Acknowledgements: The Spanish Ministry of Science, Innovation and University from the Spanish State Agency (MICIU/AEI/10.13039/501100011033) is acknowledged for funding project HERMES (ref. PID2024-162087NB-C21). This work was also supported by the Portuguese Foundation for Science and Technology (FCT) through the MICROCENO project (PTDC/CTA-AMB/0608/2020).