Planetary Protection Assessment of Radioisotope Thermoelectric Generator (RTG)-Powered Landed Missions to Ocean Worlds: Application to Enceladus.

Landed missions to icy worlds with a subsurface liquid water ocean must meet planetary protection requirements and ensure a sufficiently small likelihood of any microorganism-bearing part of the landed element reaching the ocean. A higher bound on this likelihood is set by the potential for radioisotope thermoelectric generator (RTG) power sources, the hottest possible landed element, to melt through the ice shell and reach the ocean. In this study, we quantify this potential as a function of three key parameters: surface temperature, ice shell thickness (i.e., heat flux through the shell), and thickness of a porous (insulating) snow or regolith cover. Although the model we describe can be applied to any ocean world, we present results in the context of a landed mission concept to the south polar terrain of Saturn's moon Enceladus. In this particular context, we discuss planetary protection considerations for landing site selection. The likelihood of forward microbial contamination of Enceladus' ocean by an RTG-powered landed mission can be made sufficiently low to not undermine compliance with the planetary protection policy.

Science Objectives for Flagship-Class Mission Concepts for the Search for Evidence of Life at Enceladus.

Cassini revealed that Saturn's Moon Enceladus hosts a subsurface ocean that meets the accepted criteria for habitability with bio-essential elements and compounds, liquid water, and energy sources available in the environment. Whether these conditions are sufficiently abundant and collocated to support life remains unknown and cannot be determined from Cassini data. However, thanks to the plume of oceanic material emanating from Enceladus' south pole, a new mission to Enceladus could search for evidence of life without having to descend through kilometers of ice. In this article, we outline the science motivations for such a successor to Cassini, choosing the primary science goal to be determining whether Enceladus is inhabited and assuming a resource level equivalent to NASA's Flagship-class missions. We selected a set of potential biosignature measurements that are complementary and orthogonal to build a robust case for any life detection result. This result would be further informed by quantifications of the habitability of the environment through geochemical and geophysical investigations into the ocean and ice shell crust. This study demonstrates that Enceladus' plume offers an unparalleled opportunity for in situ exploration of an Ocean World and that the planetary science and astrobiology community is well equipped to take full advantage of it in the coming decades.

Astrobiology of life on Earth.

Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub-fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low-temperature environments and chemically diverse saline- and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock-associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints.

Life on Mars: Independent Genesis or Common Ancestor?

The possibility of biological transfer between planetary bodies is seldom factored into life detection strategies, although the actuality of such an event would have profound implications for how we interpret potential biosignatures found on other worlds. This article addresses the possibility of life on Mars in the context of a biological transfer and an independent genesis of life. The phylogenetic tree of life on Earth is used as a blueprint to interpret evidence of life and as a guideline to determine the likelihood that potential biosignatures could be expressed by martian organisms. Several transfer scenarios are considered, depending on the timing of transfer with respect to the evolution of life on Earth. The implications of each transfer scenario and an independent genesis of life on the biochemical nature of the resulting martian organisms are discussed. The analysis highlights how conceding the possibility of a biological transfer has practical implications for how we search for evidence of life, both in terms of the quality of potential biosignatures and the likelihood that certain biosignatures might be expressed. It is concluded that a degree of uncertainty on the origin of martian organisms might be unavoidable, particularly in the absence of a biochemical context.

Effects of Gamma and Electron Radiation on the Structural Integrity of Organic Molecules and Macromolecular Biomarkers Measured by Microarray Immunoassays and Their Astrobiological Implications.

High-energy ionizing radiation in the form of solar energetic particles and galactic cosmic rays is pervasive on the surface of planetary bodies with thin atmospheres or in space facilities for humans, and it may seriously affect the chemistry and the structure of organic and biological material. We used fluorescent microarray immunoassays to assess how different doses of electron and gamma radiations affect the stability of target compounds such as biological polymers and small molecules (haptens) conjugated to large proteins. The radiation effect was monitored by measuring the loss in the immunoidentification of the target due to an impaired ability of the antibodies for binding their corresponding irradiated and damaged epitopes (the part of the target molecule to which antibodies bind). Exposure to electron radiation alone was more damaging at low doses (1 kGy) than exposure to gamma radiation alone, but this effect was reversed at the highest radiation dose (500 kGy). Differences in the dose-effect immunoidentification patterns suggested that the amount (dose) and not the type of radiation was the main factor for the cumulative damage on the majority of the assayed molecules. Molecules irradiated with both types of radiation showed a response similar to that of the individual treatments at increasing radiation doses, although the pattern obtained with electrons only was the most similar. The calculated radiolysis constant did not show a unique pattern; it rather suggested a different behavior perhaps associated with the unique structure of each molecule. Although not strictly comparable with extraterrestrial conditions because the irradiations were performed under air and at room temperature, our results may contribute to understanding the effects of ionizing radiation on complex molecules and the search for biomarkers through bioaffinity-based systems in planetary exploration.

