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Water present on early Mars is often assumed to have been habitable. In this study, experiments were performed to investigate the habitability of well-defined putative martian fluids and to identify the accompanying potential formation of biosignatures. Simulated martian environments were developed by combining martian fluid and regolith simulants based on the chemistry of the Rocknest sand shadow at Gale Crater. The simulated chemical environment was inoculated with terrestrial anoxic sediment from the Pyefleet mudflats (United Kingdom). These enrichments were cultured for 28 days and subsequently subcultured seven times to ensure that the microbial community was solely grown on the defined, simulated chemistry. The impact of the simulated chemistries on the microbial community was assessed by cell counts and sequencing of 16S rRNA gene profiles. Associated changes to the fluid and precipitate chemistries were established by using ICP-OES, IC, FTIR, and NIR. The fluids were confirmed as habitable, with the enriched microbial community showing a reduction in abundance and diversity over multiple subcultures relating to the selection of specific metabolic groups. The final community comprised sulfate-reducing, acetogenic, and other anaerobic and fermentative bacteria. Geochemical characterization and modeling of the simulant and fluid chemistries identified clear differences between the biotic and abiotic experiments. These differences included the elimination of sulfur owing to the presence of sulfate-reducing bacteria and more general changes in pH associated with actively respiring cells that impacted the mineral assemblages formed. This study confirmed that a system simulating the fluid chemistry of Gale Crater could support a microbial community and that variation in chemistries under biotic and abiotic conditions can be used to inform future life-detection missions.

The reliable identification of biosignatures is key to the search for life elsewhere. On ocean worlds like Enceladus or Europa, this can be achieved by impact ionization mass spectrometers, such as the SUrface Dust Analyzer (SUDA) on board NASA's upcoming Europa Clipper mission. During spacecraft flybys, these instruments can sample ice grains formed from subsurface water and emitted by these moons. Previous laboratory analog experiments have demonstrated that SUDA-type instruments could identify amino acids, fatty acids, and peptides in ice grains and discriminate between their abiotic and biotic origins. Here, we report experiments simulating impact ionization mass spectra of ice grains containing DNA, lipids, and metabolic intermediates extracted from two bacterial cultures

Planetary protection guidance for martian exploration has become a notable point of discussion over the last decade. This is due to increased scientific interest in the habitability of the red planet with updated techniques, missions becoming more attainable by smaller space agencies, and both the private sector and governments engaging in activities to facilitate commercial opportunities and human-crewed missions. The international standards for planetary protection have been developed through consultation with the scientific community and the space agencies by the Committee on Space Research's (COSPAR) Panel on Planetary Protection, which provides guidance for compliance with the Outer Space Treaty of 1967. In 2021, the Panel evaluated recent scientific data and literature regarding the planetary protection requirements for Mars and the implications of this on the guidelines. In this paper, we discuss the COSPAR Planetary Protection Policy for Mars, review the new scientific findings and discuss the next steps required to enable the next generation of robotic missions to Mars.

International audience; Enceladus is the first planetary object for which direct sampling of a subsurface water reservoir, likely habitable, has been performed. Over a decade of flybys and seven flythroughs of its watery plume, the Cassini spacecraft determined that Enceladus possesses all the ingredients for life. The existence of active eruptions blasting fresh water into space, makes Enceladus the easiest target in the search for life elsewhere in the Solar System. Flying again through the plume with more advanced instruments, landing at the surface near active sources and collecting a sample for return to Earth are the natural next steps for assessing whether life emerges in this active world. Characterizing this habitable world also requires detailed mapping and monitoring of its tidally-induced activity, from the orbit as well as from the surface using complementary platforms. Such ambitious goals may be achieved in the future in the framework of ESA large or medium-class missions in partnership with other international agencies, in the same spirit of the successful Cassini-Huygens mission. For all these reasons, exploring habitable ocean worlds, with Enceladus as a primary target, should be a priority topic of the ESA Voyage 2050 programme.

This work describes a new high-altitude Mars analogue site and the stratification of microbial communities with depth within lake sediments. Understanding how microbial communities at this site change with depth may give insights into the type of metabolism that may have dominated paleolake environments on early Mars and the resulting biosignatures. Introduction: High-Altitude Andean lakes (HAALs) are poly-extreme environments which mimic many of the physiochemical conditions present on Mars during the Noachian (4.1 – 3.7 Gya) and Hesperian Mars (3.7 – 3.0 Gya)1,2. This combination of physiochemical conditions has led to HAALs being identified as natural laboratories for studying life under conditions analogous to early Earth and early Mars1,3. Present-day Mars is a cold, hyper-arid planet with surface conditions incapable of supporting liquid water in most locations. However, orbiter, rover, and lander missions have uncovered extensive hydrated mineral deposits and widespread geomorphological evidence of long-lasting fluvial and lacustrine environments, which dried up in the Hesperian4. Following the collapse of the martian magnetosphere, conditions on the surface would have become increasingly inclement. However, lake sediments may have offered refuge against desiccation, intense ultra-violet radiation (UV), large diurnal temperature fluctuations, and reduced atmospheric pressures with biosignatures potentially being preserved following lithificaytion5. For this reason, the study of analogue environments and the nascent microbiome is crucial in informing targets for biosignature detection. Despite their relevance to early Hesperian Mars, the lakes of the Argentinian Puna (a plateau between 3000 – 6000 m) remain understudied6. Here we present the geochemistry and microbial communities associated with the lake water and sediments from the previously undescribed Laguna de Antofagasta (LDA; Catamarca, Argentina, -26.111148, -67.407338) were analysed. We demonstrated that these communities change with depth, which will aid in identifying relevant metabolisms and biosignatures to inform current and future life-detection missions. Methods: Lake water and cores (5 x 30 cm) were collected from five sample points located around the perimeter of LDA in April 2022 (Figure 1). For each microbial core, a sister core was collected for geochemical analyses (pH, temperature, and dissolved oxygen (DO) readings) to avoid contamination of microbial cores. Each core was divided evenly into an upper, middle, and bottom section before being stored under anaerobic conditions. The lake water samples were collected and filtered using 0.22 µm Sterivex filters, and all samples were transported to The Open University, UK at 4˚C. Pore and lake water were analysed using ICP-OES and IC analysis to collect bulk chemical and ionic concentrations. DNA was extracted using the XS buffer method7, and 16S rRNA gene sequencing was performed to characterise the microbial communities. In addition, Postgate B media was used during culture-dependent analysis to isolate sulphate-reducing bacteria (SRB). Figure 1: Laguna de Antofagasta (LDA) is situated in the Argentinian Puna in the Andes (A). LDA sits at the base of the Volcán La Alumbrera lava field, Antofagasta volcanic field, Catamarca, Argentina (Image B). Image credit: Gerardo Antonio Stoppiello. Results: There were clear differences in physicochemical conditions as well as microbial communities between the sites. Depth correlated with temperature stability, e.g., the upper sections 0-10 cm (n=5) ranged from 2.5 – 13.0 and the bottom 20-30 cm (n=5) fluctuated between 6. 7 and 8.3˚C. pH ranged from 6.8 to 8.3 but appeared to be stable across the depth profiles (Figure 2). Microbial communities also shifted with depth; genera associated with sulfate-reduction became dominant in the bottom sections. Subsequent culture-independent analysis successfully enriched for sulfate-reducing microorganisms. Canonical Correspondence Analysis (CCA) indicated that dissolved oxygen levels, location, and pH had the greatest impact on microbial community composition. Figure 2: Cores were separated into sections before and data were collected on temperature, pH and DO. Implications: In line with previous works6,8,9, the results from this study highlight sulfate reduction as a key metabolism within HAALs and Mars analogue environments. This is significant as biosignatures, and their preservation may be influenced by depth. This work also highlights that physical conditions in the shallow subsurface remain stable, avoiding many of the challenges presented to life by extreme UV, desiccation, and diurnal temperature fluctuation. Future work: To identify how microbial communities may have stratified in martian paleolakes, simulation experiments will be performed. To replicate geochemical conditions, a novel simulant will be used in a column and microbial community structure will be monitored across depth and time. References [1] Cabrol et al. Geophys Res Biogeosciences. 2009;114(4):1–20. [2] Farías et al. J Geophys Res Biogeosciences. 2009;114(G2):0–04. [3] Mlewski et al. Front Microbiol. 2018;9(MAY):996. [4] Carr & Head JW. Earth Planet Sci Lett. 2010;294(3–4):185–203. [5] Grotzinger et al. Science (80- ). 2014;343(6169). [6] Gomez et al. PALAIOS. 2014;29(5):233–49. [7] Tillett & Neilan. J Phycol. 2000;36(1):251–8. [8] Nixon et al. Astron Geophys. 2013;54(1):13–6. [9] Westall et al. Astrobiology. 2015;15(11):998–1029.

