Preservation of Bacillus subtilis' cellular liquid state at deep sub-zero temperatures in perchlorate brines.

Although a low temperature limit for life has not been established, it is thought that there exists a physical limit imposed by the onset of intracellular vitrification, typically occurring at ~-20 °C for unicellular organisms. Here, we show, through differential scanning calorimetry, that molar concentrations of magnesium perchlorate can depress the intracellular vitrification point of Bacillus subtilis cells to temperatures much lower than those previously reported. At 2.5 M Mg(ClO4)2, the peak vitrification temperature was lowered to -83 °C. Our results show that inorganic eutectic salts can in principle maintain liquid water in cells at much lower temperatures than those previously claimed as a lower limit to life, raising the prospects of active biochemical processes in low temperature natural settings. Our results may have implications for the habitability of Mars, where perchlorate salts are pervasive and potentially other terrestrial and extraterrestrial, cryosphere environments.

The Concept of Life on Venus Informs the Concept of Habitability.

An enduring question in astrobiology is how we assess extraterrestrial environments as being suitable for life. We suggest that the most reliable assessments of the habitability of extraterrestrial environments are made with respect to the empirically determined limits to known life. We discuss qualitatively distinct categories of habitability: empirical habitability that is constrained by the observed limits to biological activity; habitability sensu stricto, which is defined with reference to the known or unknown limits to the activity of all known organisms; and habitability sensu lato (habitability in the broadest sense), which is circumscribed by the limit of all possible life in the universe, which is the most difficult (and perhaps impossible) to determine. We use the cloud deck of Venus, which is temperate but incompatible with known life, as an example to elaborate and hypothesize on these limits.

The minimum energy required to build a cell.

Understanding the energy requirements for cell synthesis accurately and comprehensively has been a longstanding challenge. We introduce a computational model that estimates the minimum energy necessary to build any cell from its constituent parts. This method combines omics and internal cell compositions from various sources to calculate the Gibbs Free Energy of biosynthesis independently of specific metabolic pathways. Our public tool, Synercell, can be used with other models for minumum species-specific energy estimations in any well-sequenced species. The energy for synthesising the genome, transcriptome, proteome, and lipid bilayer of four cell types: Escherichia coli, Saccharomyces cerevisiae, an average mammalian cell and JCVI-syn3A were estimated. Their modelled minimum synthesis energies at 298 K were 9.54 × 10 - 11 J/cell, 4.99 × 10 - 9 J/cell, 3.71 × 10 - 7 J/cell and 3.69 × 10 - 12 respectively. Gram-for-gram synthesis of lipid bilayers requires the most energy, followed by the proteome, genome, and transcriptome. The average per gram cost of biomass synthesis is in the 300s of J/g for all four cells. Implications for the generalisability of cell construction and applications to biogeosciences, cellular biology, biotechnology, and astrobiology are discussed.

Life on Earth can grow on extraterrestrial organic carbon.

The universe is a vast store of organic abiotic carbon that could potentially drive heterotrophy on habitable planets. Meteorites are one of the transporters of this carbon to planetary surfaces. Meteoritic material was accumulating on early Earth when life emerged and proliferated. Yet it is not known if this organic carbon from space was accessible to life. In this research, an anaerobic microbial community was grown with the CM2 carbonaceous chondrite Aguas Zarcas as the sole carbon, energy and nutrient source. Using a reversed 13C-stable isotope labelling experiment in combination with optical photothermal infrared (O-PTIR) spectroscopy of single cells, this paper demonstrates the direct transfer of carbon from meteorite into microbial biomass. This implies that meteoritic organics could have been used as a carbon source on early Earth and other habitable planets, and supports the potential for a heterotrophic metabolism in early living systems.

Bridging Place-Based Astrobiology Education with Genomics, Including Descriptions of Three Novel Bacterial Species Isolated from Mars Analog Sites of Cultural Relevance.

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T).

Linking Methanogenesis in Low-Temperature Hydrothermal Vent Systems to Planetary Spectra: Methane Biosignatures on an Archean-Earth-like Exoplanet.

