Experiments for the Identification of Bacterial Cell Material in Single Ice Grains Emitted by Enceladus and Europa
Authors: Klenner Fabian , Bönigk Janine , Napoleoni Maryse , Dannenmann Marie , Pavlista Miriam , Khawaja Nozair , Hillier Jon , Olsson-Francis Karen , Postberg Frank 
Published: September 2022

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&lt;p&gt;&lt;strong&gt;Introduction&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;The reliable identification and quantification of biosignatures on extraterrestrial ocean worlds is key to the search for life in our Solar System. Saturn&amp;#8217;s moon Enceladus, and potentially Jupiter&amp;#8217;s moon Europa, emit plumes of gas and ice grains formed from subsurface water into space (Spahn et al. 2006, Roth et al. 2014). The ice grains can be sampled 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 &amp; Pappalardo 2020), or the ENceladus Ice Analyzer (ENIA), proposed for future Enceladus missions (Reh et al. 2016).&lt;/p&gt;
&lt;p&gt;CDA data collected in the Saturnian System revealed that Enceladus&amp;#8217;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), some of which could potentially act as amino acid precursors. Our recent detection of phosphates in ice grains emitted by Enceladus (Postberg et al. 2022) further enhances Enceladus&amp;#8217;s potential as a habitable environment, possibly able to support microbial life in its subsurface ocean. However, biosignatures have not yet been identified on extraterrestrial ocean worlds.&lt;/p&gt;
&lt;p&gt;Impact ionization detectors, such as SUDA or ENIA, are the only instruments capable of determining the compositions of single &amp;#181;m-sized ice grains emitted into plumes. Cassini CDA results showed that refractory organics occur in only a few % of plume ice grains. These are thought to form from an organic film covering the oceanic surface (Postberg et al., 2018).&lt;/p&gt;
&lt;p&gt;Cell material, if present, would likely reside in such grains and would only be present in an even smaller number of ice grains, maybe just one in a thousand or less. 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 &amp;#8211; 5 orders of magnitude higher than in the bulk water phase (Franklin et al. 2005). After lifting, organics and cells from this layer can initiate ice crystal formation in clouds (Pratt et al. 2009).&lt;/p&gt;
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&lt;p&gt;&lt;strong&gt;Methods and Results&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;To simulate such a scenario, where biosignatures are only present in a small fraction of emitted grains, but with a high cell density, we conducted laboratory analogue experiments with &lt;em&gt;Sphingopyxis alaskensis&lt;/em&gt;, an ultrasmall bacterium, extracted from various cold marine environments (Ting et al. 2010) and potentially capable of fitting into &amp;#181;m-sized ice grains. We simulated the case of an ice grain of 15&amp;#181;m in diameter - constituting the worst case for Enceladus where ice grains are generally much smaller (1-5 &amp;#181;m in diameter) - formed from a nucleation core of one single bacterial cell.&lt;/p&gt;
&lt;p&gt;We used Laser Induced Liquid Beam Ion Desorption (LILBID) &amp;#8211; a proven technique to simulate accurately impact ionization mass spectra of ice grains recorded in space (Klenner et al. 2019, 2020, 2020a). Recent LILBID experiments predict that 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 &amp;#8211; LILBID experiments with otherwise untreated lysed bacterial cells simulating the appearance of these microbial life forms in impact ionization mass spectra.&lt;/p&gt;
&lt;p&gt;In both polarity mass spectra, we clearly identify signatures of &lt;em&gt;S. alaskensis&lt;/em&gt;. Cation mass spectra exhibit features due to protonated amino acids, either fragments of the bacteria&amp;#8217;s proteins or metabolic intermediates. Deprotonated fatty acids, fragments of bacterial lipids, are identifiable in anion mass spectra. Our experiments show that even if less than 0.1% of the cell constituents would form a nucleation core of a 15&amp;#181;m ice grain, the bacterial signature would be apparent in the data. The recorded spectra are part of a comprehensive database containing analogue data for impact ionization mass spectrometers on board spacecraft (Klenner et al. 2022, under review).&lt;/p&gt;
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&lt;p&gt;&lt;strong&gt;Conclusions&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;Our results show that biosignatures deriving from a single bacterial cell &amp;#8211; or small fractions of it - embedded as a nucleation core in an 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 &amp;#8211; 100,000 ice grain spectra (cations and anions) during a single plume passage, allowing to assess biosignatures that are 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.&lt;/p&gt;
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&lt;p&gt;&lt;strong&gt;References&lt;/strong&gt;&lt;/p&gt;
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