Constraints on the Metabolic Activity of Microorganisms in Atacama Surface Soils Inferred from Refractory Biomarkers: Implications for Martian Habitability and Biomarker Detection.

Dryness is one of the main environmental challenges to microbial survival. Understanding the threshold of microbial tolerance to extreme dryness is relevant to better constrain the environmental limits of life on Earth and critically evaluate long-term habitability models of Mars. Biomolecular proxies for microbial adaptation and growth were measured in Mars-like hyperarid surface soils in the Atacama Desert that experience only a few millimeters of precipitation per decade, and in biologically active soils a few hundred kilometers away that experience two- to fivefold more precipitation. Diversity and abundance of lipids and other biomolecules decreased with increasing dryness. Cyclopropane fatty acids (CFAs), which are indicative of adaptive response to environmental stress and growth in bacteria, were only detected in the wetter surface soils. The ratio of trans to cis isomers of an unsaturated fatty acid, another bacterial stress indicator, decreased with increasingly dry conditions. Aspartic acid racemization ratios increased from 0.01 in the wetter soils to 0.1 in the driest soils, which is indicative of racemization rates comparable to de novo biosynthesis over long timescales (∼10,000 years). The content and integrity of stress proteins profiled by immunoassays were additional indicators that biomass in the driest soils is not recycled at significant levels. Together, our results point to minimal or no in situ microbial growth in the driest surface soils of the Atacama, and any metabolic activity is likely to be basal for cellular repair and maintenance only. Our data add to a growing body of evidence that the driest Atacama surface soils represent a threshold for long-term habitability (i.e., growth and reproduction). These results place constraints on the potential for extant life on the surface of Mars, which is 100-1000 times drier than the driest regions in the Atacama. Key Words: Atacama Desert-Dryness-Growth-Habitability-Biomarker-Mars. Astrobiology 18, 955-966.

Corrosion of bare carbon steel as a passive sensor to assess moisture availability for biological activity in Atacama Desert soils.

Here we consider that the corrosion of polished bared metal coupons can be used as a passive sensor to detect or identify the lower limit of water availability suitable for biological activity in Atacama Desert soils or solid substrates. For this purpose, carbon steel coupons were deposited at selected sites along a west-east transect and removed at predetermined times for morphological inspection. The advantage of this procedure is that the attributes of the oxide layer (corrosion extent, morphology and oxide phases) can be considered as a fingerprint of the atmospheric moisture history at a given time interval. Two types of coupons were used, long rectangular shaped ones that were half-buried in a vertical position, and square shaped ones that were deposited on the soil surface. The morphological attributes observed by SEM inspection were found to correlate to the so-called humectation time which is determined from local meteorological parameters. The main finding was that the decreasing trend of atmospheric moisture along the transect was closely related to corrosion behaviour and water soil penetration. For instance, at the coastal site oxide phases formed on the coupon surface rapidly evolve into well-crystallized species, while at the driest inland site Lomas Bayas only amorphous oxide was observed on the coupons.

Transitory microbial habitat in the hyperarid Atacama Desert.

Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity.

Mineral paragenesis on Mars: The roles of reactive surface area and diffusion.

