Multi-Technique Characterization of 3.45 Ga Microfossils on Earth: A Key Approach to Detect Possible Traces of Life in Returned Samples from Mars.

The NASA Mars 2020 Perseverance rover is actively exploring Jezero crater to conduct analyses on igneous and sedimentary rock targets from outcrops located on the crater floor (Máaz and Séítah formations) and from the delta deposits, respectively. The rock samples collected during this mission will be recovered during the Mars Sample Return mission, which plans to bring samples back to Earth in the 2030s to conduct in-depth studies using sophisticated laboratory instrumentation. Some of these samples may contain traces of ancient martian life that may be particularly difficult to detect and characterize because of their morphological simplicity and subtle biogeochemical expressions. Using the volcanic sediments of the 3.45 Ga Kitty's Gap Chert (Pilbara, Australia), containing putative early life forms (chemolithotrophs) and considered as astrobiological analogues for potential early Mars organisms, we document the steps required to demonstrate the syngenicity and biogenicity of such biosignatures using multiple complementary analytical techniques to provide information at different scales of observation. These include sedimentological, petrological, mineralogical, and geochemical analyses to demonstrate macro- to microscale habitability. New approaches, some unavailable at the time of the original description of these features, are used to verify the syngenicity and biogenicity of the purported fossil chemolithotrophs. The combination of elemental (proton-induced X-ray emission spectrometry) and molecular (deep-ultraviolet and Fourier transform infrared) analyses of rock slabs, thin sections, and focused ion beam sections reveals that the carbonaceous matter present in the samples is enriched in trace metals (e.g., V, Cr, Fe, Co) and is associated with aromatic and aliphatic molecules, which strongly support its biological origin. Transmission electron microscopy observations of the carbonaceous matter documented an amorphous nanostructure interpreted to correspond to the degraded remains of microorganisms and their by-products (extracellular polymeric substances, filaments…). Nevertheless, a small fraction of carbonaceous particles has signatures that are more metamorphosed. They probably represent either reworked detrital biological or abiotic fragments of mantle origin. This study serves as an example of the analytical protocol that would be needed to optimize the detection of fossil traces of life in martian rocks.

Farming on Mars: Treatment of basaltic regolith soil and briny water simulants sustains plant growth.

A fundamental challenge in human missions to Mars is producing consumable foods efficiently with the in situ resources such as soil, water, nutrients and solar radiation available on Mars. The low nutrient content of martian soil and high salinity of water render them unfit for direct use for propagating food crops on Mars. It is therefore essential to develop strategies to enhance nutrient content in Mars soil and to desalinate briny water for long-term missions on Mars. We report simple and efficient strategies for treating basaltic regolith simulant soil and briny water simulant for suitable resources for growing plants. We show that alfalfa plants grow well in a nutrient-limited basaltic regolith simulant soil and that the alfalfa biomass can be used as a biofertilizer to sustain growth and production of turnip, radish and lettuce in the basaltic regolith simulant soil. Moreover, we show that marine cyanobacterium Synechococcus sp. PCC 7002 effectively desalinates the briny water simulant, and that desalination can be further enhanced by filtration through basalt-type volcanic rocks. Our findings indicate that it is possible to grow food crops with alfalfa treated basaltic regolith martian soil as a substratum watered with biodesalinated water.

Microbial induced calcite precipitation can consolidate martian and lunar regolith simulants.

We demonstrate that Microbial Induced Calcite Precipitation (MICP) can be utilized for creation of consolidates of Martian Simulant Soil (MSS) and Lunar Simulant Soil (LSS) in the form of a 'brick'. A urease producer bacterium, Sporosarcina pasteurii, was used to induce the MICP process for the both simulant soils. An admixture of guar gum as an organic polymer and NiCl2, as bio- catalyst to enhance urease activity, was introduced to increase the compressive strength of the biologically grown bricks. A casting method was utilized for a slurry consisting of the appropriate simulant soil and microbe; the slurry over a few days consolidated in the form of a 'brick' of the desired shape. In case of MSS, maximum strength of 3.3 MPa was obtained with 10mM NiCl2 and 1% guar gum supplementation whereas in case of LSS maximum strength of 5.65 Mpa was obtained with 1% guar gum supplementation and 10mM NiCl2. MICP mediated consolidation of the simulant soil was confirmed with field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and thermogravimetry (TG). Our work demonstrates a biological approach with an explicit casting method towards manufacturing of consolidated structures using extra-terrestrial regolith simulant; this is a promising route for in situ development of structural elements on the extra-terrestrial habitats.

Science and Curation Considerations for the Design of a Mars Sample Return (MSR) Sample Receiving Facility (SRF).