In this work, the viability of the detection of methane produced by microbial activity in low-temperature hydrothermal vents on an Archean-Earth-like exoplanet in the habitable zone is explored via a simplified bottom-up approach using a toy model. By simulating methanogens at hydrothermal vent sites in the deep ocean, biological methane production for a range of substrate inflow rates was determined and compared to literature values. These production rates were then used, along with a range of ocean floor vent coverage fractions, to determine likely methane concentrations in the simplified atmosphere. At maximum production rates, a vent coverage of 4-15 × 10-4 % (roughly 2000-6500 times that of modern Earth) is required to achieve 0.25% atmospheric methane. At minimum production rates, 100% vent coverage is not enough to produce 0.25% atmospheric methane. NASA's Planetary Spectrum Generator was then used to assess the detectability of methane features at various atmospheric concentrations. Even with future space-based observatory concepts (such as LUVOIR and HabEx), our results show the importance of both mirror size and distance to the observed planet. Planets with a substantial biomass of methanogens in hydrothermal vents can still lack a detectable, convincingly biological methane signature if they are beyond the scope of the chosen instrument. This work shows the value of coupling microbial ecological modeling with exoplanet science to better understand the constraints on biosignature gas production and its detectability.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

Detecting Microbiology in the Upper Atmosphere: Relative-Velocity Filtered Sampling.

The purpose of this article is to reopen from a practical perspective the question of the extent in altitude of Earth's biosphere. We make a number of different suggestions for how searches for biological material could be conducted in the mesosphere and lower thermosphere, colloquially referred to as the "ignore-osphere" because it has been generally ignored in the meteorological community compared to other regions. Relatively recent technological advances such as CubeSats in very low Earth orbit or more standard approaches such as the rocket-borne MAGIC meteoric smoke particle sampler are shown as potentially viable for sampling biological material in the ignore-osphere. The issue of contamination is discussed, and a potential solution to the problem is proposed by means of a new detector design that filters for particles based on their size and relative velocity to the detector.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Limitations of microbial iron reduction under extreme conditions.

Microbial iron reduction is a widespread and ancient metabolism on Earth, and may plausibly support microbial life on Mars and beyond. Yet, the extreme limits of this metabolism are yet to be defined. To investigate this, we surveyed the recorded limits to microbial iron reduction in a wide range of characterized iron-reducing microorganisms (n = 141), with a focus on pH and temperature. We then calculated Gibbs free energy of common microbially mediated iron reduction reactions across the pH-temperature habitability space to identify thermodynamic limits. Comparing predicted and observed limits, we show that microbial iron reduction is generally reported at extremes of pH or temperature alone, but not when these extremes are combined (with the exception of a small number of acidophilic hyperthermophiles). These patterns leave thermodynamically favourable combinations of pH and temperature apparently unoccupied. The empty spaces could be explained by experimental bias, but they could also be explained by energetic and biochemical limits to iron reduction at combined extremes. Our data allow for a review of our current understanding of the limits to microbial iron reduction at extremes and provide a basis to test more general hypotheses about the extent to which biochemistry establishes the limits to life.

Structural Responses of Nucleic Acids to Mars-Relevant Salts at Deep Subsurface Conditions.

High pressure deep subsurface environments of Mars may harbor high concentrations of dissolved salts, such as perchlorates, yet we know little about how these salts influence the conditions for life, particularly in combination with high hydrostatic pressure. We investigated the effects of high magnesium perchlorate concentrations compared to sodium and magnesium chloride salts and high pressure on the conformational dynamics and stability of double-stranded B-DNA and, as a representative of a non-canonical DNA structure, a DNA-hairpin (HP), whose structure is known to be rather pressure-sensitive. To this end, fluorescence spectroscopies including single-molecule FRET methodology were applied. Our results show that the stability both of the B-DNA as well as the DNA-HP is largely preserved at high pressures and high salt concentrations, including the presence of chaotropic perchlorates. The perchlorate anion has a small destabilizing effect compared to chloride, however. These results show that high pressures at the kbar level and perchlorate anions can modify the stability of nucleic acids, but that they do not represent a barrier to the gross stability of such molecules in conditions associated with the deep subsurface of Mars.

Meteorites as Food Source on Early Earth: Growth, Selection, and Inhibition of a Microbial Community on a Carbonaceous Chondrite.