Geochemical models of secondary mineral precipitation on Mars generally assume semiopen systems (open to the atmosphere but closed at the water-sediment interface) and equilibrium conditions. However, in natural multicomponent systems, the reactive surface area of primary minerals controls the dissolution rate and affects the precipitation sequences of secondary phases, and simultaneously, the transport of dissolved species may occur through the atmosphere-water and water-sediment interfaces. Here we present a suite of geochemical models designed to analyze the formation of secondary minerals in basaltic sediments on Mars, evaluating the role of (i) reactive surface areas and (ii) the transport of ions through a basalt sediment column. We consider fully open conditions, both to the atmosphere and to the sediment, and a kinetic approach for mineral dissolution and precipitation. Our models consider a geochemical scenario constituted by a basin (i.e., a shallow lake) where supersaturation is generated by evaporation/cooling and the starting point is a solution in equilibrium with basaltic sediments. Our results show that cation removal by diffusion, along with the input of atmospheric volatiles and the influence of the reactive surface area of primary minerals, plays a central role in the evolution of the secondary mineral sequences formed. We conclude that precipitation of evaporites finds more restrictions in basaltic sediments of small grain size than in basaltic sediments of greater grain size.

Xeropreservation of functionalized lipid biomarkers in hyperarid soils in the Atacama Desert.

Our understanding of long-term organic matter preservation comes mostly from studies in aquatic systems. In contrast, taphonomic processes in extremely dry environments are relatively understudied and are poorly understood. We investigated the accumulation and preservation of lipid biomarkers in hyperarid soils in the Yungay region of the Atacama Desert. Lipids from seven soil horizons in a 2.5 m vertical profile were extracted and analyzed using GC-MS and LC-MS. Diagnostic functionalized lipids and geolipids were detected and increased in abundance and diversity with depth. Deeper clay units contain fossil organic matter (radiocarbon dead) that has been protected from rainwater since the onset of hyperaridity. We show that these clay units contain lipids in an excellent state of structural preservation with functional groups and unsaturated bonds in carbon chains. This indicates that minimal degradation of lipids has occurred in these soils since the time of their deposition between >40,000 and 2 million years ago. The exceptional structural preservation of biomarkers is likely due to the long-term hyperaridity that has minimized microbial and enzymatic activity, a taphonomic process we term xeropreservation (i.e. preservation by drying). The degree of biomarker preservation allowed us to reconstruct major changes in ecology in the Yungay region that reflect a shift in hydrological regime from wet to dry since the early Quaternary. Our results suggest that hyperarid environments, which comprise 7.5% of the continental landmass, could represent a rich and relatively unexplored source of paleobiological information on Earth.

The Last Possible Outposts for Life on Mars.

The evolution of habitable conditions on Mars is often tied to the existence of aquatic habitats and largely constrained to the first billion years of the planet. Here, we propose an alternate, lasting evolutionary trajectory that assumes the colonization of land habitats before the end of the Hesperian period (ca. 3 billion years ago) at a pace similar to life on Earth. Based on the ecological adaptations to increasing dryness observed in dryland ecosystems on Earth, we reconstruct the most likely sequence of events leading to a late extinction of land communities on Mars. We propose a trend of ecological change with increasing dryness from widespread edaphic communities to localized lithic communities and finally to communities exclusively found in hygroscopic substrates, reflecting the need for organisms to maximize access to atmospheric sources of water. If our thought process is correct, it implies the possibility of life on Mars until relatively recent times, perhaps even the present.

Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert.

The Atacama Desert, northern Chile, is one of the driest deserts on Earth and, as such, a natural laboratory to explore the limits of life and the strategies evolved by microorganisms to adapt to extreme environments. Here we report the exceptional adaptation strategies of chlorophototrophic and eukaryotic algae, and chlorophototrophic and prokaryotic cyanobacteria to the hyperarid and extremely high solar radiation conditions occurring in this desert. Our approach combined several microscopy techniques, spectroscopic analytical methods, and molecular analyses. We found that the major adaptation strategy was to avoid the extreme environmental conditions by colonizing cryptoendolithic, as well as, hypoendolithic habitats within gypsum deposits. The cryptoendolithic colonization occurred a few millimeters beneath the gypsum surface and showed a succession of organized horizons of algae and cyanobacteria, which has never been reported for endolithic microbial communities. The presence of cyanobacteria beneath the algal layer, in close contact with sepiolite inclusions, and their hypoendolithic colonization suggest that occasional liquid water might persist within these sub-microhabitats. We also identified the presence of abundant carotenoids in the upper cryptoendolithic algal habitat and scytonemin in the cyanobacteria hypoendolithic habitat. This study illustrates that successful lithobiontic microbial colonization at the limit for microbial life is the result of a combination of adaptive strategies to avoid excess solar irradiance and extreme evapotranspiration rates, taking advantage of the complex structural and mineralogical characteristics of gypsum deposits-conceptually called "rock's habitable architecture." Additionally, self-protection by synthesis and accumulation of secondary metabolites likely produces a shielding effect that prevents photoinhibition and lethal photooxidative damage to the chlorophototrophs, representing another level of adaptation.