The most important single element of the "ground system" portion of a Mars Sample Return (MSR) Campaign is a facility referred to as the Sample Receiving Facility (SRF), which would need to be designed and equipped to receive the returned spacecraft, extract and open the sealed sample container, extract the samples from the sample tubes, and implement a set of evaluations and analyses of the samples. One of the main findings of the first MSR Sample Planning Group (MSPG, 2019a) states that "The scientific community, for reasons of scientific quality, cost, and timeliness, strongly prefers that as many sample-related investigations as possible be performed in PI-led laboratories outside containment." There are many scientific and technical reasons for this preference, including the ability to utilize advanced and customized instrumentation that may be difficult to reproduce inside in a biocontained facility, and the ability to allow multiple science investigators in different labs to perform similar or complementary analyses to confirm the reproducibility and accuracy of results. It is also reasonable to assume that there will be a desire for the SRF to be as efficient and economical as possible, while still enabling the objectives of MSR to be achieved. For these reasons, MSPG concluded, and MSPG2 agrees, that the SRF should be designed to accommodate only those analytical activities that could not reasonably be done in outside laboratories because they are time- or sterilization-sensitive, are necessary for the Sample Safety Assessment Protocol (SSAP), or are necessary parts of the initial sample characterization process that would allow subsamples to be effectively allocated for investigation. All of this must be accommodated in an SRF, while preserving the scientific value of the samples through maintenance of strict environmental and contamination control standards. Executive Summary The most important single element of the "ground system" portion of a Mars Sample Return (MSR) Campaign is a facility referred to as the Sample Receiving Facility (SRF), which would need to be designed and equipped to enable receipt of the returned spacecraft, extraction and opening of the sealed sample container, extraction of the samples from the sample tubes, and a set of evaluations and analyses of the samples-all under strict protocols of biocontainment and contamination control. Some of the important constraints in the areas of cost and required performance have not yet been set by the necessary governmental sponsors, but it is reasonable to assume there will be a desire for the SRF to be as efficient and economical as is possible, while still enabling the objectives of MSR science to be achieved. Additionally, one of the main findings of MSR Sample Planning Group (MSPG, 2019a) states "The scientific community, for reasons of scientific quality, cost, and timeliness, strongly prefers that as many sample-related investigations as possible be performed in PI-led laboratories outside containment." There are many scientific and technical reasons for this preference, including the ability to utilize advanced and customized instrumentation that may be difficult to reproduce inside a biocontained facility. Another benefit is the ability to enable similar or complementary analyses by multiple science investigators in different laboratories, which would confirm the reproducibility and accuracy of results. For these reasons, the MSPG concluded-and the MSR Science Planning Group Phase 2 (MSPG2) agrees-that the SRF should be designed to accommodate only those analytical activities inside biocontainment that could not reasonably be done in outside laboratories because such activities are time-sensitive, sterilization-sensitive, required by the Sample Safety Assessment Protocol (SSAP), or are necessary parts of the initial sample characterization process that would allow subsamples to be effectively allocated for investigation. All activities within the SRF must be done while preserving the scientific value of the samples through maintenance of strict environmental and contamination control standards. The SRF would need to provide a unique environment that consists of both Biosafety Level 4 (BSL-4) equivalent containment and a very high level of contamination control. The SRF would also need to accommodate the following activities: (1)Receipt of the returned spacecraft, presumably in a sealed shipping container (2)De-integration (i.e., disassembly) and assessment of the returned system, beginning with the spacecraft exterior and ending with accessing and isolating all Mars material (gas, dust, regolith, and rock) (3)Initial sample characterization, leading to development of a sample catalog sufficient to support sample allocation (see Tait et al., 2022) (4)Science investigations necessary to complete the SSAP (see Kminek et al., 2021) (5)Certain science investigations that are both time- and sterilization-sensitive (see Tosca et al., 2022; Velbel et al., 2022) (6)A managed transition to post-SRF activities that would include analysis of samples (either sterilized or not) outside biocontainment and the transfer of some or all samples to one or more uncontained curation facilities The MSPG2 has produced a compilation of potential design requirements for the SRF, based on the list of activities noted above, that can be used in cost and schedule planning. The text of this report is meant to serve as an overview and explanation of these proposed SRF Design Requirements that have been compiled by the MSPG2 SRF Requirements Focus Group (Supplement 1). Summary of Findings FINDING SRF-1: The quality of the science that can be achieved with the MSR samples will be negatively impacted if they are not protected from contamination and inappropriate environmental conditions. A significant amount of SRF infrastructure would therefore be necessary to maintain and monitor appropriate levels of cleanliness, contamination control, and environmental conditions. FINDING SRF-2: Although most MSR sample investigations would take place outside of the SRF, the SRF needs to include significant laboratory capabilities with advanced instruments and associated sample preparation systems to enable the MSR science objectives to be successfully achieved. FINDING SRF-3: Preliminary studies of different operational scenarios should be started as soon as possible to enable analysis of the trade-offs between the cost and size of the SRF and the amount of time needed to prepare the samples for allocation and analysis. FINDING SRF-4: The ability to add additional analytical capabilities within biocontainment should be preserved to address the contingency scenario in which unsterilized material is not cleared to be analyzed outside of biocontainment. If potential evidence of martian life were to be detected in the samples, for example, it would be a high priority to conduct further investigations related to any putative lifeforms, as well as to enable other sterilization-sensitive science investigations to be conducted in biocontainment.

Planning Implications Related to Sterilization-Sensitive Science Investigations Associated with Mars Sample Return (MSR).

