Abstract EANA2025-68

Enceladus, an icy moon of Saturn, has emerged as a key target in the search for extraterrestrial life due to its subsurface ocean, proposed hydrothermal activity, and geochemically promising conditions. Cassini mission data suggest that this ocean contains molecular hydrogen (H₂), carbon dioxide (CO₂), and methane (CH₄), as well as moderate salinity (0.5-4 % NaCl), alkaline pH (8.5-11), and temperatures close to 0 °C. These findings support the hypothesis of ongoing serpentinization processes and point toward an environment potentially habitable for certain types of anaerobic microorganisms — particularly hydrogenotrophic methanogens.

In this project, we investigate the viability and potential metabolic activity of four methanogenic archaea — Methanococcoides burtonii, Methanosarcina barkeri, Methanosarcina soligelidi, and Methanobacterium subterraneum — under Enceladus-analog conditions. These species were selected based on their known physiological tolerances, including psychrotolerance, halotolerance, alkaliphily, and anaerobic metabolism.

Growth experiments are conducted in minimal media, adjusted to simulate Enceladus-like salinity (2-4% NaCl), pH (7.0-9.0), and temperature (4-15 °C), using a fractional factorial design to reduce experimental complexity while covering the relevant environmental space. Methane production, as a proxy for metabolic activity, is measured via headspace pressure changes. Survival is assessed by colony-forming unit (CFU) counts following serial dilution and plating.

In addition to habitability studies, multifactorial stress experiments investigate organismal survival after desiccation, freeze-thaw cycles, and optionally ionizing radiation. As comparative benchmarks, the radiation- and freeze-tolerant bacterium Planococcus halocryophilus and the desiccation-tolerant halophile Salinisphaera shabanensis are included as control strains.

This study contributes to our understanding of the limits of life under extraterrestrial conditions and informs future life-detection missions and planetary protection strategies. Anaerobic archaea, particularly methanogens, have received little attention in planetary protection protocols despite their high environmental resilience and potential for survival under space-relevant stress. This research supports a more comprehensive assessment of microbial contamination risks associated with space missions.

In the broader context, insights from this work may also benefit biotechnological applications. The robustness of methanogenic archaea under cold, saline, and alkaline conditions offers potential for the development of industrial biocatalysts suitable for low-energy or resource-limited settings.