Detecting biosignatures of B. subtilis spores in ice grains ejected from icy moons

The subsurface oceans of icy moons in our Solar System, such as Jupiter’s moon Europa and Saturn’s moon Enceladus, are prime targets in the search for extraterrestrial life. At Enceladus’ south pole, ice grains of ocean water are ejected into space in a plume and can be detected by instruments on flyby missions [1,2]. The Cosmic Dust Analyzer (CDA) [3], an impact ionization mass spectrometer on the Cassini spacecraft, analyzed the composition of ejected ice grains and thereby the ocean, that is moderately salty [1], alkaline [2,4,5] and contains organic compounds [6,7]. Although less is currently known about the conditions in the subsurface of Europa, this will soon change when it is investigated by the Europa Clipper and JUICE missions in the next decade [8, 9]. Europa Clipper is equipped with a next generation impact ionization mass spectrometer, the SUrface Dust Analyser (SUDA), to analyze the composition of ice grains ejected from the moon’s surface or interior [10]. Interpretation of CDA data was aided by an analogue laboratory experiment that employs the Laser Induced Liquid Beam Ion Desorption (LILBID) technique and simulates impact ionization with laser desorption [11]. LILBID can also predict the detectability of molecular biosignatures enclosed in ice grains for future mass spectrometers, such as SUDA [12-15].

Potential extraterrestrial microorganisms adapted to one site within the oceans of the icy moons may be exposed to adverse environmental conditions when dispersed to different locations, which could induce sporulation in putative spore-forming microorganisms [16]. At the interface of the ocean and rocky core of the icy moons, hydrothermally heated seawater (e.g., >90°C for Enceladus [17]) mixes with the cold ocean generating temperature gradients. Under similar conditions on Earth, spore-forming bacteria have been isolated from warm hydrothermal vent environments that form endospores to withstand temperature fluctuations [18]. The resilient endospores of such bacteria can disperse widely to cold waters and survive over long timescales [19]. The lower end of transport times within the ocean of Enceladus has recently been estimated to range from hours to weeks [20]. These or longer time scales are consistent with sporulation times of common spore formers on Earth, such as Bacillus subtilis [21]. Spores are highly resistant to environmental stressors [22] and have been found to survive exposure to surface conditions of Enceladus and Europa including severe radiation [23]. This renders them promising targets for the analysis of surface material by Europa Clipper's SUDA or lander missions, such as ESA’s planned L4 mission [24].

We analyzed the molecular biosignatures of B. subtilis spores and vegetative cells in a water matrix with the LILBID technique to determine their detectability in ice grains by impact ionization mass spectrometers.

We found that biosignatures of B. subtilis spores (1.80 mg/mL) and vegetative cells (4.77 mg/mL) would be detectable in the ice grains. Mass spectral features of the biological material included mass peaks of various amino acids and small organic compounds that, while not necessarily a biotic identifier on their own, can indicate the presence of life if detected in combination. For spores specifically, the amino acids arginine in cation spectra and glutamic acid in anion spectra were readily detectable at low concentrations. Dipicolinic acid (DPA) also yielded characteristic mass peaks and fragments of the compound could be identified in both cation and anion mass spectra of the spores. This would be a unique identifier for sporulation, since DPA is a characteristic component of various bacterial spores but absent in vegetative cells [25]. The analogue spectra are collected in a database [26] that will aid interpretation of mass spectra obtained on future space missions.

We show that future impact ionization mass spectrometers, such as Europa Clipper’s SUDA, would be able not just to identify characteristic biomolecules derived from B. subtilis spores and vegetative cells, but could specify their differing biosignatures. Our work highlights the value of impact ionization mass spectrometers in the exploration of icy worlds and the predictive capabilities of the LILBID technique for future space missions to Europa or Enceladus, like the recently announced L4 mission by ESA [23] or a New Frontiers mission by NASA [27].

[1] Postberg, F. et al. (2009). Nature 459(7250), 1098-1101.

[2] Postberg, F. et al. (2011). Nature 474(7353), 620-622.

[3] Srama, R., et al. (2004). Space Sci Rev 114, 465–518.

[4] Glein, C. R. et al. (2018). In Enceladus and the Icy Moons of Saturn, p. 39.

[5] Postberg, F. et al. (2023). Nature 618, 489-493.

[6] Postberg, F. et al. (2018). Nature 558(7711), 564-568.

[7] Khawaja, N. et al. (2019). MNRAS 489(4), 5231-5243.

[8] Howell, S.M. & Pappalardo, R.T (2020). Nat Commun 11, 1311.

[9] Grasset, O. et al. (2013). Planet Space Sci 78, 1-12.

[10] Kempf, S. et al. (2025). Space Sci Rev 221, 10.

[11] Klenner, F. et al. (2019). Rapid Commun Mass Spectrom 33(22), 1751-1760.

[12] Klenner, F. et al. (2020a). Astrobiology 20(2), 179-189.

[13] Klenner, F. et al. (2020b). Astrobiology 20(10), 1168-1184.

[14] Dannenmann, M. et al. (2023). Astrobiology 23(1), 60-75.

[15] Klenner, F. et al. (2024). Sci Adv 10(12), eadl0849.

[16] Piggot, P. J., & Hilbert, D. W. (2004). Curr Opin Microbiol 7(6), 579-586.

[17] Hsu, H.-W. et al. (2015). Nature 519(7542), 207- 210.

[18] Elsgaard, L., & Prieur, D. (2011). J Gt Lakes Res 37(1), 203-206.

[19] De Rezende, J. R. et al. (2013). The ISME journal, 7(1), 72-84.

[20] Bouffard, M. et al. (2025) Nat Astron (2025).

[21] Driks, A. (2002). Cell Mol Life Sci 59, 389–391.

[22] Setlow, P. (2016). In The bacterial spore: from molecules to systems, 201-215.

[23] Vincent, L.N. et al., (2024). Commun Earth Environ 5, 688.

[24] Martins, Z. et al., Report of the Expert Committee for the Large-class mission in ESA’s Voyage 2050 plan.

[25] Perry, J.J. & Foster J.W. (1955) J Bacteriol 69, 337.

[26] Klenner, F. et al. (2022). Earth Space Sci 9, e2022EA002313.

[27] NASA Planetary Science and Astrobiology Decadal Survey 2023-2032.