The NASA/ESA Mars Sample Return (MSR) Campaign seeks to establish whether life on Mars existed where and when environmental conditions allowed. Laboratory measurements on the returned samples are useful if what is measured is evidence of phenomena on Mars rather than of the effects of sterilization conditions. This report establishes that there are categories of measurements that can be fruitful despite sample sterilization and other categories that cannot. Sterilization kills living microorganisms and inactivates complex biological structures by breaking chemical bonds. Sterilization has similar effects on chemical bonds in non-biological compounds, including abiotic or pre-biotic reduced carbon compounds, hydrous minerals, and hydrous amorphous solids. We considered the sterilization effects of applying dry heat under two specific temperature-time regimes and the effects of γ-irradiation. Many measurements of volatile-rich materials are sterilization sensitive-they will be compromised by either dehydration or radiolysis upon sterilization. Dry-heat sterilization and γ-irradiation differ somewhat in their effects but affect the same chemical elements. Sterilization-sensitive measurements include the abundances and oxidation-reduction (redox) states of redox-sensitive elements, and isotope abundances and ratios of most of them. All organic molecules, and most minerals and naturally occurring amorphous materials that formed under habitable conditions, contain at least one redox-sensitive element. Thus, sterilization-sensitive evidence about ancient life on Mars and its relationship to its ancient environment will be severely compromised if the samples collected by Mars 2020 rover Perseverance cannot be analyzed in an unsterilized condition. To ensure that sterilization-sensitive measurements can be made even on samples deemed unsafe for unsterilized release from containment, contingency instruments in addition to those required for curation, time-sensitive science, and the Sample Safety Assessment Protocol would need to be added to the Sample Receiving Facility (SRF). Targeted investigations using analogs of MSR Campaign-relevant returned-sample types should be undertaken to fill knowledge gaps about sterilization effects on important scientific measurements, especially if the sterilization regimens eventually chosen are different from those considered in this report. Executive Summary A high priority of the planned NASA/ESA Mars Sample Return Campaign is to establish whether life on Mars exists or existed where and when allowed by paleoenvironmental conditions. To answer these questions from analyses of the returned samples would require measurement of many different properties and characteristics by multiple and diverse instruments. Planetary Protection requirements may determine that unsterilized subsamples cannot be safely released to non-Biosafety Level-4 (BSL-4) terrestrial laboratories. Consequently, it is necessary to determine what, if any, are the negative effects that sterilization might have on sample integrity, specifically the fidelity of the subsample properties that are to be measured. Sample properties that do not survive sterilization intact should be measured on unsterilized subsamples, and the Sample Receiving Facility (SRF) should support such measurements. This report considers the effects that sterilization of subsamples might have on the science goals of the MSR Campaign. It assesses how the consequences of sterilization affect the scientific usefulness of the subsamples and hence our ability to conduct high-quality science investigations. We consider the sterilization effects of (a) the application of dry heat under two temperature-time regimes (180°C for 3 hours; 250°C for 30 min) and (b) γ-irradiation (1 MGy), as provided to us by the NASA and ESA Planetary Protection Officers (PPOs). Measurements of many properties of volatile-rich materials are sterilization sensitive-they would be compromised by application of either sterilization mode to the subsample. Such materials include organic molecules, hydrous minerals (crystalline solids), and hydrous amorphous (non-crystalline) solids. Either proposed sterilization method would modify the abundances, isotopes, or oxidation-reduction (redox) states of the six most abundant chemical elements in biological molecules (i.e., carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulphur, CHNOPS), and of other key redox-sensitive elements that include iron (Fe), other first-row transition elements (FRTE), and cerium (Ce). As a result of these modifications, such evidence of Mars' life, paleoenvironmental history, potential habitability, and potential biosignatures would be corrupted or destroyed. Modifications of the abundances of some noble gases in samples heated during sterilization would also reset scientifically important radioisotope geochronometers and atmospheric-evolution measurements. Sterilization is designed to render terminally inactive (kill) all living microorganisms and inactivate complex biological structures (including bacterial spores, viruses, and prions). Sterilization processes do so by breaking certain pre-sterilization chemical bonds (including strong C-C, C-O, C-N, and C-H bonds of predominantly covalent character, as well as weaker hydrogen and van der Waals bonds) and forming different bonds and compounds, disabling the biological function of the pre-sterilization chemical compound. The group finds the following: No sterilization process could destroy the viability of cells whilst still retaining molecular structures completely intact. This applies not only to the organic molecules of living organisms, but also to most organic molecular biosignatures of former life (molecular fossils). As a matter of biological principle, any sterilization process would result in the loss of biological and paleobiological information, because this is the mechanism by which sterilization is achieved. Thus, almost all life science investigations would be compromised by sterilizing the subsample by either mode. Sterilization by dry heat at the proposed temperatures would lead to changes in many of the minerals and amorphous solids that are most significant for the study of paleoenvironments, habitability, potential biosignatures, and the geologic context of life-science observations. Gamma-(γ-)irradiation at even sub-MGy doses induces radiolysis of water. The radiolysis products (e.g., free radicals) react with redox-sensitive chemical species of interest for the study of paleoenvironments, habitability, and potential biosignatures, thereby adversely affecting measurements of those species. Heat sterilization and radiation also have a negative effect on CHNOPS and redox-sensitive elements. MSPG2 was unable to identify with confidence any measurement of abundances or oxidation-reduction states of CHNOPS elements, other redox-sensitive elements (e.g., Fe and other FRTE; Ce), or their isotopes that would be affected by only one, but not both, of the considered sterilization methods. Measurements of many attributes of volatile-rich subsamples are sterilization sensitive to both heat and γ-irradiation. Such a measurement is not useful to Mars science if what remains in the subsample is evidence of sterilization conditions and effects instead of evidence of conditions on Mars. Most measurements relating to the detection of evidence for extant or extinct life are sterilization sensitive. Many measurements other than those for life-science seek to retrieve Mars' paleoenvironmental information from the abundances or oxidation-reduction states of CHNOPS elements, other redox-sensitive elements, or their isotopes (and some noble gases) in returned samples. Such measurements inform scientific interpretations of (paleo)atmosphere composition and evolution, (paleo)surface water origin and chemical evolution, potential (paleo)habitability, (paleo)groundwater-porewater solute chemistry, origin and evolution, potential biosignature preservation, metabolic element or isotope fractionation, and the geologic, geochronological, and geomorphic context of life-sciences observations. Most such measurements are also sterilization sensitive. The sterilization-sensitive attributes cannot be meaningfully measured in any such subsample that has been sterilized by heat or γ-irradiation. Unless such subsamples are deemed biohazard-safe for release to external laboratories in unsterilized form, all such measurements must be made on unsterilized samples in biocontainment. An SRF should have the capability to carry out scientific investigations that are sterilization-sensitive to both PPO-provided sterilization methods (Figure SE1). The following findings have been recognized in the Report. Full explanations of the background, scope, and justification precede the presentation of each Finding in the Section identified for that Finding. One or more Findings follow our assessment of previous work on the effects of each provided sterilization method on each of three broad categories of measurement types-biosignatures of extant or ancient life, geological evidence of paleoenvironmental conditions, and gases. Findings are designated Major if they explicitly refer to both PPO-provided sterilization methods or have specific implications for the functionalities that need to be supported within an SRF. FINDING SS-1: More than half of the measurements described by iMOST for investigation into the presence of (mostly molecular) biosignatures (iMOST Objectives 2.1, 2.2 and 2.3) in returned martian samples are sterilization-sensitive and therefore cannot be performed with acceptable analytical precision or sensitivity on subsamples sterilized either by heat or by γ-irradiation at the sterilization parameters supplied to MSPG2. That proportion rises to 86% of the measurements specific to the investigation of extant or recent life (iMOST Objective 2.3) (see Section 2.5). This Finding supersedes Finding #4 of the MSPG Science in Containment report (MSPG, 2019). FINDING SS-2: Almost three quarters (115 out of 160; 72%) of the measurements described by iMOST for science investigations not associated with Objective 2 but associated with Objectives concerning geological phenomena that include past interactions with the hydrosphere (Objectives 1 and 3) and the atmosphere (Objective 4) are sterilization-tolerant and therefore can (generally) be performed with acceptable analytical precision or sensitivity on subsamples sterilized either by heat or by γ-irradiation at the sterilization parameters supplied to MSPG2 (see Section 2.5). This Finding supports Finding #6 of the MSPG Science in Containment report (MSPG, 2019). MSPG2 endorses the previously proposed strategy of conducting as many measurements as possible outside the SRF where the option exists. FINDING SS-3: Suggested strategies for investigating the potential for extant life in returned martian samples lie in understanding biosignatures and, more importantly, the presence of nucleic acid structures (DNA/RNA) and possible agnostic functionally similar information-bearing polymers. A crucial