Meteoritic material accumulated on the surface of the anoxic early Earth during the Late Heavy Bombardment around 4.0 Gya and may have provided Earth's surface with extraterrestrial nutrients and energy sources. This research investigates the growth of an anaerobic microbial community from pond sediment on native and pyrolyzed (heat-treated) carbonaceous chondrite Cold Bokkeveld. The community was grown anaerobically in liquid media. Native Cold Bokkeveld supported the growth of a phylogenetically clustered subset of the original pond community by habitat filtering. The anaerobic community on meteorite was dominated by the Deltaproteobacteria Geobacteraceae and Desulfuromonadaceae. Members of these taxa are known to use elemental sulfur and ferric iron as electron acceptors, and organic compounds as electron donors. Pyrolyzed Cold Bokkeveld, however, was inhibitory to the growth of the microbial community. These results show that carbonaceous chondrites can support and select for a specific anaerobic microbial community, but that pyrolysis, for example by geothermal activity, could inhibit microbial growth and toxify the material. This research shows that extraterrestrial meteoritic material can shape the abundance and composition of anaerobic microbial ecosystems with implications for early Earth. These results also provide a basis to design anaerobic material processing of asteroidal material for future human settlement.

The smallest space miners: principles of space biomining.

As we aim to expand human presence in space, we need to find viable approaches to achieve independence from terrestrial resources. Space biomining of the Moon, Mars and asteroids has been indicated as one of the promising approaches to achieve in-situ resource utilization by the main space agencies. Structural and expensive metals, essential mineral nutrients, water, oxygen and volatiles could be potentially extracted from extraterrestrial regolith and rocks using microbial-based biotechnologies. The use of bioleaching microorganisms could also be applied to space bioremediation, recycling of waste and to reinforce regenerative life support systems. However, the science around space biomining is still young. Relevant differences between terrestrial and extraterrestrial conditions exist, including the rock types and ores available for mining, and a direct application of established terrestrial biomining techniques may not be a possibility. It is, therefore, necessary to invest in terrestrial and space-based research of specific methods for space applications to learn the effects of space conditions on biomining and bioremediation, expand our knowledge on organotrophic and community-based bioleaching mechanisms, as well as on anaerobic biomining, and investigate the use of synthetic biology to overcome limitations posed by the space environments.

Bridging the gap between microbial limits and extremes in space: space microbial biotechnology in the next 15 years.

The establishment of a permanent human settlement in space is one of humanity's ambitions. To achieve this, microorganisms will be used to carry out many functions such as recycling, food and pharmaceutical production, mining and other processes. However, the physical and chemical extremes in all locations beyond Earth exceed known growth limits of microbial life. Making microbes more tolerant of a greater range of extraterrestrial extremes will not produce organisms that can grow in unmodified extraterrestrial environments since in many of them not even liquid water can exist. However, by narrowing the gap, the engineering demands on bioindustrial processes can be reduced and greater robustness can be incorporated into the biological component. I identify and describe these required microbial biotechnological modifications and speculate on long-term possibilities such as microbial biotechnology on Saturn's moon Titan to support a human presence in the outer Solar System and bioprocessing of asteroids. A challenge for space microbial biotechnology in the coming decades is to narrow the microbial gap by systemically identifying the genes required to do this and incorporating them into microbial systems that can be used to carry out bioindustrial processes of interest.

Habitability Is Binary, But It Is Used by Astrobiologists to Encompass Continuous Ecological Questions.

The term "habitability" is pervasive throughout the space sciences and astrobiology literature and is broadly used to describe an environment's ability to support life. Here, we argue that, while it is fundamentally a binary matter whether an organism can persist in an environment or not, these binary assessments lead to continuous ecological measurements that are often collected under the umbrella term "habitability" by astrobiologists. Although the use of habitability in this way has provided a framework for those studying the potential of environments to support life, including comparative analyses between terrestrial and extraterrestrial environments, it can also generate confusion and limit interdisciplinary understanding. Namely, differing ecological metrics used as proxies for habitability can yield differing conclusions depending upon the metrics chosen. Therefore, we suggest that in this continuous sense, the terms habitable and habitability lose meaning unless the specific scientific question and biological metric chosen to address it are defined. As a corollary, the search for universal single metrics to make habitability assessments is not to be encouraged, and as we argue, attempting to do so would oversimply analyses of the ability of environments to support life.