In situ metabolism in halite endolithic microbial communities of the hyperarid Atacama Desert.

The Atacama Desert of northern Chile is one of the driest regions on Earth, with areas that exclude plants and where soils have extremely low microbial biomass. However, in the driest parts of the desert there are microorganisms that colonize the interior of halite nodules in fossil continental evaporites, where they are sustained by condensation of atmospheric water triggered by the salt substrate. Using a combination of in situ observations of variable chlorophyll fluorescence and controlled laboratory experiments, we show that this endolithic community is capable of carbon fixation both through oxygenic photosynthesis and potentially ammonia oxidation. We also present evidence that photosynthetic activity is finely tuned to moisture availability and solar insolation and can be sustained for days, and perhaps longer, after a wetting event. This is the first demonstration of in situ active metabolism in the hyperarid core of the Atacama Desert, and it provides the basis for proposing a self-contained, endolithic community that relies exclusively on non-rainfall sources of water. Our results contribute to an increasing body of evidence that even in hyperarid environments active metabolism, adaptation, and growth can occur in highly specialized microhabitats.

Tracking the weathering of basalts on Mars using lithium isotope fractionation models.

Lithium (Li), the lightest of the alkali elements, has geochemical properties that include high aqueous solubility (Li is the most fluid mobile element) and high relative abundance in basalt-forming minerals (values ranking between 0.2 and 12 ppm). Li isotopes are particularly subject to fractionation because the two stable isotopes of lithium-7Li and 6Li-have a large relative mass difference (∼15%) that results in significant fractionation between water and solid phases. The extent of Li isotope fractionation during aqueous alteration of basalt depends on the dissolution rate of primary minerals-the source of Li-and on the precipitation kinetics, leading to formation of secondary phases. Consequently, a detailed analysis of Li isotopic ratios in both solution and secondary mineral lattices could provide clues about past Martian weathering conditions, including weathering extent, temperature, pH, supersaturation, and evaporation rate of the initial solutions in contact with basalt rocks. In this paper, we discuss ways in which Martian aqueous processes could have lead to Li isotope fractionation. We show that Li isotopic data obtained by future exploration of Mars could be relevant to highlighting different processes of Li isotopic fractionation in the past, and therefore to understanding basalt weathering and environmental conditions early in the planet's history.

What can in situ ion chromatography offer for Mars exploration?

The successes of the Mars exploration program have led to our unprecedented knowledge of the geological, mineralogical, and elemental composition of the martian surface. To date, however, only one mission, the Phoenix lander, has specifically set out to determine the soluble chemistry of the martian surface. The surprising results, including the detection of perchlorate, demonstrated both the importance of performing soluble ion measurements and the need for improved instrumentation to unambiguously identify all the species present. Ion chromatography (IC) is the state-of-the-art technique for soluble ion analysis on Earth and would therefore be the ideal instrument to send to Mars. A flight IC system must necessarily be small, lightweight, low-power, and have low eluent consumption. We demonstrate here a breadboard system that addresses these issues by using capillary IC at low flow rates with an optimized eluent generator and suppressor. A mix of 12 ions known or plausible for the martian soil, including 4 (oxy)chlorine species, has been separated at flow rates ranging from 1 to 10 µL/min, requiring as little as 200 psi at 1.0 µL/min. This allowed the use of pneumatic displacement pumping from a pressurized aluminum eluent reservoir and the elimination of the high-pressure pump entirely (the single heaviest and most energy-intensive component). All ions could be separated and detected effectively from 0.5 to 100 µM, even when millimolar concentrations of perchlorate were present in the same mixtures.

Chance and necessity in biochemistry: implications for the search for extraterrestrial biomarkers in Earth-like environments.