observation is that exposure of microorganisms to temperatures associated with sterilization above those typical of a habitable surface or subsurface environment results in a loss of biological information. If extant life is a target for subsample analysis, sterilization of material via dry heat would likely compromise any such analysis (see Section 3.2). FINDING SS-4: Suggested strategies for investigating the potential for extant life in returned martian samples lie in understanding biosignatures, including the presence of nucleic acid structures (DNA/RNA) and possible agnostic functionally similar information-bearing polymers. A crucial observation is that exposure of microorganisms to γ-radiation results in a loss of biological information through molecular damage and/or destruction. If extant life is a target for subsample analysis, sterilization of material via γ-radiation would likely compromise any such analysis (see Section 3.3). FINDING SS-5: Suggested strategies for investigating biomolecules in returned martian samples lie in detection of a variety of complex molecules, including peptides, proteins, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), as well as compounds associated with cell membranes such as lipids, sterols, and fatty acids and their geologically stable reaction products (hopanes, steranes, etc.) and possible agnostic functionally similar information-bearing polymers. Exposure to temperatures above MSR Campaign-Level Requirements for sample temperature, up to and including sterilization temperatures, results in a loss of biological information. If the presence of biosignatures is a target for subsample analysis, sterilization of material via dry heat would likely compromise any such analysis (see Section 4.2). FINDING SS-6: Suggested strategies for investigating biomolecules in returned martian samples lie in detection of a variety of complex molecules, including peptides, proteins, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and compounds associated with cell membranes such as lipids, sterols and fatty acids and their geologically stable reaction products (hopanes, steranes, etc.) and possible agnostic functionally similar information-bearing polymers. Exposure to radiation results in a loss of biological information. If the presence of biosignatures is a target for subsample analysis, sterilization of material via γ-irradiation would likely compromise any such analysis (see Section 4.3). [Figure: see text] MAJOR FINDING SS-7: The use of heat or γ-irradiation sterilization should be avoided for subsamples intended to be used for organic biosignature investigations (for extinct or extant life). Studies of organic molecules from extinct or extant life (either indigenous or contaminants, viable or dead cells) or even some organic molecules derived from abiotic chemistry cannot credibly be done on subsamples that have been sterilized by any means. The concentrations of amino acids and other reduced organic biosignatures in the returned martian samples may also be so low that additional heat and/or γ-irradiation sterilization would reduce their concentrations to undetectable levels. It is a very high priority that these experiments be done on unsterilized subsamples inside containment (see Section 4.4). FINDING SS-8: Solvent extraction and acid hydrolysis at ∼100°C of unsterilized martian samples will inactivate any biopolymers in the extract and would not require additional heat or radiation treatment for the subsamples to be rendered sterile. Hydrolyzed extracts should be safe for analysis of soluble free organic molecules outside containment and may provide useful information about their origin for biohazard assessments; this type of approach, if approved, is strongly preferred and endorsed (see Section 4.4). FINDING SS-9: Minerals and amorphous materials formed by low temperature processes on Mars are highly sensitive to thermal alteration, which leads to irreversible changes in composition and/or structure when heated. Exposure to temperatures above MSR Campaign-Level Requirements for sample temperature, up to and including sterilization temperatures, has the potential to alter them from their as-received state. Sterilization by dry heat at the proposed sterilization temperatures would lead to changes in many of the minerals that are most significant for the study of paleoenvironments, habitability, and potential biosignatures or biosignature hosts. It is crucial that the returned samples are not heated to temperatures above which mineral transitions occur (see Section 5.3). FINDING SS-10: Crystal structure, major and non-volatile minor element abundances, and stoichiometric compositions of minerals are unaffected by γ-irradiation of up to 0.3-1 MGy, but crystal structures are completely destroyed at 130 MGy. Measurements of these specific properties cannot be acquired from subsamples γ-irradiated at the notional 1 MGy dose-they are sterilization-sensitive (see Section 5.4). FINDING SS-11: Sterilization by γ-irradiation (even at sub-MGy doses) results in significant changes to the redox state of elements bound within a mineral lattice. Redox-sensitive elements include Fe and other first-row transition elements (FRTE) as well as C, H, N, O, P and S. Almost all minerals and naturally occurring amorphous materials that formed under habitable conditions, including the ambient paleotemperatures of Mars' surface or shallow subsurface, contain at least one of these redox-sensitive elements. Therefore, measurements and investigations of the listed properties of such geological materials are sterilization sensitive and should not be performed on γ-irradiated subsamples (see Section 5.4). FINDING SS-12: A significant fraction of investigations that focus on high-temperature magmatic and impact-related processes, their chronology, and the chronology of Mars' geophysical evolution are sterilization-tolerant. While there may be a few analyses involved in such investigations that could be affected to some degree by heat sterilization, most of these analyses would not be affected by sterilization involving γ-irradiation (see Section 5.6). MAJOR FINDING SS-13: Scientific investigations of materials containing hydrous or otherwise volatile-rich minerals and/or X-ray amorphous materials that formed or were naturally modified at low (Mars surface-/near-surface) temperature are sterilization-sensitive in that they would be compromised by changes in the abundances, redox states, and isotopes of CHNOPS and other volatiles (e.g., noble gases for chronometry), FRTE, and Ce, and cannot be performed on subsamples that have been sterilized by either dry heat or γ-irradiation (see Section 5.7). MAJOR FINDING SS-14: It would be far preferable to work on sterilized gas samples outside of containment, if the technical issues can all be worked out, than to build and operate a large gas chemistry laboratory inside containment. Depending on their reactivity (or inertness), gases extracted from sample tubes could be sterilized by dry heat or γ-irradiation and analyzed outside containment. Alternatively, gas samples could be filtered through an inert grid and the filtered gas analyzed outside containment (see Section 6.5). MAJOR FINDING SS-15: It is fundamental to the campaign-level science objectives of the Mars Sample Return Campaign that the SRF support characterization of samples returned from Mars that contain organic matter and/or minerals formed under habitable conditions that include the ambient paleotemperatures of Mars' surface or subsurface (<∼200°C)-such as most clays, sulfates, and carbonates-in laboratories on Earth in their as-received-at-the-SRF condition (see Section 7.1). MAJOR FINDING SS-16: The search for any category of potential biosignature would be adversely affected by either of the proposed sterilization methods (see Section 7.1). MAJOR FINDING SS-17: Carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus, and other volatiles would be released from a subsample during the sterilization step. The heat and γ-ray sterilization chambers should be able to monitor weight loss from the subsample during sterilization. Any gases produced in the sample headspace and sterilization chamber during sterilization should be captured and contained for future analyses of the chemical and stable isotopic compositions of the evolved elements and compounds for all sterilized subsamples to characterize and document fully any sterilization-induced alteration and thereby recover some important information that would otherwise be lost (see Section 7.2). This report shows that most of the sterilization-sensitive iMOST measurement types are among either the iMOST objectives for life detection and life characterization (half or more of the measurements for life-science sub-objectives are critically sterilization sensitive) or the iMOST objectives for inferring paleoenvironments, habitability, preservation of potential biosignatures, and the geologic context of life-science observations (nearly half of the measurements for sub-objectives involving geological environments, habitability, potential biosignature preservation, and gases/volatiles are critically sterilization sensitive) (Table 2; see Beaty et al., 2019 for the full lists of iMOST objectives, goals, investigations, and sample measurement types). Sterilization-sensitive science about ancient life on Mars and its relationship to its ancient environment will be severely impaired or lost if the samples collected by Perseverance cannot be analyzed in an unsterilized condition. Summary: ○The SRF should have the capability to carry out or otherwise support scientific investigations that are sensitive to both PPO-provided sterilization methods. ○Measurements of most life-sciences and habitability-related (paleoenvironmental) phenomena are sensitive to both PPO-provided sterilization modes. (Major Finding SS-7, SS-15, SS-16 and Finding SS-1, SS-3, SS-4, SS-5, SS-6, SS-9, SS-11, SS-13) If subsamples for sterilization-sensitive measurement cannot be deemed safe for release, then additional contingency analytical capabilities are needed in the SRF to complete MSR Campaign measurements of sterilization-sensitive sample properties on unsterilized samples in containment (Figure SE1, below). ○Measurements of high-temperature (low-volatile) phenomena are tolerant of both PPO-provided sterilization modes (Finding SS-12). Subsamples for such measurements may be sterilized and released to laboratories outside containment without compromising the scientific value of the measurements. ○Capturing, transporting, and analyzing gases is important and will require careful design of apparatus. Doing so for volatiles present as headspace gases and a dedicated atmosphere sample will enable important atmospheric science (Major Finding SS-14). Similarly, capturing and analyzing gases evolved during subsample sterilization (i.e., gas from the sterilization chamber) would compensate for some sterilization-induced loss of science data from volatile-rich solid (geological) subsamples (Finding SS-14, SS-17; other options incl. SS-8).