Introduction The reliable identification and quantification 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 (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 & Pappalardo 2020), or the ENceladus Ice Analyzer (ENIA), proposed for future Enceladus missions (Reh et al. 2016). CDA data 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), 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’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. Impact ionization detectors, such as SUDA or ENIA, are the only instruments capable of determining the compositions of single µm-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). 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 – 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). Methods and Results 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 Sphingopyxis alaskensis, an ultrasmall bacterium, extracted from various cold marine environments (Ting et al. 2010) and potentially capable of fitting into µm-sized ice grains. We simulated the case of an ice grain of 15µm in diameter - constituting the worst case for Enceladus where ice grains are generally much smaller (1-5 µm in diameter) - formed from a nucleation core of one single bacterial cell. We used Laser Induced Liquid Beam Ion Desorption (LILBID) – 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 – LILBID experiments with otherwise untreated lysed bacterial cells simulating the appearance of these microbial life forms in impact ionization mass spectra. In both polarity mass spectra, we clearly identify signatures of S. alaskensis. Cation mass spectra exhibit features due to protonated amino acids, either fragments of the bacteria’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 15µm 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). Conclusions Our results show that biosignatures deriving from a single bacterial cell – 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 – 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. 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) AbSciCon Abstract, 505-01. 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.

Introduction. The Cassini spacecraft’s encounters with ice grains within the plumes of Enceladus have implied that they originate as droplets of ocean fluid (aerosols), produced by vigorous bubbling at the liquid-vapour interface [1],[2]. Seawater aerosols on Earth, also formed through bubble-bursting, exhibit strong size-dependent differences in their composition and organic content [3],[4]. Compositionally distinct populations of grains detected by Cassini suggest that analogous processes may operate to segregate ocean constituents within the plumes [1]. We used a purpose-built experimental plume aerosol facility to investigate the size-dependent flux and composition of aerosols produced via bubble-bursting under conditions relevant to Enceladus. Our findings show how rates of volatile exsolution influence aerosol size distributions, revealing mechanisms that shape plume stratification. Ongoing work is establishing the efficiency of transfer of organic compounds into aerosols under biological and non-biological scenarios, and how this varies across size fractions relevant to the ice grain profiles encountered by Cassini. Methods. Plume simulation facilities. Aerosols are generated in a temperature-controlled reaction vessel that holds between 5 and 40 litres of fluid, through which mixes of gases are bubbled (Fig. 1). Fluid and gas compositions can be adjusted to simulate any desired chemical conditions. Gas flow rates and bubble injection dynamics can be varied between endmember states of small diffuse bubbles, representative of steady exsolution of volatiles, and vigorous jets of large bubbles, representative of violent boiling. Aerosols are fed into the inlet of a Dekati ELPI+ cascade impactor with real-time enumeration via a gas stream. Particle numbers and size distributions are recorded per second. Aerosols are physically separated into 14 size fractions with diameters between 0.01 and 10 µm and collected onto polycarbonate or aluminium foils, which are then recovered for downstream analyses. Experimental approach. We conducted experiments using both simple NaCl fluids, and fluids designed to fully simulate Enceladus ocean chemistry [5]. To investigate endmember ‘controlled boiling’ scenarios [6], bubble injection dynamics were altered between diffuse and violent bubble jets, while absolute gas flow rate was maintained at 14.2 litres s-1 m-2 of surface area. Aerosol size distributions were monitored for 25 minutes. In ongoing work, collected aerosols fractions are recovered and analysed for their inorganic and organic composition and biomass content, and compared across biological and non-biological simulations. Extracellular polysaccharides produced by the facultative anaerobic bacterium Halomonas ventosae [7] are used as a biological analogue of the insoluble organic layer inferred to exist at the surface of the liquid reservoir at Enceladus [1]. This allows us to investigate how the presence of such a layer affects the flux and size-dependent organic content of aerosols. Results and discussion. Our preliminary data show that contrasting bubble dynamics can result in dramatically different aerosol profiles (Fig. 2). Both bubbling styles led to the production of high numbers of extremely small (< 0.031 µm) particles. In the peak size range of ice grains encountered by Cassini (1 – 4 µm), bubbling styles produced aerosols with similar efficiencies [8]. However, between 0.031 and 1.006 µm aerosols produced by violent bubble jets exceeded those produced by diffuse bubbling by at least an order of magnitude. Because Cassini did not encounter high numbers of such particle sizes, this finding suggests that diffuse bubbling is a more likely plume origin, or that some other process within the plumes efficiently removes particles in this size range. Our experiments provide new insights into the size-dependent flux of aerosols produced at the liquid/vapour interface at Enceladus, and how this ultimately defines the composition of ice-grains ejected to the altitude of spacecraft fly-throughs. Ongoing work is investigating the efficiency of transfer of organics into plume aerosols under biological and non-biological scenarios. Findings will enable new insights into Cassini plume fly-through data and can be leveraged to predict plume behaviour and composition at other planetary bodies with active plumes, such as Europa. References. [1] Postberg F, et al. Nature. (2018) 558(7711):564-568; [2] Porco CC, et al. Astrobiology (2017) 17(9):876-901; [3] Salter ME, et al. Geophys Res Lett. (2016) 43(15):8277-8285; [4] Rastelli E, et al. Sci Reports (2017) 7(1):1-10; [5] Fox‐Powell MG, Cousins CR. J. Geophys Res. Planets. (2021) doi:10.1029/2020je006628; [6] Ingersoll AP, Nakajima M. Icarus (2016) 272:319-326; [7] Mata JA, et al. Res Microbiol (2006) 157(9):827-835; [8] Spahn F, et al. Science (2006) 311(5766):1416-1418 Figure 1. Schematic of experimental plume aerosol facility. Figure 2. Aerosol size distributions recorded over 25-minute experimental runs. Data points represent median values, error bars indicate minimum and maximum counts.

