From Microorganisms to Biosignatures: Subcritical Water Extraction as a Sample Preparation Technique for Future Life Detection Missions

Abstract

The discovery of potentially habitable conditions on Europa and Enceladus has led to an increased interest in searching for extant life in these locations (Chyba & Phillips, 2002;Porco et al., 2017). There is a consensus that multiple lines of evidence will be required to provide the extraordinary proof required to support any claims of the detection of extraterrestrial life (Hand et al., 2017). Among the possible lines of evidence, chemical biosignatures are considered key because of the way that life is expected to leave an imprint on chemical distributions, or by the presence of chemical complexity, that is difficult to explain via abiotic processes (Lovelock, 1965;McKay, 2004). Amino acids are among the targets that could be used to determine the presence of life beyond Earth (Neveu et al., 2018). Amino acids are the core of terrestrial life as the building blocks of proteins (Aerts et al., 2020), but are also found in meteorites, which indicates they do not have an Earth-specific origin (Glavin et al., 2010;Pizzarello et al., 2012). For this reason, the detection of one or more of the typical "meteoritic/abiotic" amino acids is not indicative of life, although detecting multiple more complex members of this compound class would suggest biotic processes. Fortunately, instead of relying only on the detection of certain amino acids, we can use their relative distributions to establish their biotic or abiotic origin. The first relative distribution is related to chiral forms of amino acids and expressed by enantiomeric excess. Life on Earth uses mostly left-handed amino acids, while abiotic samples contain mixtures of both chiral forms. The second distribution is related to the overall complexity of amino acids, as expressed by the ratio of each amino acid to the simplest one, glycine. While abiotic processes tend to produce simpler, easier to synthesize amino acids like glycine, amino acids produced biotically tend to be more complex as a requirement for protein functionality. Therefore, the determination of both enantiomeric excesses and relative abundances of amino acids could provide strong evidence of biogenicity (Creamer et al., 2017;McKay, 2004).A key challenge for amino acid analysis is their release from cells or proteins present in the sample. The gold standard method to achieve this in terrestrial laboratories is acid hydrolysis. While some alteration of amino acids can occur during this treatment (Kaiser & Benner, 2005;Pickering & Newton, 1990), it is overall a robust method for assessing amino acid distributions. However, implementing an extraction that requires 6 N HCl heated up to 110°C for 24 hr involves considerable developmental and operational risks which would significantly complicate

A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry

“…The high temperature and pressure conditions used in subcritical water extraction leads to cell lysis and hydrolysis of macromolecules into its building blocks. We have recently demonstrated the use of SCWE for the release of amino acids from microorganisms . Here, we demonstrate that SCWE also releases fatty acids by hydrolysis of lipids.…”

“…We have recently demonstrated the use of SCWE for the release of amino acids from microorganisms. 28 Here, we demonstrate that SCWE also releases fatty acids by hydrolysis of lipids. Because of the low solubility of fatty acids in water, a precipitate is formed after SCWE.…”

Effect of pH on the Release of Amino Acids from Microorganisms via Subcritical Water Extraction

“…18 Our work showed that moderate acidification of the sample with 10 mM HCl results in higher extraction/hydrolysis yields without a negative impact on the subsequent measurement of amino acid biosignatures. 15,18 To date, though, no work has explored the effect of SCWE on amino acid distributions when performed under alkaline conditions. The effect of alkaline conditions is important because models based on data observed by the Cassini spacecraft suggest that Enceladus' ocean is a Na−Cl−CO 3 buffer with an alkaline pH of ∼8−12.…”

In situ organic biosignature detection techniques for space applications

“…It is noteworthy that the recoveries of most amino acids did improve with higher temperature and longer extraction times; however, some amino acids, such as aspartic acid and glycine, degraded. SCWE is also suitable for lysing cells and spores, while simultaneously hydrolyzing proteins that can subsequently be analyzed by, for example, CE (Cieslarova et al, 2022). Urey, an instrument based on SCWE and microchip electrophoresis (Mars Organic Analyzer, MOA, see Section 5.1), was initially under development for the Rosalind Franklin rover; however, it was descoped before the first prototype was manufactured (Amashukeli et al, 2008;Aubrey et al, 2008;Bada et al, 2008).…”