Bacterial Growth in Brines Formed by the Deliquescence of Salts Relevant to Cold Arid Worlds.

Hygroscopic salts at Mars' near-surface (MgSO4, (per)chlorates, NaCl) may form brines by absorbing moisture from the atmosphere at certain times through the process of deliquescence. We have previously shown strong bacterial growth in saturated MgSO4 (∼67% w/v as epsomite) at room temperature, and growth was observed at the MgSO4 eutectic point (43% w/v at -4°C). Here, we have investigated the growth of salinotolerant microbes (Halomonas, Marinococcus, Planococcus) from Hot Lake, Washington; Basque Lake, British Columbia; and Great Salt Plains, Oklahoma under deliquescing conditions. Bacterial cultures were grown to mid-log phase in SP medium supplemented with 50% MgSO4 (as epsomite), 20% NaClO3, or 10% NaCl (w/v), and small aliquots in cups were dried by vacuum desiccation. When the dried culture was rehydrated by the manual addition of water, the culture resumed growth in the reconstituted brine. When desiccated cultures were maintained in a sealed container with a brine reservoir of the matching growth medium controlling the humidity of the headspace, the desiccated microbial culture evaporites formed brine by deliquescence using humidity alone. Bacterial cultures resumed growth in all three salts once rehydrated by deliquescence. Cultures of Halomonas sp. str. HL12 showed robust survival and growth when subjected to several cycles of desiccation and deliquescent or manual rehydration. Our laboratory demonstrations of microbial growth in deliquescent brines are relevant to the surface and near-subsurface of cold arid worlds like Mars. When conditions become wetter, hygroscopic evaporite minerals can deliquesce to produce the earliest habitable brines. Survival after desiccation and growth in deliquescent brines increases the likelihood that microbes from Earth, carried on spacecraft, pose a contamination risk to Mars.

