One is Enough: Identifying Cell Material in a Single µm-sized Ice Grain Emitted by Enceladus or Europa

Introduction

The reliable identification of biosignatures on extraterrestrial ocean worlds is key to the search for life in our Solar System. Saturn's moon Enceladus, and potentially Jupiter's moon Europa, emit plumes of gas and ice grains formed from subsurface water into space (e.g. Spahn et al. 2006, Roth et al. 2014). The compositions of individual ice grains in such a plume can be analyzed during spacecraft flybys by impact ionization mass spectrometers, such as the Cosmic Dust Analyzer (CDA; Srama et al. 2004) on board the past Cassini mission, the SUrface Dust Analyzer (SUDA; Kempf et al. 2014) on board the upcoming Europa Clipper mission (Howell & Pappalardo 2020), or the ENceladus Ice Analyzer (ENIA), proposed for future Enceladus missions (Reh et al. 2016).

Analysis of CDA mass spectra collected in the Saturnian System revealed that Enceladus's ocean is salty (Postberg et al. 2009) and contains a variety of organic material, including complex macromolecules (Postberg et al. 2018) as well as low-mass volatile compounds (Khawaja et al. 2019). Our recent detection of phosphates in ice grains emitted by Enceladus (Postberg et al. 2022, under review) further enhances Enceladus's potential as a habitable environment, possibly able to support microbial life in its subsurface ocean.

Impact ionization detectors, such as SUDA or ENIA, are the only instruments capable of determining the compositions of single µm- or sub-µm-sized ice grains emitted by ocean world plumes. Cassini CDA results showed that refractory organics occur in only a few percent of plume ice grains and are believed to form from an organic film covering the oceanic surface (Postberg et al., 2018). If cell material is indeed present, it would likely reside in even fewer ice grains, potentially as many as just one in a thousand.

On Earth, 70% of the planetary surface is covered by a biofilm, the surface microlayer on top of the ocean water (Flemming and Wuertz, 2019), which hosts a distinct microbial community at cell densities 3 – 5 orders of magnitude higher than in the bulk water phase (Franklin et al. 2005). After lifting, organics and cells from this surface layer can initiate ice crystal formation in clouds (Pratt et al. 2009).

Methods and Results

To simulate a scenario, in which biosignatures are present only in a small fraction of emitted grains, but with relatively high cell densities, we conducted laboratory analogue experiments with Sphingopyxis alaskensis, an ultrasmall (volume of <0.1 µm3) bacterium, extracted from various cold marine environments (Ting et al. 2010). We simulated the case of an individual ice grain of 15 µm in diameter - constituting an extremely low cell density case for Enceladus where ice grains are generally much smaller (1-5 µm in diameter) - formed from a nucleation core of a single inactivated bacterial cell.

We used Laser Induced Liquid Beam Ion Desorption (LILBID) – a proven technique to accurately simulate impact ionization mass spectra of ice grains recorded byin space (Klenner et al. 2019, 2020, 2020a). Recent LILBID experiments demonstrated that trace amounts of DNA and lipids extracted from bacterial cultures will produce characteristic signals in the mass spectra of ice grains emitted from ocean worlds (Dannenmann et al. 2022, under review). Here we present the next steps – LILBID experiments with untreated lysed bacterial cells, simulating the appearance of these microbial life forms in impact ionization mass spectra of single 15 µm ice grains.

In both polarity mass spectra, we clearly identify signatures of S. alaskensis. Cationic mass spectra exhibit features due to protonated amino acids, either fragments of the bacteria's proteins or metabolic intermediates. Sequences of deprotonated fatty acids, fragments of bacterial lipids, are identifiable in anionic mass spectra. Our experiments show that even if less than 0.1% of the constituents of a single cell would form a nucleation core of a 15µm ice grain, the bacterial signature would be apparent in the data. The signal-to-noise ratio would be even higher in smaller ice grains.

Conclusions

Our results show that biosignatures deriving from a single bacterial cell – or small fractions thereof - embedded as a nucleation core in a single µm ice grain will be clearly identifiable in impact ionization mass spectra from SUDA-type instruments. This demonstrates the advantage of analyzing individual ice grains in a plume over analyzing the average composition of all plume material encountered during a flyby. A modern impact ionization instrument like SUDA or ENIA would be capable of recording 10,000 – 100,000 ice grain spectra (cationic and anionic) during a single plume passage, allowing the detection of biosignatures present in only 1 out of 100,000 grains during a multiple flyby mission. Such low abundances would be out of reach for other analytical methods that measure the integrated - and thus extremely diluted - concentrations of such biosignatures in a plume. The recorded spectra complement a comprehensive database containing analogue data for impact ionization mass spectrometers on board spacecraft (Klenner et al. 2022, under review), thereby aiding planning for future space missions to Enceladus or Europa.

References

Dannenmann, M. et al. (2022) Astrobiology, under review.

Flemming, H.-C. & Wuerz, S. (2019) Nat Rev Microbiol 17, 247–260.

Franklin, M.P. et al. (2005) Environ Microbiol 7, 723–736.

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

Kempf, S. et al. (2014) EPSC 9, EPSC2014–229.

Khawaja, N. et al. (2019) Mon Not R Astron Soc 489, 5231–5243.

Klenner, F. et al. (2019) Rapid Commun Mass Spectrom 33, 1751–1760.

Klenner, F. et al. (2020a) Astrobiology 20, 179–189.

Klenner, F. et al. (2020b) Astrobiology 20, 1168–1184.

Klenner, F. et al. (2022) Earth Space Sci, under review.

Postberg, F. et al. (2009) Nature 459, 1098–1101.

Postberg, F. et al. (2018) Nature 558, 564–568.

Postberg, F. et al. (2022) Nature, under review.

Pratt, K.A. et al. (2009) Nat Geosci 2, 398–401.

Reh, K. et al (2016) IEEE AeroConf Abstract, 1–8.

Roth, L. et al. (2014) Science 343, 171–174.

Spahn, F. et al. (2006) Science 311, 1416–1418.

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

Ting, L. et al. (2010) Environ Microbiol 12, 2658–267.