In this paper, we examine a restricted subset of the question of possible alien biochemistries. That is, we look into how different life might be if it emerged in environments similar to that required for life on Earth. We advocate a principle of chance and necessity in biochemistry. According to this principle, biochemistry is in some fundamental way the sum of two processes: there is an aspect of biochemistry that is an endowment from prebiotic processes, which represents the necessity, plus an aspect that is invented by the process of evolution, which represents the chance. As a result, we predict that life originating in extraterrestrial Earth-like environments will share biochemical motifs that can be traced back to the prebiotic world but will also have intrinsic biochemical traits that are unlikely to be duplicated elsewhere as they are combinatorially path-dependent. Effective and objective strategies to search for biomarkers, and evidence for a second genesis, on planets with Earth-like environments can be built based on this principle.

Salt deliquescence drives photosynthesis in the hyperarid Atacama Desert.

Endolithic cyanobacteria are found in halite nodules in the hyperarid core of the Atacama Desert. Using Pulse Amplitude Modulated Fluorometry, we show here that photosynthetic systems of these cyanobacteria become active when the relative humidity rises above 70% and the salt becomes wet by way of deliquescence. This is the first evidence of active metabolism in the hyperarid core of the Atacama, and supports the view of a microbial community sustained by deliquescence. Our results expand the water activity envelope of life on Earth.

Radiocarbon evidence of active endolithic microbial communities in the hyperarid core of the Atacama Desert.

The hyperarid core of the Atacama Desert is one of the driest and most inhospitable places on Earth, where life is most commonly found in the interior of rocks (i.e., endolithic habitats). Due to the extreme dryness, microbial activity in these habitats is expected to be low; however, the rate of carbon cycling within these microbial communities remains unknown. We address this issue by characterizing the isotopic composition ((13)C and (14)C) of phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) in colonized rocks from four different sites inside the hyperarid core. δ(13)C results suggest that autotrophy and/or quantitative conversion of organic matter to CO2 are the dominant processes occurring with the rock. Most Δ(14)C signatures of PLFA and GLFA were consistent with modern atmospheric CO2, indicating that endoliths are using atmospheric carbon as a primary carbon source and are also cycling carbon quickly. However, at one site the PLFA contained (14)C from atmospheric nuclear weapons testing that occurred during the 1950s and 1960s, indicating a decadal rate of carbon cycling. At the driest site (Yungay), based on the relative abundance and (14)C content of GLFA and PLFA, there was evidence of possible preservation. Hence, in low-moisture conditions, glycolipids may persist while phospholipids are preferentially hydrolyzed.

The Icebreaker Life Mission to Mars: a search for biomolecular evidence for life.

The search for evidence of life on Mars is the primary motivation for the exploration of that planet. The results from previous missions, and the Phoenix mission in particular, indicate that the ice-cemented ground in the north polar plains is likely to be the most recently habitable place that is currently known on Mars. The near-surface ice likely provided adequate water activity during periods of high obliquity, ≈ 5 Myr ago. Carbon dioxide and nitrogen are present in the atmosphere, and nitrates may be present in the soil. Perchlorate in the soil together with iron in basaltic rock provides a possible energy source for life. Furthermore, the presence of organics must once again be considered, as the results of the Viking GCMS are now suspect given the discovery of the thermally reactive perchlorate. Ground ice may provide a way to preserve organic molecules for extended periods of time, especially organic biomarkers. The Mars Icebreaker Life mission focuses on the following science goals: (1) Search for specific biomolecules that would be conclusive evidence of life. (2) Perform a general search for organic molecules in the ground ice. (3) Determine the processes of ground ice formation and the role of liquid water. (4) Understand the mechanical properties of the martian polar ice-cemented soil. (5) Assess the recent habitability of the environment with respect to required elements to support life, energy sources, and possible toxic elements. (6) Compare the elemental composition of the northern plains with midlatitude sites. The Icebreaker Life payload has been designed around the Phoenix spacecraft and is targeted to a site near the Phoenix landing site. However, the Icebreaker payload could be supported on other Mars landing systems. Preliminary studies of the SpaceX Dragon lander show that it could support the Icebreaker payload for a landing either at the Phoenix site or at midlatitudes. Duplicate samples could be cached as a target for possible return by a Mars Sample Return mission. If the samples were shown to contain organic biomarkers, interest in returning them to Earth would be high.

Mitochondria and the evolutionary roots of cancer.

Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell.