Growing a putative inhabitant of Enceladus ocean surface

Saturns icy moon Enceladus is a prime target in the search for extraterrestrial life in our Solar System. Its subsurface ocean fulfils essential criteria for life as we know it on Earth: liquid water 1, organic chemistry 2, 3 and a potential energy source from hydrothermal activity induced by tidal heating 4, 5. At its south pole, ice grains form from the ocean and are ejected through cracks in the ice shell, transporting the ocean water to space where it can be detected by flyby missions 6, 7.The Cosmic Dust Analyzer (CDA) 8, an impact ionization mass spectrometer onboard the Cassini-Huygens spacecraft, analyzed the composition of single ejected ice grains, providing insights into the composition of the moderately salty and alkaline ocean 4, 6, 7. Interpretation of CDA data was aided by complementary analogue experiments with a Laser Induced Liquid Beam Ion Desorption Mass Spectrometer (LILBID-MS) that simulates impact ionization with laser desorption 9. The LILBID technique can also predict the mass spectral appearance of bacterial biosignatures in ice grains detected by future impact ionization mass spectrometers 10, 11. However, past experiments have not considered the influence of ambient environmental conditions on the growth of putative extraterrestrial cells and the biosignatures they produce.To be enclosed in ice grains, cells have to be present at the ocean surface 2. Thus, they either need to grow in proximity to the ice-ocean interface or be transported upwards from deeper habitats. Here, we investigate the first scenario of viable cells at the ocean surface, represented by Rhodonellum psychrophilum, an alkaliphilic psychrophile, as a model organism.At its surface, the ocean is likely stratified by a thin salt-depleted layer with NaCl concentrations of 0.05 M 6, 12 and pH 9-11 7, 13, 14. This lies within the range of the optimal growth conditions for R. psychrophilum: 0 0.1 M NaCl and pH 10 15. Water temperatures reach the freezing point at the ice-ocean interface, ca. -0.1 C for an aqueous 0.05 M NaCl solution, and increase by approximately 2 C over the stratified layer 12. Although optimal growth of R. psychrophilum occurs at 5 C, it can sustain temperatures down to 0 C 15. As a strictly aerobic chemoheterotroph, it would rely on transport of radiolytic oxygen from the irradiated icy surface 16 and organic carbon from hydrothermal sites at the ocean floor 2, 3.We first grew R. psychrophilum at 6 C in its optimal growth medium (R2-A medium 15) adjusted to pH 10 with Na2CO3 to demonstrate its suitability as a representative of putative alkaliphilic cells in Enceladus ocean. In ongoing experiments, we now aim to set up the culture in an Enceladus ocean water simulant based on CDA data and existing models of the ocean that consists of the following inorganic compounds: 0.05 M NaCl, 0.01 M KCl, up to 0.1 M Na2CO3 (variable for pH adjustment), 0.002 M Na3PO4, 0.001 M SiO2, and 0.001 M NH3 4, 6, 7, 13, 14, 17, 18, 19. Organic carbon, sulfur and additional nitrogen sources will first be supplied by the addition of R2-A medium but finally substituted by organic compounds detected in the ocean 2, 3, 18. We will incubate R. psychrophilum in the dark at 6 C to obtain optimal biomass yield 15 and at 0 C to simulate growth in surface waters. Finally, the cells will be analyzed by LILBID-MS to predict their mass spectral appearance in ejected ice grains.Cultivating bacteria in simulated Enceladus ocean water will illustrate its potential for supporting life and yield biosignatures reflective of authentic environmental settings, increasing the predictive power of biosignature detection experiments.1 Thomas, P. C. et al. (2016). lcarus, 264, 37-47.2 Postberg, F. et al. (2018). Nature, 558(7711), 564-568.3 Khawaja, N. et al. (2019). MNRAS, 489(4), 5231-5243.4 Hsu, H.-W. et al. (2015). Nature, 519(7542), 207- 210.5 Waite, J. H. et al. (2017). Science, 356(6334), 155-159.6 Postberg, F. et al. (2009). Nature, 459(7250), 1098-1101.7 Postberg, F. et al. (2011). Nature, 474(7353), 620-622.8 Srama, R., et al. (2004). Space Sci. Rev. 114, 465518.9 Klenner, F. et al. (2019). Rapid Commun. Mass Spectrom., 33(22), 1751-1760.10 Dannenmann, M. et al. (2023). Astrobiology, 23(1), 60-75.11 Klenner, F. et al. (2024). Sci. Adv., 10(12), eadl0849.12 Bouffard, M., et al. (2023). preprint https://doi.org/10.21203/rs.3.rs-2398898/v113 Glein, C. R. et al. (2018). In Enceladus and the Icy Moons of Saturn, Schenk P. M. et al. (eds). University of Arizona Press, Tucson, AZ, p. 39.14 Postberg, F. et al. (2023). Nature 618, 489-493.15 Schmidt, M. et al. (2006). INT J SYST EVOL MICR, 56(12), 2887-2892.16 Ray, C. et al. (2021). Icarus, 364, 114248.17 Postberg et al. (2021). In AGU Fall Meeting Abstracts, Vol. 2021, pp. P32A-05.18 Peter, J. S. et al. (2024). Nat. Astron., 8(2), 164-173.19 Fifer, L. M. et al. (2022). Planet. Sci. J., 3(8), 191.