The Scientific Importance of Returning Airfall Dust as a Part of Mars Sample Return (MSR).

Dust transported in the martian atmosphere is of intrinsic scientific interest and has relevance for the planning of human missions in the future. The MSR Campaign, as currently designed, presents an important opportunity to return serendipitous, airfall dust. The tubes containing samples collected by the Perseverance rover would be placed in cache depots on the martian surface perhaps as early as 2023-24 for recovery by a subsequent mission no earlier than 2028-29, and possibly as late as 2030-31. Thus, the sample tube surfaces could passively collect dust for multiple years. This dust is deemed to be exceptionally valuable as it would inform our knowledge and understanding of Mars' global mineralogy, surface processes, surface-atmosphere interactions, and atmospheric circulation. Preliminary calculations suggest that the total mass of such dust on a full set of tubes could be as much as 100 mg and, therefore, sufficient for many types of laboratory analyses. Two planning steps would optimize our ability to take advantage of this opportunity: (1) the dust-covered sample tubes should be loaded into the Orbiting Sample container (OS) with minimal cleaning and (2) the capability to recover this dust early in the workflow within an MSR Sample Receiving Facility (SRF) would need to be established. A further opportunity to advance dust/atmospheric science using MSR, depending upon the design of the MSR Campaign elements, may lie with direct sampling and the return of airborne dust. Summary of Findings FINDING D-1: An accumulation of airfall dust would be an unavoidable consequence of leaving M2020 sample tubes cached and exposed on the surface of Mars. Detailed laboratory analyses of this material would yield new knowledge concerning surface-atmosphere interactions that operate on a global scale, as well as provide input to planning for the future robotic and human exploration of Mars. FINDING D-2: The detailed information that is possible from analysis of airfall dust can only be obtained by investigation in Earth laboratories, and thus this is an important corollary aspect of MSR. The same information cannot be obtained from orbit, from in situ analyses, or from analyses of samples drilled from single locations. FINDING D-3: Given that at least some martian dust would be on the exterior surfaces of any sample tubes returned to Earth, the capability to receive and curate dust in an MSR Sample Receiving Facility (SRF) is essential. SUMMARY STATEMENT: The fact that any sample tubes cached on the martian surface would accumulate some quantity of martian airfall dust presents a low-cost scientifically valuable opportunity. Some of this dust would inadvertently be knocked off as part of tube manipulation operations, but any dust possible should be loaded into the OS along with the sample tubes. This dust should be captured in an SRF and made available for detailed scientific analysis.

Rationale and Proposed Design for a Mars Sample Return (MSR) Science Program.