Introduction: Constraining the composition of Europa’s ocean is critical to understanding whether it could support microbial life, and provides an insight into the satellite’s evolution. However, there is much uncertainty about the ocean as it sits beneath a layer of ice and is thus not directly accessible for observation or to orbiting spacecraft. Salts on Europa’s surface, such as MgSO4 and NaCl, cannot be explained entirely by exogenous delivery, and so must originate from the ocean [1, 2] indicating that the ocean interacts with a silicate interior to liberate solutes [3]. If so, this means a “bottom-up” approach, that explores water-rock interactions occurring between the ocean and silicate interior, can be used to assess potential ocean compositions. Previous studies have taken a similar approach [4–7] but have focused on silicate compositions based on carbonaceous chondrites. However, computer modelling has shown that a composition between a L and LL-type ordinary chondrites correlated with Europa’s moment of inertia, and therefore, could also be a candidate composition for Europa’s silicate interior [8]. Moreover, these previous studies have focused on water-rock interactions that use either pure H2O or HCl initial fluids [4–7, 9], derived from ices during Europa’s accretion. However, the europan surface shows evidence of cratering, with 90% of craters of cometary origin [10]. Comets could, therefore, have made a significant contribution to Europa’s water inventory and influenced the composition of the ocean, which warrants further investigation. Methods: The computer code CHIM-XPT [11] was used to explore possible ocean compositions that could arise from water-rock interactions occurring within the europan interior. Four silicate compositions based on L, LL and CV (both oxidised and reduced endmembers) chondrites (Table 1) were reacted with two fluids: pure H2O and a cometary melt (based on the volatile composition of 67P/Churyumov-Gerasimenko [12]). These fluids represent plausible endmember compositions that may have contributed to the ocean water inventory. Initial models were run to mimic water-rock interactions occurring at an anticipated pressure and temperature of 250 MPa and 333.15 K [13]. These fluids were then cooled and depressurised to mimic the ascent of fluids from the interior to the ocean reservoir. Models were run at water-rock ratios (W/R) of 1-100 to reflect the degree of alteration experienced within the silicate interior [9, 14]. Table 1. Silicate compositions (based on data from [15]) used for models. Results and Discussion: Here, results from models performed at a W/R of 1 are presented, which is reflective of the degree of alteration expected today for the europan silicate interior [9]. Variations in secondary mineral assemblages were produced from the initial fluid compositions. Pure H2O models were dominated by serpentine and greenalite for all four silicate compositions. Conversely, cometary models were dominated by talc, carbonates, serpentine, greenalite and chlorite. The high abundance of carbonates in cometary models reflect the high C content found in the initial fluid composition. Overall, the evolved ocean compositions were dominated (concentrations > 10-3.mol.kg-1) by Cl, C, Si, K and Na regardless of initial fluid endmember compositions (Figure 1). Differences in ocean composition between the four silicate compositions was restricted to variations in Fe and/or S concentrations. The absolute concentrations of Fe and S were strongly influenced by initial silicate composition in the pure H2O models, leading to variations greater than one order of magnitude (Figure 1). Only S showed a variation of greater that one order of magnitude for cometary melt fluids, where all S was removed from the ocean for CVoxi by mineral precipitation. Figure 1. Proposed ocean compositions from water-rock interactions and the ascent of fluids from depth. Although the dominant elements were similar between pure H2O and cometary melt models, differences in the absolute concentration of Fe were observed (Figure 2). Fe concentrations were at least four orders of magnitude greater in pure H2O models than in cometary melt models. This is significant because the variation of Fe within the europan ocean could influence its ability to support life; Fe is a redox active element that is used by chemotrophic organisms for energy. A variation of four orders of magnitude was also observed for Mg concentrations, with Mg greater in cometary melt models than in pure H2O models. Given the presence of Mg-salts within Europa’s icy crust, it is possible that the variation in concentration between the two initial fluids could be diagnostic of source fluids and be an indication that cometary ices may have made a significant contribution to Europa’s ocean. Figure 2. Fe and Mg concentrations for pure H2O and cometary melt fluids and silicate compositions [15]. Conclusions: The modelling indicates that the initial fluid would have a stronger influence on the resultant europan ocean composition than proposed candidate silicate compositions. Hence, a wider range of candidate fluid compositions should be explored to assess their possible influence on the composition of Europa’s ocean. Furthermore, results showed a variation in absolute Mg concentrations between both initial fluid compositions. If surface Mg-salts did form as the underlying ocean froze then this Mg variation could be used to identify the composition of the ocean. However, to fully understand the link between the ocean and salts in the ice shell requires further investigations into the salt precipitation sequence as potential ocean compositions freeze. References: [1] McCord et al. (1998) Science, 1242. [2] Trumbo et al. (2019) Sci. Adv., 5. [3] Hand (2018) Halogens on and Within the Ocean Worlds of the Outer Solar System. [4] Zolotov & Mironenko (2007) Ices, Oceans, and Fire: Satellites of the Outer Solar System, Abstract# 6065. [5] Zolotov & Shock (2001) J. Geophys. Res. E Planets, 106. [6] Kargel et al. (2000) Icarus, 148. [7] Daswani et al. (2021) Geophys. Res. Let, 48. [8] Kuskov & Kronrod (2005) Icarus, 177. [9] Zolotov (2012) Icarus, 220. [10] Zahnle et al. (1998) Icarus, 136. [11] Reed (1982) Geochim. Cosmochim. Acta, 46. [12] Rubin et al. (2019) Mon. Not. R. Astron. Soc., 489. [13] Vance et al. (2007) Astrobiology, 7. [14] Spivack & Edmond (1987) Geochim. Cosmochim. Acta, 51. [15] Jarosewich (1990) Meteoritics, 25.

The identification of novel terrestrial sites that are analogous for other planetary bodies is an active area of research within astrobiology, because of the logistical and financial difficulties in obtaining extraterrestrial samples for analysis. Characterisation of potential analogue sites is undertaken to assess how accurately they represent a specific extraterrestrial environment. Analysing their physicochemical conditions and microbial communities are key components of these studies to understand what metabolisms would be viable in such environments. One such novel analogue environment is the salt plains of Western Sahara. Western Sahara is one of the driest regions on Earth. It is located on the northwest coast of West Africa and is characterised by high UV exposure, low annual precipitation and water activity, subsurface water and high annual temperatures. These features make Western Sahara a potential analogue site for Mars during the Noachian-Hesperian transition period (3.5 – 3.8 Ga), when the atmosphere began to thin and surface water started evaporating (Warner et al., 2010), similar to other terrestrial deserts, such as the Atacama Desert and the McMurdo Dry Valleys. The hypersalinity, aridity and high UV radiation levels of the Western Sahara salt plains would also be appropriate to study whether dissimilatory sulfur metabolisms would be viable in a Noachian-Hesperian Mars analogue environment. Dissimilatory sulfur cycling refers to the use of inorganic sulfur compounds for energy conservation and it has been recognised as a metabolic strategy of interest for putative martian life (Macey et al., 2020). On Earth, evidence from stable sulfur isotope fractionation has suggested this metabolism emerged early in the history of life (~3.5 Ga). During this period, the conditions on Mars were predicted as being more habitable than present-day, with an active magnetic field, thicker atmosphere and liquid water on the surface. In this study, molecular and geochemical techniques were used to give first insights into the potential of the Western Sahara salt plains to serve as an analogue of Mars during the Noachian-Hesperian transition period. The microbiology was investigated through cultivation-independent and culture-dependent analyses of salt crystals, sediment and water samples obtained at three sites near Llaayoune (Fig. 1). The chemical nature of the samples was analysed through ion chromatography (IC) and inductively coupled plasma - optical emission spectrometry (ICP-OES). The geochemical characterisation confirmed the high salinity of the samples and identified that sodium, potassium, magnesium and sulfur were the most enriched elements within all samples. Cultivation-dependent work resulted in the enrichment of a wide range of metabolic strategies from the samples including aerobic heterotrophs, phototrophs and sulfate-reducers. The enrichments from the salt were dominated by strains of Bacillus, whereas sulfate-reducing strains of Clostridium were isolated from the sediment samples. Microscope analysis of phototroph-selective media also indicated that algae and Cyanobacteria were successfully enriched from the samples. 16S rRNA amplicon sequencing results will also be presented to gain further in-depth understanding of the microbial community composition. Additionally, results from quantitative polymerase chain reaction (qPCR) experiment targeting sox and dsr genes will be presented to identify the abundance of genes specific for dissimilatory sulfur metabolisms within the samples. Preliminary data shows that sulfur cycling is occurring in Western Sahara salt plains. Future characterisation of this environment will involve metagenomic analysis of the samples and genome sequencing of the isolates to identify the key metabolisms underpinning the survival and viability of the microbial community. Comparative studies with other Mars analogue environments will then be undertaken to identify metabolisms that may have been thermodynamically viable in ancient martian aqueous environments. Figure 1. Location of the sample collection sites. Images were generated with Google Earth Pro. References Macey, M. C., Fox-Powell, M., Ramkissoon, N. K., Stephens, B. P., Barton, T., Schwenzer, S. P., . . . Olsson-Francis, K. (2020). The identification of sulfide oxidation as a potential metabolism driving primary production on late Noachian Mars. Scientific Reports, 10(1), 10941. https://doi.org/10.1038/s41598-020-67815-8 Warner, N., Gupta, S., Lin, S.-Y., Kim, J.-R., Muller, J.-P., & Morley, J. (2010). Late Noachian to Hesperian climate change on Mars: Evidence of episodic warming from transient crater lakes near Ares Vallis [https://doi.org/10.1029/2009JE003522]. Journal of Geophysical Research: Planets, 115(E6). https://doi.org/https://doi.org/10.1029/2009JE003522