The Role of Minerals in Events That Led to the Origin of Life.

The role of minerals in the events that led to the origin of life is discussed with regard to (1) their catalytic role for the formation of RNA-like oligomers from their monomers and (2) their protective role for organic molecules formed in space that were delivered to planetary surfaces. Results obtained in the laboratory demonstrate that minerals do catalyze the oligomerization of ribonucleic acid (RNA) monomers to produce short RNA chains. Furthermore, and more importantly, these synthetic RNA chains formed by mineral catalysis serve as a template for the formation of complementary RNA chains, which is a significant finding that demonstrates the role of minerals in the origin of life. Simulation experiments run under Mars-like conditions have also shown that Mars analog minerals can shield the precursors of RNA and proteins against the harmful effects of UV and gamma radiation at the martian surface and 5 cm below the surface.

The Detection of Elemental Signatures of Microbes in Martian Mudstone Analogs Using High Spatial Resolution Laser Ablation Ionization Mass Spectrometry.

The detection and identification of biosignatures on planetary bodies such as Mars in situ is extremely challenging. Current knowledge from space exploration missions suggests that a suite of complementary instruments is required in situ for a successful identification of past or present life. For future exploration missions, new and innovative instrumentation capable of high spatial resolution chemical (elemental and isotope) analysis of solids with improved measurement capabilities is of considerable interest because a multitude of potential signatures of extinct or extant life have dimensions on the micrometer scale. The aim of this study is to extend the current measurement capabilities of a miniature laser ablation ionization mass spectrometer (LIMS) designed for space exploration missions to detect signatures of microbial life. In total, 14 martian mudstone analogue samples were investigated regarding their elemental composition. Half the samples were artificially inoculated with a low number density of microbes, and half were used as abiotic controls. The samples were treated in a number of ways. Some were cultured anaerobically and some aerobically; some abiotic samples were incubated with water, and some remained dry. Some of the samples were exposed to a large dose of γ radiation, and some were left un-irradiated. While no significant elemental differences were observed between the applied sample treatments, the instrument showed the capability to detect biogenic element signatures of the inoculated microbes by monitoring biologically relevant elements, such as hydrogen, carbon, sulfur, iron, and so on. When an enrichment in carbon was measured in the samples but no simultaneous increase in other biologically relevant elements was detected, it suggests, for example, a carbon-containing inclusion; when the enrichment was in carbon and in bio-relevant elements, it suggests the presences of microbes. This study presents first results on the detection of biogenic element patterns of microbial life using a miniature LIMS system designed for space exploration missions.

Infrared Spectroscopic Detection of Biosignatures at Lake Tírez, Spain: Implications for Mars.

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.

Mineralogy, Structure, and Habitability of Carbon-Enriched Rocky Exoplanets: A Laboratory Approach.

Carbon-enriched rocky exoplanets have been proposed to occur around dwarf stars as well as binary stars, white dwarfs, and pulsars. However, the mineralogical make up of such planets is poorly constrained. We performed high-pressure high-temperature laboratory experiments (P = 1-2 GPa, T = 1523-1823 K) on chemical mixtures representative of C-enriched rocky exoplanets based on calculations of protoplanetary disk compositions. These P-T conditions correspond to the deep interiors of Pluto- to Mars-sized planets and the upper mantles of larger planets. Our results show that these exoplanets, when fully differentiated, comprise a metallic core, a silicate mantle, and a graphite layer on top of the silicate mantle. Graphite is the dominant carbon-bearing phase at the conditions of our experiments with no traces of silicon carbide or carbonates. The silicate mineralogy comprises olivine, orthopyroxene, clinopyroxene, and spinel, which is similar to the mineralogy of the mantles of carbon-poor planets such as the Earth and largely unaffected by the amount of carbon. Metals are either two immiscible iron-rich alloys (S-rich and S-poor) or a single iron-rich alloy in the Fe-C-S system with immiscibility depending on the S/Fe ratio and core pressure. We show that, for our C-enriched compositions, the minimum carbon abundance needed for C-saturation is 0.05-0.7 wt% (molar C/O ∼0.002-0.03). Fully differentiated rocky exoplanets with C/O ratios more than that needed for C-saturation would contain graphite as an additional layer on top of the silicate mantle. For a thick enough graphite layer, diamonds would form at the bottom of this layer due to high pressures. We model the interior structure of Kepler-37b and show that a mere 10 wt% graphite layer would decrease its derived mass by 7%, which suggests that future space missions that determine both radius and mass of rocky exoplanets with insignificant gaseous envelopes could provide quantitative limits on their carbon content. Future observations of rocky exoplanets with graphite-rich surfaces would show low albedos due to the low reflectance of graphite. The absence of life-bearing elements other than carbon on the surface likely makes them uninhabitable.

Trivalent Phosphorus and Phosphines as Components of Biochemistry in Anoxic Environments.