The Mars Sample Return (MSR) Campaign represents one of the most ambitious scientific endeavors ever undertaken. Analyses of the martian samples would offer unique science benefits that cannot be attained through orbital or landed missions that rely only on remote sensing and in situ measurements, respectively. As currently designed, the MSR Campaign comprises a number of scientific, technical, and programmatic bodies and relationships, captured in a series of existing and anticipated documents. Ensuring that all required scientific activities are properly designed, managed, and executed would require significant planning and coordination. Because there are multiple scientific elements that would need to be executed to achieve MSR Campaign success, it is critical to ensure that the appropriate management, oversight, planning, and resources are made available to accomplish them. This could be achieved via a formal MSR Science Management Plan (SMP). A subset of the MSR Science Planning Group 2 (MSPG2)-termed the SMP Focus Group-was tasked to develop inputs for an MSR Campaign SMP. The scope is intended to cover the interface to the Mars 2020 mission, science elements in the MSR flight program, ground-based science infrastructure, MSR science opportunities, and the MSR sample and science data management. In this report, a comprehensive MSR Science Program is proposed that comprises specific science bodies and/or activities that could be implemented to address the science functionalities throughout the MSR Campaign. The proposed structure was designed by taking into consideration previous management review processes, a set of guiding principles, and key lessons learned from previous robotic exploration and sample return missions. Executive Summary The Mars Sample Return (MSR) Campaign represents one of the most ambitious scientific endeavors ever undertaken. Analyses of the martian samples would offer unique science benefits that cannot be attained through orbital or landed missions that rely only on remote sensing and in situ measurements, respectively. Ensuring that all required scientific activities are properly designed, managed, and executed would require significant planning and coordination. As currently designed, the MSR Campaign comprises a number of scientific, technical, and programmatic bodies and relationships, captured in a series of existing and anticipated documents. Because there are so many scientific elements that would need to be executed to achieve MSR Campaign success, it is critical to ensure that the appropriate management, oversight, planning, and resources are made available to accomplish them. To date, however, no dedicated budget lines within NASA and ESA have been made available for these purposes, and no formal MSR Science Management Plan (SMP) has yet been established. It is thus evident that: A joint ESA/NASA MSR Science Program, along with the necessary funding and resources, will be required to accomplish the end-to-end scientific objectives of MSR. To aid in planning, the MSR Science Program requires an overarching SMP to fully describe how it could be implemented to meet the MSR scientific objectives and maximize the overall science return. A subset of the MSR Science Planning Group 2 (MSPG2)-termed the SMP Focus Group-was tasked to develop inputs for the MSR Campaign SMP. The scope covers the interface to the Mars 2020 mission, science elements in the MSR flight program, ground-based science infrastructure, MSR science opportunities, and the MSR sample and science data management. Some of the required bodies and activities already exist; the remainder require definition. In this report, a comprehensive MSR Science Program is proposed, comprising specific science bodies and/or activities that could be implemented to address the science functionalities throughout the MSR Campaign. The proposed structure was designed by taking into consideration previous management review processes, a set of guiding principles, and key lessons learned from previous robotic exploration and sample return missions. While we acknowledge that the proposal is non-unique, that is, other implementations could meet the overall needs of the MSR Campaign, we have striven to optimize efficiencies and eliminate unnecessary overlap wherever possible to reduce the potential cost and complexity of the MSR Science Program. Many elements of the proposed Science Program are interdependent, as the decision to trigger certain bodies or activities depend on reaching key milestones throughout the MSR Campaign. Although the timing of certain elements may be flexible depending on the anticipated date of samples arriving on Earth, it is crucial that others are implemented as soon as is feasible. As a first step, formalizing the Science Program's management structure as soon as possible would ensure that impending time-sensitive trades are conducted, and the resulting decisions are made with adequate scientific input. Summary of Findings FINDING SMP-1: A joint science management structure and documented agreements among the MSR Partners are required to coordinate the MSR Science Program elements that are not currently defined in existing structures or documents. FINDING SMP-2: A long-term ESA/NASA MSR Science Program, along with the necessary funding and human resources, will be required to accomplish the end-to-end scientific objectives of MSR. FINDING SMP-3: The MSR Science Management Plan should be linked to, but not encompass, other required functionalities within the MSR Campaign. Input will be needed to produce formal plans for (at a minimum) curation, planetary protection, data management, and public engagement. FINDING SMP-4: The guiding principles proposed in the MSR Science Planning Group (MSPG) Framework document (2019c) remain appropriate and relevant and should be utilized in drafting the MSR Science Memorandum of Understanding (MOU) and Science Management Plan. FINDING SMP-5 (a): MSR scientific return would be maximized if participation in the MSR Science Program is not limited to scientists sponsored by existing MSR Partners; rather, opportunities should be provided to scientists from around the world. (b) All programmatic decision-making power (e.g., selection of competitive proposals) would still rest with the Partners. FINDING SMP-6: At the implementation level, the MSR Science Program should, wherever possible, leverage structures, programs, and lessons-learned from previous mission organization to benefit from their experiences to engender familiarity among both decision-makers and the science community. FINDING SMP-7: The MSR Science Program requires the establishment of scientific bodies to meet management, science operations, and public participation needs. These bodies require dedicated funding, addressing scientific functionalities that span the entirety of the MSR Campaign. FINDING SMP-8: Some elements of the MSR Science Program cannot be delayed in the event of an MSR Program schedule delay, as they are linked to key decisions or operations of the Mars 2020 mission.

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.