Introduction. The plumes emanating from the south polar region of Saturn’s moon Enceladus were studied by the Cassini mission and have revealed evidence for ongoing hydrothermal activity [1], organic chemistry [2], and redox conditions favorable for microbial metabolism [3] in the ocean below. Salt-rich icy particles encountered by Cassini were interpreted to originate as rapidly frozen droplets of ocean spray [4]. If extant microbial communities exist within Enceladus’s ocean, cells may become incorporated into these particles and ejected into space where they can be sampled by future spacecraft [5]. However, the potential fate of biomass within frozen icy particles is not understood. Here, we used cryogenic imaging alongside sublimation-extraction techniques to study the interaction of microorganisms with precipitated inorganic phases during freezing of simulated Enceladus ocean fluids, at contrasting cooling rates. Our findings reveal how potential microbial biomass may be expressed in the Enceladus plumes, and carry implications for its potential detection using current and future spacecraft sampling strategies Methods: Cells taken from four phylogenetically and morphologically distinct pure cultures, as well as a mixed community, were suspended in simulated Enceladus ocean fluid [6], and droplets were cooled at two contrasting rates: rapidly quench cooling in liquid nitrogen (101-102 K s-1), and gradual cooling (0.01 K s-1). Partitioning of microorganisms alongside ice and non-ice phases within frozen droplet interiors was imaged at 120 K via cryo-scanning electron microscopy. Specific associations between microorganisms and cryogenic mineral phases were analyzed using cryo-electron microscopy, and the mineralogy of crystalline precipitates was determined via infrared spectroscopy and X-ray diffraction (XRD) following extraction from the ice matrix by sublimation. Results and discussion: Under both freezing scenarios, ice crystallisation produces an interconnected network of solidified solute-rich brine veins accumulated at ice grain boundaries, within which intact microorganisms can become entombed (Fig. 1). The presence of microbial cells also leads to the formation of small, solitary inclusions of brine fully encased by ice, which are disconnected from the main brine vein network. Microscale crystallization textures, previously identified to be diagnostic of cooling rate [6], are disrupted and modified by the presence of biomass, with distinct differences arising between brine veins and solitary inclusions. XRD analyses of cryogenic salt phases shows that the presence of microbial biomass during freezing also influences bulk mineralogy (Fig. 2). In the absence of biomass, nahcolite (NaHCO3) is kinetically inhibited during flash freezing. However, in the presence of biomass, nahcolite does form during flash freezing, and the resulting mineralogy instead resembles that of gradually cooled control fluids. This indicates that the presence of microorganisms kinetically favors the precipitation of nahcolite. Moreover, this finding provides a route by which the presence of biomass at Enceladus could influence the mineralogy of icy particles in the plumes. Given that mineralogy is also sensitive to cooling rate and fluid pH [6], analyses of mineralogy within plume particles at Enceladus should be prioritized in future observations. Our results demonstrate how microbial biomass may be preserved within icy particles relevant to the Enceladus plumes, revealing complex, cooling rate-dependent interactions between microbial biomass and rapidly precipitating inorganic phases. The association of microbial cells with specific inorganic phases has implications for interpreting secondary ion fragments in spacecraft impact-ionization mass spectra [7], and in the planning for future plume sampling missions that target in situ analyses, or more ambitiously, cryogenic sample return. References: [1] Waite, J. H. et al. Science 356, 155–159 (2017). [2] Postberg, F. et al. Nature 558, 564–568 (2018). [3] Ray, C. et al. Icarus 364, 114248 (2021). [4] Postberg et al. Science 459, 1098-1101 (2009). [5] Porco, C.C., et al. Astrobiology, 17, 876-901 (2017). [6] Fox-Powell & Cousins, JGR: Planets 126(1), e2020JE006628 (2021). [7] Klenner et al. Astrobiology 20, 1168-1184 (2020). Figure 1. (Cryo) electron micrographs of co-partitioned cryogenic salt phases and microbial biomass. A, B., Cryo-SEM images of brine vein network with preserved E. coli cell. C., Solitary inclusion. D., brine vein crystallisation textures. Figure 2. X-ray diffraction patterns from salt mineral assemblages produced by freezing in the presence and absence of microbial cells from community enrichments. ‘Control’ trace represents samples cooled gradually in the absence of biomass. H: halite (NaCl); T: Trona (Na2CO3•NaHCO3•2H2O); N: Nahcolite (NaHCO3).

Nitrate-dependent Fe

Modern-day Mars is a dry, harsh environment with no surface water, but billions of years ago it might have been water-rich, like Earth. Scientists believe that, as water was lost on Mars, the remaining water would have become increasingly salty and rich in sulphur. By researching environments on Earth with a similar chemistry, we can develop hypotheses about whether life could have existed on Mars. In our research, we studied Colour Peak, a sulphur-rich, salty spring system in the Canadian High Arctic. This environment contains bacteria that use sulphur to get their energy. By studying water and sediment samples from this region, we confirmed that bacteria that used sulphur as an energy source could potentially have lived on Mars. These results can help scientists who are analysing data from Mars missions to identify evidence of former life on Mars.

Volcanic features and impact craters are ubiquitous features on Mars, and hydrothermal systems associated with the production of these features should have been abundant in Mars’s early history. These hydrothermal systems represent potentially habitable environments and are therefore a high priority for continued investigations of the Martian crust. Here we present a Mars analog study where basaltic magma intruded water-bearing sediments to produce a high-temperature (as high as ∼700°C) hydrothermal system, which we use to constrain the potential habitability of similar systems on Mars via mineralogy and geochemistry including S, C, and O isotopic systematics. Our analog site suggests evidence for a habitable environment once the system cooled below 120°C and the potential presence of microbial activity based on the combination of dolomite and C-isotopic systems in the same sample. These findings highlight the importance of future missions to investigate the interface of sediments with magmas and/or late-stage impact melts where microbial life may have taken hold when temperature conditions allowed.