Phosphorus is an essential element for all life on Earth, yet trivalent phosphorus (e.g., in phosphines) appears to be almost completely absent from biology. Instead phosphorus is utilized by life almost exclusively as phosphate, apart from a small contingent of other pentavalent phosphorus compounds containing structurally similar chemical groups. In this work, we address four previously stated arguments as to why life does not explore trivalent phosphorus: (1) precedent (lack of confirmed instances of trivalent phosphorus in biochemicals suggests that life does not have the means to exploit this chemistry), (2) thermodynamic limitations (synthesizing trivalent phosphorus compounds is too energetically costly), (3) stability (phosphines are too reactive and readily oxidize in an oxygen (O2)-rich atmosphere), and (4) toxicity (the trivalent phosphorus compounds are broadly toxic). We argue that the first two of these arguments are invalid, and the third and fourth arguments only apply to the O2-rich environment of modern Earth. Specifically, both the reactivity and toxicity of phosphines are specific to aerobic life and strictly dependent on O2-rich environment. We postulate that anaerobic life persisting in anoxic (O2-free) environments may exploit trivalent phosphorus chemistry much more extensively. We review the production of trivalent phosphorus compounds by anaerobic organisms, including phosphine gas and an alkyl phosphine, phospholane. We suggest that the failure to find more such compounds in modern terrestrial life may be a result of the strong bias of the search for natural products toward aerobic organisms. We postulate that a more thorough identification of metabolites of the anaerobic biosphere could reveal many more trivalent phosphorus compounds. We conclude with a discussion of the implications of our work for the origin and early evolution of life, and suggest that trivalent phosphorus compounds could be valuable markers for both extraterrestrial life and the Shadow Biosphere on Earth.

A touch of awe: crafting meaning from the wonder of the cosmos.

From the birth of galaxies to the self-organizing dynamics of our planet to the ongoing expansion of the universe, the more we discover about the evolution of the cosmos, the more acutely we realize the enormity of what remains to be known. Just this year astrophysicists at the University of Nottingham confirmed that there are at least two trillion galaxies in the cosmos, 10 times more than had been previously thought. What guidance or wisdom can the study of cosmology and astrophysics offer us in our search for meaning and purpose? In conversation with Steve Paulson, executive producer and host of To the Best of Our Knowledge, theoretical physicists Paul Davies and Ard Louis, and astrophysicist Lucianne Walkowicz share their perceptions based on years of gazing upward and beyond our own intimate planet.

Heart in space: effect of the extraterrestrial environment on the cardiovascular system.

National space agencies and private corporations aim at an extended presence of humans in space in the medium to long term. Together with currently suboptimal technology, microgravity and cosmic rays raise health concerns about deep-space exploration missions. Both of these physical factors affect the cardiovascular system, whose gravity-dependence is pronounced. Heart and vascular function are, therefore, susceptible to substantial changes in weightlessness. The altered cardiovascular function in space causes physiological problems in the postflight period. A compromised cardiovascular system can be excessively vulnerable to space radiation, synergistically resulting in increased damage. The space radiation dose is significantly lower than in patients undergoing radiotherapy, in whom cardiac damage is well-documented following cancer therapy in the thoracic region. Nevertheless, epidemiological findings suggest an increased risk of late cardiovascular disease even with low doses of radiation. Moreover, the peculiar biological effectiveness of heavy ions in cosmic rays might increase this risk substantially. However, whether radiation-induced cardiovascular effects have a threshold at low doses is still unclear. The main countermeasures to mitigate the effect of the space environment on cardiac function are physical exercise, antioxidants, nutraceuticals, and radiation shielding.

Critical Assessment of Analytical Techniques in the Search for Biomarkers on Mars: A Mummified Microbial Mat from Antarctica as a Best-Case Scenario.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

Kombucha Multimicrobial Community under Simulated Spaceflight and Martian Conditions.

Kombucha microbial community (KMC) produces a cellulose-based biopolymer of industrial importance and a probiotic beverage. KMC-derived cellulose-based pellicle film is known as a highly adaptive microbial macrocolony-a stratified community of prokaryotes and eukaryotes. In the framework of the multipurpose international astrobiological project "BIOlogy and Mars Experiment (BIOMEX)," which aims to study the vitality of prokaryotic and eukaryotic organisms and the stability of selected biomarkers in low Earth orbit and in a Mars-like environment, a cellulose polymer structural integrity will be assessed as a biomarker and biotechnological nanomaterial. In a preflight assessment program for BIOMEX, the mineralized bacterial cellulose did not exhibit significant changes in the structure under all types of tests. KMC members that inhabit the cellulose-based pellicle exhibited a high survival rate; however, the survival capacity depended on a variety of stressors such as the vacuum of space, a Mars-like atmosphere, UVC radiation, and temperature fluctuations. The critical limiting factor for microbial survival was high-dose UV irradiation. In the tests that simulated a 1-year mission of exposure outside the International Space Station, the core populations of bacteria and yeasts survived and provided protection against UV; however, the microbial density of the populations overall was reduced, which was revealed by implementation of culture-dependent and culture-independent methods. Reduction of microbial richness was also associated with a lower accumulation of chemical elements in the cellulose-based pellicle film, produced by microbiota that survived in the post-test experiments, as compared to untreated cultures that populated the film. Key Words: BIOlogy and Mars Experiment (BIOMEX)-Kombucha multimicrobial community-Biosignature-Biofilm-Bacterial cellulose. Astrobiology 17, 459-469.

Weathering Profiles in Phosphorus-Rich Rocks at Gusev Crater, Mars, Suggest Dissolution of Phosphate Minerals into Potentially Habitable Near-Neutral Waters.