The identification of biosignatures on extraterrestrial ocean worlds is key to the search for life on these bodies. Saturn’s icy moon Enceladus, and possibly Jupiter’s moon Europa, eject plumes containing gas and ice grains formed from subsurface salty water into space [1,2,3,4]. The emitted ice grains can be analyzed by impact ionization mass spectrometers, such as the Cosmic Dust Analyzer (CDA) onboard the earlier Cassini-Huygens mission [5] or the Surface Dust Analyzer (SUDA) onboard the upcoming Europa Clipper mission [6] rendering the ocean’s composition accessible for analysis during spacecraft flybys [7]. CDA data collected in the Saturnian system revealed that Enceladus’ ocean is salty [8] and contains a variety of organic material, including complex macromolecules [9] and low mass volatile compounds [10]. These low mass compounds can potentially act as amino acid precursors and could be capable of interacting within or near Enceladus’ hydrothermal vent systems [11], or Enceladus’ porous rocky core [12]. These findings enhance Enceladus’ relevance as a habitable environment potentially able to support microbial life in its subsurface ocean. However, biosignatures have not yet been identified in extraterrestrial environments. The Laser Induced Liquid Beam Ion Desorption (LILBID) experiment is proven to accurately reproduce impact ionization mass spectra of ice grains recorded in space [13]. Previous analogue experiments using this setup have shown that amino acids, fatty acids and peptides could successfully be detected in simulated ocean world scenarios [14,15]. The next step is to investigate whether the characteristic mass spectral fingerprints of bacterial life forms, if enclosed in ice grains, could also be detected and identified by SUDA-type instruments. We therefore conducted high sensitivity LILBID experiments on extracts of two bacteria, Sphingopyxis alaskensis (S.alaskensis) and Escherichia coli (E.coli), to simulate their characteristic spectral features in cationic and anionic impact ionization mass spectra. E. coli is a well-studied model bacterium, and S. alaskensis is a model oligotroph, abundant in marine environments. The predictable, small size of S. alaskensis (3) and its ability to utilize low concentrations of nutrients [16] makes it a suitable analogue bacterium for potential lifeforms on extraterrestrial oceans. Laboratory spectra of extracted bacterial DNA, lipids and the corresponding aqueous phases produced during their extraction - potentially containing polar molecules - were performed. To simulate the salty Enceladean or Europan ocean water, the extracts have been investigated in background solutions with increasingly high NaCl concentrations. In the mass spectra, we observe mass lines identified as microbial fragments, corresponding to fatty acids derived from the bacteria’s membrane lipids. In the S. alaskensis and E. coli DNA mass spectra we also identify nucleobases and compounds produced from the phosphate deoxyribose backbone. The recorded mass spectra are stored in a growing database of laboratory LILBID spectra (Klenner et al., in prep.), designed to aid in the interpretation of results from earlier missions, such as Cassini, as well as help planning future missions to icy ocean worlds in the Solar System, such as Europa Clipper [17]. References [1] M. K. Dougherty et al. (2006) Identification of a Dynamic Atmosphere at Enceladus with the Cassini Magnetometer. Science 311:1406-1409. [2] F. Spahn et al. (2006) Cassini dust measurements at Enceladus and implications for the origin of the E ring. Science 311:1416-8. [3] L. Roth et al. (2014) Transient Water Vapor at Europa’s South Pole. Science 343:171-174. [4] W. B. Sparks et al. (2016) Probing for evidence of plumes on Europa with HST/STIS. ApJ 829:121. [5] R. Srama et al. (2004) The Cassini Cosmic Dust Analyzer. Space Sci Rev 114:465-518. [6] S. Kempf et al. (2014) SUDA: A Dust Mass Spectrometer for Compositional Surface Mapping for a Mission to Europa. Eur Planet Sci Congr 9:EPSC2014-229. [7] F. Postberg et al. (2011) A salt-water reservoir as the source of a compositionally stratified plume on Enceladus. Nature 474:620–622. [8] F. Postberg et al. (2009) Sodium salts in E-ring ice grains from an ocean below the surface of Enceladus. Nature 459:1098–1101. [9] F. Postberg (2018) Macromolecular organic compounds from the depths of Enceladus. Nature 558:564–568. [10] N. Khawaja et al. (2019) Low-mass nitrogen-, oxygen-bearing, and aromatic compounds in Enceladean ice grains. Mon Not R Astron Soc 489:5231–5243. [11] H.-W. Hsu et al. (2015) Ongoing hydrothermal activities within Enceladus. Nature 519:207–210. [12] G. Choblet at al. (2017) Powering prolonged hydrothermal activity inside Enceladus. Nat Astron 1:841-847. [13] F. Klenner et al. (2019) Analogue spectra for impact ionization mass spectra of water ice grains obtained at different impact speeds in space. Rapid Commun Mass Spectrom 33:1751–1760. [14] F. Klenner et al. (2020a) Analog Experiments for the Identification of Trace Biosignatures in Ice Grains from Extraterrestrial Ocean Worlds. Astrobiology 20:179–189. [15] F. Klenner et al. (2020b) Discriminating Abiotic and Biotic Fingerprints of Amino Acids and Fatty Acids in Ice Grains Relevant to Ocean Worlds, Astrobiology 20:1168-1184. [16] T. Williams et al. (2009) Carbon and nitrogen substrate utilization in the marine bacterium Sphingopyxis alaskensis strain RB2256. ISME J 3:1036-1052. [17] S.M. Howell and R.T. Pappalardo (2020) NASA’s Europa Clipper—a mission to a potentially habitable ocean world. Nat Commun 11:1311.

Hydrothermal systems that formed as a result of impact events possess all the key requirements for life: liquid water, a supply of bio‐essential elements, and potential energy sources. Therefore, they are prime locations in the search for life on other planets. Here, we apply thermochemical modeling to determine secondary mineral formation within an impact‐generated hydrothermal system, using geochemical data returned for two soils on Mars found in regions that have previously experienced alteration. The computed mineral reaction pathways provide a basis for Gibbs energy calculations that enable both the identification of available geochemical energy, obtained from Fe‐based redox reactions, that could be utilized by potential microbial life within these environments, and an estimate of potential cell numbers. Our results suggest that water–rock interactions occurring within impact‐generated hydrothermal systems could support a range of Fe‐based redox reactions. The geochemical energy produced from these reactions would be substantial and indicates that crater environments have the potential to support microbial cell numbers similar to what has been identified in terrestrial environments.

Detecting and identifying biosignatures is key to the search for life on extraterrestrial ocean worlds. Saturn’s moon Enceladus emits a plume of gas and water ice grains, formed from its subsurface ocean, into space. A similar phenomenon is suspected to occur on Jupiter’s moon Europa. Impact Ionization mass spectrometers, such as the Cosmic Dust Analyzer (CDA) onboard the past Cassini mission or the Surface Dust Analyzer (SUDA) on board the upcoming Europa Clipper mission, can sample the emitted ice grains, rendering the ocean accessible for compositional analysis by spacecraft flybys. The CDA data collected in the Saturnian system showed that Enceladus’ ocean is salty [1] and contains a variety of organic material, such as complex macromolecules [2] and low mass volatile compounds, the latter of which potentially act as amino acid precursors and are capable of interacting within or near Enceladus’ hydrothermal vent system [4], or Enceladus’ porous rocky core [5]. Although these findings enhance Enceladus’ relevance as a potential habitable environment, biosignatures have so far not been identified. Interpreting the space-based icy grain data requires on-ground calibration via analogue experiments. The Laser Induced Liquid Beam Ion Desorption (LILBID) technique is capable of accurately reproducing the mass spectra of ice grains recorded in space [6]. Previous LILBID experiments have shown that bioessential molecules, namely amino acids, fatty acids, and peptides can be detected in the ice grains [7], and that abiotic and biotic formation processes of these molecules can be distinguished from each other [8]. The next steps are to investigate whether building blocks of bacteria, such as membrane lipids – indicators for earthlike microbial life - can also be detected in ice grains and characterized using future impact ionization mass spectrometers. To predict their spectral appearance in impact ionization mass spectra, high sensitivity LILBID experiments on extracts from Escherichia Coli and Sphingopyxis alaskensis were performed. Spectra of lipids, and the corresponding aqueous phases produced during their extraction, potentially containing polar molecules, were produced using increasingly NaCl-rich matrices, designed to mimic the salty ocean of Enceladus or Europa. In the mass spectra, we identify fragments characteristic for the building blocks of bacteria, such as fatty acids deriving from the bacteria’s membrane lipids. Sensitivity to lipid fragments and polar molecules decreases with rising salt concentration. These spectra, as well as those of other biosignatures, have been incorporated into a comprehensive database, to provide comparable analogue data of a wide range of compounds applicable to future impact ionization mass spectrometers. References [1] Postberg et al. (2009) Nature 459:1098–1101, [2] Postberg et al. (2018) Nature 558:564–568, [3] Khawaja et al. (2019) Mon Not R Astron Soc 489:5231–5243, [4] Hsu et al. (2015) Nature 519:207– 210, [5] Choblet at al. (2017) Nat Astron 1:841-847, [6] Klenner et al. (2019) Rapid Commun Mass Spectrom 33:1751–1760, [7] Klenner et al. (2020) Astrobiology 20:179–189, [8] Klenner et al. (2020) Astrobiology 20: 1168–1184.

Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genome sequences (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center’s Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival., Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center’s Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival.

NASA’s search for habitable environments has focused on alteration mineralogy of the Martian crust and the formation of hydrous minerals, because they reveal information about the fluid and environmental conditions from which they precipitated. Extensive work has focused on the formation of alteration minerals at low temperatures, with limited work investigating metamorphic or high-temperature alteration. We have investigated such a site as an analog for Mars: a mafic dike on the Colorado Plateau that was hydrothermally altered from contact with groundwater as it was emplaced in the porous and permeable Jurassic Entrada sandstone. Our results show evidence for fluid mobility removing Si and K but adding S, Fe, Ca, and possibly Mg to the system as alteration progresses. Mineralogically, all samples contain calcite, hematite, and kaolinite; with most samples containing minor anatase, barite, halite, and dolomite. The number of alteration minerals increase with alteration. The hydrothermal system that formed during interaction of the magma (heat source) and groundwater would have been a habitable environment once the system cooled below ∼120 °C. The mineral assemblage is similar to alteration minerals seen within the Martian crust from orbit, including those at Gusev and Jezero Craters. Therefore, based on our findings, and extrapolating them to the Martian crust, these sites may represent habitable environments which would call for further exploration and sample return of such hydrothermally altered igneous materials.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124–155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars., European Union’s Horizon 2020 research and innovation program under Grant Agreement No 654208

Introduction The subsurface environment of Enceladus is potentially habitable: there is a global subsurface ocean [1], energy from hydrothermal activity [2] and bioessential elements [3]. Carbon, as a fundamental bioessential element, is critical for life, so understanding how it is processed within the Enceladus environment is crucial in assessing this moon’s potential habitability. Evidence from the south polar plumes suggests that carbon is likely to be bound within the silicate interior [4] and liberated through water-rock (silicate-ocean) interactions. Methods We have undertaken thermochemical modelling (CHIM-XPT) [5] of these interactions and tested different hypotheses for the formation of Enceladus. Both models reacted the silicate interior (with a CI chondrite composition [6]) with a fluid representative of the subsurface ocean: model 1) a dilute sodium chloride solution, based upon the assumption that the subsurface ocean originated as almost pure water [7]; model 2) a solution with a cometary composition (using data collected from 67P [8]), based upon the assumption that the ocean originated from melted cometary ice [9]. We have explored the full temperature and pressure ranges anticipated at the rock-water interface [2]. We have modelled the rock-water interactions: at the rock-water interface in the areas of hydrothermal activity; within the silicate interior, and in cooler areas of the ocean floor. In addition, we have run thermochemical models to cool the subsurface ocean fluid as it ascends from the ocean floor to the water-ice boundary; these results should be more comparable to the Cassini observations, as the plume material emanates from the ocean surface. Figure 1 - A cross section of the subsurface of Enceladus' South Polar Region, highlighting the area of study. The stars represent the different regions of study for the water-rock interaction models; each location represents different temperatures and pressure regimes. The arrows represent cooling and change in the chemical composition of the fluid as it moves from the ocean floor to the ice/ocean interface. Image Credit: R. E. Hamp Results/ Discussion With regards to the plausible chemistry of the Enceladus ocean, the model output agrees with the observations from the Cassini data; the dominant chemical species are sodium chloride, potassium chloride and sodium carbonates. The model has also revealed a possible formation mechanism for the silica nanoparticles within the subsurface ocean that could account for the detection of such particles in the Enceladus plumes [2]. This, in turn, validates our model and the simulant chemistry inputted. The cooling model results show that the pH of the ocean at the water/rock interface was significantly lower than that at the ocean/ice interface (8 compared with 10.5). Furthermore, the concentration of carbonates and sulfates decrease as they ascend though the water column, but the concentration of silica particles increases. The output from this work provides results very closely aligned to the concentrations of the chemical species detected by the Cassini Spacecraft. This work has also determined that a carbon cycle may operate at the rock-water interface, which could include a carbonate pump system and the formation of hydrocarbons up to C4. The availability of these chemical species and the potential for further ongoing reactions supports the evidence that the subsurface environment of Enceladus may be habitable. Summary We will present the outcomes from this modelling, which includes a theoretical composition for a modern day subsurface ocean, potential carbon cycling pathways and the effect of carbon species on the pH of the subsurface ocean fluid. We will also present the initial findings from modelling of the cooling of ocean fluids as they are transported from the ocean floor to the ocean/ice interface surface. [1] Thomas P. C. et al., (2016), Icarus, 264, 37-47 [2] Hsu H. W. et al., (2015), Nature, 519, 207-210 [3] McKay, C. P. et al., (2014) Astro-biology, 14, 352-355 [4] Glein C. R. & Waite J. H., (2020), Geophys Res Let, 47 [5] Reed, M. H., Spycher, N. F., Palandri, J., (2010) User guide for CHIM-XPT, University of Oregon, Oregon [6] Hamp R. E. et al., (2019), 50th LPSC 2019, Abstract 1091 [7] Brown R. H. et al, (2006) Science, 311, 1425-1428 [8] Hertier K. H., et al., (2017) RAS monthly notices, 469 [9] Neveu, M., et al., (2017) Geochim et Cosmochim, 212, 324-371

Introduction Owing to the limited amount of data from other planetary bodies in our Solar System, terrestrial analogues are frequently used to further the understanding of extraterrestrial environments. One of the moons of Jupiter, Europa has been selected for future exploration in our Solar System. Measurements carried out by the Galileo spacecraft suggest the existence of a tidally heated, sub-surface ocean that could harbour life [1]; yet the composition of the ocean is unknown as there have been no direct measurements [2, 3]. The information about Europa is thus limited but has been complemented by modelling surface and interior processes [4] (fig. 1). Surface features and data from Galileo initially suggested an ocean dominated by magnesium sulfate; however, recent observations suggest it is dominated by sodium chloride with a high concentration of magnesium [5, 6, 7]. Considering the limited data available about the interior of this moon, analogue studies can be used to extend our understanding of the crucial constraints on microbial habitability below the ice shell. To date, terrestrial analogue studies for Europa have focused on subglacial lakes, cold springs, and sulfate lakes. These sites, however, have been selected based on a single or few parameters. Some are of interest due to their pH level, temperature, or main ionic composition. This approach often ignores or compromises other important variables that may influence habitability, such as pressure, dissolved gases or minor ions. The aim of this work is to develop a quantitative method to evaluate the similarity between terrestrial analogue sites and models of the Europan ocean. This will improve the selection of appropriate terrestrial analogues, the consequent validity of the results from microbiological and geochemical studies, and thus, their correlation to studies of the potential habitability of Europa. Objective analogue site selection requires a quantitative method with which to evaluate the similarity between terrestrial analogue sites and models of the Europan ocean and that takes into account all major variables that could determine habitability. Given the number of possible variables, classifying analogue environments is an inherently multivariate problem. Current classification systems for aqueous environments tend to fall short as they rely on human input, use inconsistent parameters or do not account for all relevant physicochemical conditions. To address this issue, machine learning models can be used, which are self-improving algorithms able to learn what a categorised dataset looks like and then make predictions on what category new data will fit into [8]. Methods A terrestrial analogue database was constructed with published chemical (ionic composition), temperature, pressure, and pH data from potential analogue sites, including subglacial lakes, saline lakes and hydrothermal environments. Using a range of multivariate classification methods and clustering solutions, categories were defined by the full range of known physicochemical characteristics. Machine learning algorithms were trained with such categories and the highest accuracy model was selected. New samples (such as new potential analogue sites, data from a Europan model or even direct measurements) can be fed into the trained model and it will output a so-called similarity score. This score is based on how similar the model calculates this new sample is to each of the categories of the training dataset. The category with the highest similarity score contains the sites that should be considered analogous to the new sample; in our case this new sample can be any Europan composition model found in the literature. Cross validation methods and genetic algorithms were used to improve the models and thus the accuracy of the similarity scores. Cross-validation creates subsets of the database and assigns one to be used to evaluate the model and the rest to train it. The process then repeats by selecting a different subset to be used to evaluate the model, leaving the rest as part of the training set, until every subset has been used to both train the model and test it. Genetic algorithms operate on the principle of biological evolution, by generating multiple versions of the model, changing certain parameters stochastically (mutation) and selecting those with the desired metrics to populate the subsequent generation of versions of the model. These processes were applied to several machine learning algorithms to select the ones with the highest accuracy. Results We applied the model of selecting possible analogues to existing estimates of Europa’s ocean chemistry. Initial classification (accuracy 90.6%) attempts suggest the use of low temperature sites: either sulphate-saline dominated lakes like Basque Lake (Canada) or Last Chance (Greenland) (similarity 96.0%); or calcium-rich lakes like Don Juan Pond (Antarctica) or El Chichón (Mexico) (similarity 99.9%). The similarity between the physicochemical data of the modelled Europan ocean and the terrestrial analogue sites can be calculated with high accuracy, justifying the use of microbial communities or geochemical samples from these environments for studying the habitability of extra-terrestrial environments. Ongoing work is improving the predictive accuracy of our machine learning model. The results from this work demonstrate that machine learning algorithms can improve the selection process of field sites for the study of microbial ecology and astrobiology. References [1] Russell et al. (2017). Astrobiology, 17(12), 1265–1273. [2] Kivelson et al. (2000). Science, 289(5483), 1340–1343. [3] Pappalardo et al. (1999). Journal of Geophysical Research E: Planets, 104(E10), 24015–24055. [4] Jin & Ji (2012). Science China: Physics, Mechanics and Astronomy, 55(1), 156–161. [5] Trumbo et al. (2019). Science Advances, 5(6), 2–6. [6] Zolotov & Shock (2001). Journal of Geophysical Research E: Planets, 106(E12), 32815–32827. [7] Ligier et al. (2016). The Astronomical Journal, 151(6), 163. [8] Mander et al. (2013). Proceedings of the Royal Society B: Biological Sciences, 280(1770).

The environmental conditions on present-day Mars are detrimental for life as we know it; however cumulative evidence suggests that early Noachian Mars (~ 4 billion Ga) had a warmer climate with a denser atmosphere1 capable of supporting surficial liquid water, and providing protection from UV and cosmic radiation. It is possible, therefore, that early Mars could have been hospitable for microorganisms. While cellular degeneration is a rapid process following cell death, microorganisms do leave behind molecular clues as to their existence, or biosignatures, such as the lipid molecules that previously comprised their cell membranes. Each group of microorganisms leaves behind a distinct lipid “fingerprint” that is relatively resistant to harsh environmental conditions and can be preserved over geological timescales2. Various robotic and remote-sensing missions have confirmed the presence of salt deposits (e.g., chlorides, sulfates) at the late Noachian to late Hesperian (~ 4 to 3.5 Ga) boundaries of present-day Martian deposits. In particular, bulk enrichments of calcium and magnesium sulfates have been reported in the Hesperian (~ 3.3–3.7 Ga) sedimentary rocks of Gale crater3, which indicate the previous presence of widespread liquid water on Mars. On Earth, such salts have been found to harbour and protect microbial life for a prolonged period, possibly over millions of years4, thus evaporite sequences provide a compelling target for life detection, as putative biosignatures could be preserved5. Currently, the beneficial or deleterious effects of sulfate chemistry on the preservation of organic matter, especially under biologically destructive modern Martian conditions is not well constrained. Given the prominent presence of sulfates on the surface of Mars, this work explores whether sulfate minerals can act as viable substrates for the long-term preservation of lipids, when exposed to Mars-like radiative and atmospheric conditions, and aims to determine whether those signs of life can be explicitly detected. To carry out this work, we use a combination of analogue fieldwork and laboratory-based simulation studies. Microorganisms isolated from a magnesium sulfate-rich analogue site (Basque Lake) and other terrestrial early Mars analogues, will be entombed within artificial sulfate crystals by evaporating an experimental brine under low atmospheric pressure and UV radiation similar to that encountered on the surface of Mars using The Open University’s Mars chamber facility. The lipids within the samples will be extracted and derivatized to make them amenable to by pyrolysis-gas chromatography-mass spectrometry (py-GC-MS); py-GC-MS is an important component of the Sample Analysis at Mars (SAM) instrument suite onboard NASA’s Mars Science Laboratory (MSL) and of the Mars Organic Molecular Organizer (MOMA) on the ESA’s Rosalind Franklin (ExoMars) rover. This work will aid in characterizing the impacts of the simulated Martian environment and Martian mineralogy on the preservation and detection of lipid biomarkers within sulfitic evaporite deposits and will inform the robotic missions targeting the search for ancient signatures of life on Mars. References: [1] Carr MH, Head JW (2010) Earth and Planetary Science Letters 294: 185-203 [2] Luo G et al., (2019) Earth-Science Reviews 189: 99-124 [3] Rapin, W et al., (2019) Nature Geosciences 12:889–895 [4] Fendrihan S et al., (2006) Reviews in Environmental Science and Biotechnology 5: 203-218 [5] Johnson SS et al., (2020) Astrobiology 2020 20:167-178

Authors acknowledge funding from the Science and Technology Facilities Council from the Grant ST/P000657/1. We would also like to acknowledge funding from a Leverhulme Trust Research Project Grant (RPG-2016-153) and thank the Polar Continental Shelf Program (Natural Resources Canada) for logistical field support in Nunavut. The transition of the martian climate from the wet Noachian era to the dry Hesperian (4.1–3.0 Gya) likely resulted in saline surface waters that were rich in sulfur species. Terrestrial analogue environments that possess a similar chemistry to these proposed waters can be used to develop an understanding of the diversity of microorganisms that could have persisted on Mars under such conditions. Here, we report on the chemistry and microbial community of the highly reducing sediment of Colour Peak springs, a sulfidic and saline spring system located within the Canadian High Arctic. DNA and cDNA 16S rRNA gene profiling demonstrated that the microbial community was dominated by sulfur oxidising bacteria, suggesting that primary production in the sediment was driven by chemolithoautotrophic sulfur oxidation. It is possible that the sulfur oxidising bacteria also supported the persistence of the additional taxa. Gibbs energy values calculated for the brines, based on the chemistry of Gale crater, suggested that the oxidation of reduced sulfur species was an energetically viable metabolism for life on early Mars. Publisher PDF

Finding evidence of life elsewhere in the Solar System is dependent on understanding biotic processes that could occur within potentially habitable environments. Here, we describe a suite of high-pressure flow-through reactors that have been developed to investigate biotic and abiotic processes within simulated sub-surface martian and icy moon environments.

The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus’ plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.

The detection of potential biosignatures with mineral matrices is part of a multifaceted approach in the search for life on other planetary bodies. The 2020 ExoMars Rosalind Franklin rover includes within its payload three IR spectrometers in the form of ISEM (Infrared Spectrometer for ExoMars), MicrOmega, and Ma-MISS (Mars Multispectral Imager for Subsurface Studies). The use of this technique in the detection and characterization of biosignatures is of great value. Organic materials are often co-deposited in terrestrial evaporites and as such have been proposed as relevant analogs in the search for life on Mars. This study focuses on Ca-sulfates collected from the hypersaline Tírez Lake in Spain. Mid infrared and visible near infrared analysis of soils, salt crusts, and crystals with green and red layering indicative of microbial colonization of the samples was acquired from across the lake and identified the main mineral to be gypsum with inputs of carbonate and silica. Organic functional groups that could be attributed to amides and carboxylic acids were identified as well as chlorophyll; however, due to the strong mineralogical absorptions observed, these were hard to unambiguously discern. Taxonomical assignment demonstrated that the archaeal community within the samples was dominated by the halophilic extremophile Halobacteriaceae while the bacterial community was dominated by the class Nocardiaceae. The results of this research highlight that sulfates on Mars are a mixed blessing, acting as an effective host for organic matter preservation but also a material that masks the presence of organic functional groups when analyzed with spectroscopic tools similar to those due to fly on the 2020 ExoMars rover. A suite of complementary analytical techniques therefore should be used to support the spectral identification of any candidate extraterrestrial biosignatures.