UNLABELLED: Abundant evidence indicates that significant surface and near-surface liquid water has existed on Mars in the past. Evaluating the potential for habitable environments on Mars requires an understanding of the chemical and physical conditions that prevailed in such aqueous environments. Among the geological features that may hold evidence of past environmental conditions on Mars are weathering profiles, such as those in the phosphorus-rich Wishstone-class rocks in Gusev Crater. The weathering profiles in these rocks indicate that a Ca-phosphate mineral has been lost during past aqueous interactions. The high phosphorus content of these rocks and potential release of phosphorus during aqueous interactions also make them of astrobiological interest, as phosphorus is among the elements required for all known life. In this work, we used Mars mission data, laboratory-derived kinetic and thermodynamic data, and data from terrestrial analogues, including phosphorus-rich basalts from Idaho, to model a conceptualized Wishstone-class rock using the reactive transport code CrunchFlow. Modeling results most consistent with the weathering profiles in Wishstone-class rocks suggest a combination of chemical and physical erosion and past aqueous interactions with near-neutral waters. The modeling results also indicate that multiple Ca-phosphate minerals are likely in Wishstone-class rocks, consistent with observations of martian meteorites. These findings suggest that Gusev Crater experienced a near-neutral phosphate-bearing aqueous environment that may have been conducive to life on Mars in the past. KEY WORDS: Mars-Gusev Crater-Wishstone-Reactive transport modeling-CrunchFlow-Aqueous interactions-Neutral pH-Habitability.

Considering space weather forces interaction on human health: the equilibrium paradigm in clinical cosmobiology - is it equal?

BACKGROUND: We are constantly affected by changes in space weather. The principal "players" are solar activity (SA), geomagnetic activity (GMA) and antagonistic to them, cosmic ray activity (CRA) and high energy proton flux. CRA is measured by neutron activity on the earth's surface in imp/min. SA and GMA are linked and serve as a shield for the earth from CRA. For a long time SA and GMA were the main areas of studies. The aim of this study was to compare some effects of the mentioned forces and discuss the temporal distribution of both groups of space weather, in relation to their effects on humans. METHODS: The time distribution of GMA storms (daily) was compared with quiet (low) GMA, with higher CRA (neutron activity). Space weather data were obtained from the USA, Russia and Finland. RESULTS: A total of 4383 days were analyzed in the years 2000-2012. A total of 71 days (1.62%) of geomagnetic storms (GS) and 2753 days (63.8%) of quiet (I0) GMA were registered. A second study was provided including the years 1983-2007 (9131 days); here 3800 days (41.62%) were quiet GMA days and 400 storm days (4.38%). According to publications in the medical literature, many phenomena are connected with the extremes of space weather. CONCLUSIONS: Despite a great number of publications and the significant role of GS, it is a relatively rare event and most medical emergencies and deaths occur on days of low GMA, accompanied by higher CRA (neutron activity). High neutron activity deserves more attention when analyzing space effects on human health and their mechanism of action.

A habitable fluvio-lacustrine environment at Yellowknife Bay, Gale crater, Mars.

The Curiosity rover discovered fine-grained sedimentary rocks, which are inferred to represent an ancient lake and preserve evidence of an environment that would have been suited to support a martian biosphere founded on chemolithoautotrophy. This aqueous environment was characterized by neutral pH, low salinity, and variable redox states of both iron and sulfur species. Carbon, hydrogen, oxygen, sulfur, nitrogen, and phosphorus were measured directly as key biogenic elements; by inference, phosphorus is assumed to have been available. The environment probably had a minimum duration of hundreds to tens of thousands of years. These results highlight the biological viability of fluvial-lacustrine environments in the post-Noachian history of Mars.

The Tissint Martian meteorite as evidence for the largest impact excavation.

High-pressure minerals in meteorites provide clues for the impact processes that excavated, launched and delivered these samples to Earth. Most Martian meteorites are suggested to have been excavated from 3 to 7 km diameter impact craters. Here we show that the Tissint meteorite, a 2011 meteorite fall, contains virtually all the high-pressure phases (seven minerals and two mineral glasses) that have been reported in isolated occurrences in other Martian meteorites. Particularly, one ringwoodite (75 × 140 µm(2)) represents the largest grain observed in all Martian samples. Collectively, the ubiquitous high-pressure minerals of unusually large sizes in Tissint indicate that shock metamorphism was widely dispersed in this sample (~25 GPa and ~2,000 °C). Using the size and growth kinetics of the ringwoodite grains, we infer an initial impact crater with ~90 km diameter, with a factor of 2 uncertainty. These energetic conditions imply alteration of any possible low-T minerals in Tissint.

Ultraviolet-radiation-induced methane emissions from meteorites and the Martian atmosphere.

Almost a decade after methane was first reported in the atmosphere of Mars there is an intensive discussion about both the reliability of the observations--particularly the suggested seasonal and latitudinal variations--and the sources of methane on Mars. Given that the lifetime of methane in the Martian atmosphere is limited, a process on or below the planet's surface would need to be continuously producing methane. A biological source would provide support for the potential existence of life on Mars, whereas a chemical origin would imply that there are unexpected geological processes. Methane release from carbonaceous meteorites associated with ablation during atmospheric entry is considered negligible. Here we show that methane is produced in much larger quantities from the Murchison meteorite (a type CM2 carbonaceous chondrite) when exposed to ultraviolet radiation under conditions similar to those expected at the Martian surface. Meteorites containing several per cent of intact organic matter reach the Martian surface at high rates, and our experiments suggest that a significant fraction of the organic matter accessible to ultraviolet radiation is converted to methane. Ultraviolet-radiation-induced methane formation from meteorites could explain a substantial fraction of the most recently estimated atmospheric methane mixing ratios. Stable hydrogen isotope analysis unambiguously confirms that the methane released from Murchison is of extraterrestrial origin. The stable carbon isotope composition, in contrast, is similar to that of terrestrial microbial origin; hence, measurements of this signature in future Mars missions may not enable an unambiguous identification of biogenic methane.