Existing martian simulants are predominantly based on the chemistry of the average ‘global’ martian regolith as defined by data on chemical and mineralogical variability detected by orbiting spacecraft, surface rovers and landers. We have therefore developed new martian simulants based on the known composition of regolith from four different martian surface environments: an early basaltic terrain, a sulfur-rich regolith, a haematite-rich regolith and a contemporary Mars regolith. Simulants have been developed so that the Fe2+/Fe3+ ratios can be adjusted, if necessary, leading to the development of four standard simulants and four Fe-modified simulants. Characterisation of the simulants confirm that all but two (both sulfur-rich) are within 5 wt% of the martian chemistries that they were based on and, unlike previous simulants, they have Fe2+/Fe3+ ratios comparable to those found on Mars. Here we outline the design, production and characterisation of these new martian regolith simulants. These are to be used initially in experiments to study the potential habitability of martian environments in which Fe may be a key energy source.

The Dallol volcano and its associated hydrothermal field are located in a remote area of the northern Danakil Depression in Ethiopia, a region only recently appraised after decades of inaccessibility due to severe political instability and the absence of infrastructure. The region is notable for hosting environments at the very edge of natural physical-chemical extremities. It is surrounded by a wide, hyperarid salt plain and is one of the hottest (average annual temperature Dallol : 36-38°C) and most acidic natural systems (pH Dallol ≈0) on Earth. Spectacular geomorphologies and mineral deposits produced by supersaturated hydrothermal waters and brines are the result of complex interactions between active and inactive hydrothermal alteration of the bedrock, sulfuric hot springs and pools, fumaroles and geysers, and recrystallization processes driven by hydrothermal waters, degassing, and rapid evaporation. The study of planetary field analog environments plays a crucial role in characterizing the physical and chemical boundaries within which life can exist on Earth and other planets. It is essential for the definition and assessment of the conditions of habitability on other planets, including the possibility for biosignature preservation and in situ testing of technologies for life detection. The Dallol area represents an excellent Mars analog environment given that the active volcanic environment, the associated diffuse hydrothermalism and hydrothermal alteration, and the vast acidic sulfate deposits are reminiscent of past hydrothermal activity on Mars. The work presented in this paper is an overview of the Dallol volcanic area and its hydrothermal field that integrates previous literature with observations and results obtained from field surveys and monitoring coupled with sample characterization. In so doing, we highlight its exceptional potential as a planetary field analog as well as a site for future astrobiological and exploration programs, This is a contribution to the Europlanet 2020 Research Infrastructure programme funded under the European Commission's Horizon 2020 programme (No. 654208

Currently, the surface of Mars cannot sustain liquid water, but there is evidence suggesting that water was present in the Noachian. Although water might exist in the subsurface of Mars, it could not sustain in the present day unless it was highly saline. Thus, saline springs in polar desert environments are analogues with which to investigate martian conditions. An example of this is Axel Heiberg Island, located in a region of continuous permafrost in the Canadian High Arctic, which hosts sulfidic and highly saline springs. In this study, cultivation-dependant and independent techniques were used to investigate the microbial diversity of a sediment sample collected from a saline cold (3–8 °C) pool at Colour Peak Springs on Axel Heiberg. Both DNA and RNA were extracted from the samples, and the microbial community was characterised using the 16S rRNA gene from the extracted nucleic acids. The metabolic profile was characterised by screening DNA and cDNA for functional genes relating to the cycling of carbon (coxL, xoxF, cbbL), nitrogen (nifH, nosZ, nod) and sulfur (dsrB, soxB). The community profiles were used to inform enrichment strategies, allowing for the isolation and characterisation of several halophilic isolates including strains of Marinobacter, Halomonas, Halanaerobium and Loktanella. Through this work we have been able to develop an in-depth characterisation of the metabolic and phylogenetic diversity that is present and viable within this analogue site. This allows us to start building an understanding of the underlying mechanisms and strategies that enable organisms to persist in these environments.

In the introduction of this book, we briefly explored the history of the exploration of Mars, where we discussed a major shift in the scientific thinking and public perception of the nature of the surface of Mars as new missions explored the Red Planet. The first observations of the surface sparked speculations of Mars being inhabited by intelligent beings, inspiring fantasy, science fiction, and, of course, further exploration. The turn came when Mariner 4 returned images of a lunar-like landscape, a monotonous basaltic world with many impact craters but no water, and therefore no prospects for life to find a habitable niche. The authors of the chapters of this book show how different our understanding of Mars is today from the historical Mariner 4 perspective!

Here, we report the draft genome sequence of a strain of Clostridium isolated from sediment collected from an estuarine environment. The strain was isolated using a minimal medium designed to select for chemoautotrophic microorganisms., Here, we report the draft genome sequence of a strain of Clostridium isolated from sediment collected from an estuarine environment. The strain was isolated using a minimal medium designed to select for chemoautotrophic microorganisms. The strain may represent a novel species within the genus Clostridium, and this genome sequence enables further investigation into the genetic and metabolic diversity of this organism.

A kombucha multimicrobial culture (KMC) was exposed to simulated Mars-like conditions in low-Earth orbit (LEO). The study was part of the Biology and Mars Experiment (BIOMEX), which was accommodated in the European Space Agency's EXPOSE-R2 facility, outside the International Space Station. The aim of the study was to investigate the capability of a KMC microecosystem to survive simulated Mars-like conditions in LEO. During the 18-month exposure period, desiccated KMC samples, represented by living cellulose-based films, were subjected to simulated anoxic Mars-like conditions and ultraviolet (UV) radiation, as prevalent at the surface of present-day Mars. Postexposure analysis demonstrated that growth of both the bacterial and yeast members of the KMC community was observed after 60 days of incubation; whereas growth was detected after 2 days in the initial KMC. The KMC that was exposed to extraterrestrial UV radiation showed degradation of DNA, alteration in the composition and structure of the cellular membranes, and an inhibition of cellulose synthesis. In the "space dark control" (exposed to LEO conditions without the UV radiation), the diversity of the microorganisms that survived in the biofilm was reduced compared with the ground-based controls. This was accompanied by structural dissimilarities in the extracellular membrane vesicles. After a series of subculturing, the revived communities restored partially their structure and associated activities.

The question of whether Earth is a unique location for life remains one of the most enduring questions of our time. Geochemical data suggests that habitable environments may exist, or may have existed, elsewhere in the Solar System with promising targets including Mars and icy bodies where liquid water is believed to exist (Kargel, 2000; Grotzinger et al., 2014; Glein et al., 2015). Furthermore, potential habitable Exoplanets have been discovered where potentially there is sufficient atmospheric pressure to maintain liquid water (Jenkins et al., 2015; Gillon et al., 2017; Orosei et al., 2018). Yet, for life to exist it is not solely dependent on liquid water as it also needs bio-essential elements, an energy source, and the environmental conditions, that are conducive to life (Nixon et al., 2013). To investigate the feasibility of life elsewhere in the Solar System a combination of field and laboratory based studies, in-situ space experiments, and theoretical modeling is required. Here, we present 14 original research papers, one mini review, and two hypothesis and theory papers highlighting the novel and diverse methods that are employed to investigate potential life beyond the Earth. The overall focus of this collection of work is to understand if terrestrial life could exist elsewhere in the Solar System, and if so, what evidence (bio-signatures) could be used to support or negate the hypothesis of life.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.