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The diversity of planktonic microorganisms in fluids from a group of flowing subterranean boreholes was monitored from the day they were drilled to as long as three and a half months after drilling as they drained into Evander Au mine. Geochemical analyses of the water, characterization of microbial communities by phospholipids fatty acid (PLFA) and DNA sequence analyses, and calculations of free energy flux indicated thatmine-introduced microbial contaminants, dominated by β and γ Proteobacteria, Cenarchaeaceae and Candidatus Nitrososphaera, were flushed from the boreholes and replaced by fracture water derived microbial communities dominated by Firmicutes, Methanosarcinalesand Thermoproteaceaea. The fracture water was a mixture of paleometeoric water and 2.0 Ga old, diagenetically altered, hydrothermal fluid. The C and H isotopic data for C1−4 indicated that the CH4 was primarily abiogenic in origin although ∼35–50% of it might have originated from microbial methanogenesis. Noble gas analyses yielded estimated residence times of some 10 million years for the fracture water, which is estimated to represent a capture cross-section of 0.25–0.50 km2. The 16S rRNA and dsrAB gene sequences indicated that the indigenous bacterial communities were predominantly comprised of sulfate reducers belonging to the genera Desulfotomaculum, Candiditus Desulforudis and Desulfofustis. The sulfur isotopic analyses of sulfate and sulfide yielded fractionation delta-34S values ranging from 16 to 22% consistent with microbial sulfate reduction.Thermodynamic analyses indicate that methanogenic reactions are inhibited by the high partial pressure of abiogenic CH4 and that sulfate-reducing reactions are more favorable, which is consistent with the abundance of 16S rRNA genes belonging to known sulfate reducing bacteria. Supplemental materials are available for this article. Go to the publisher’s online edition of Geomicrobiology Journal to view the free supplemental files.

Samples returned from Mars would be placed under quarantine at a Sample Receiving Facility (SRF) until they are considered safe to release to other laboratories for further study. The process of determining whether samples are safe for release, which may involve detailed analysis and/or sterilization, is expected to take several months. However, the process of breaking the sample tube seal and extracting the headspace gas will perturb local equilibrium conditions between gas and rock and set in motion irreversible processes that proceed as a function of time. Unless these time-sensitive processes are understood, planned for, and/or monitored during the quarantine period, scientific information expected from further analysis may be lost forever. At least four processes underpin the time-sensitivity of Mars returned sample science: (1) degradation of organic material of potential biological origin, (2) modification of sample headspace gas composition, (3) mineral-volatile exchange, and (4) oxidation/reduction of redox-sensitive materials. Available constraints on the timescales associated with these processes supports the conclusion that an SRF must have the capability to characterize attributes such as sample tube headspace gas composition, organic material of potential biological origin, as well as volatiles and their solid-phase hosts. Because most time-sensitive investigations are also sensitive to sterilization, these must be completed inside the SRF and on timescales of several months or less. To that end, we detail recommendations for how sample preparation and analysis could complete these investigations as efficiently as possible within an SRF. Finally, because constraints on characteristic timescales that define time-sensitivity for some processes are uncertain, future work should focus on: (1) quantifying the timescales of volatile exchange for core material physically and mineralogically similar to samples expected to be returned from Mars, and (2) identifying and developing stabilization or temporary storage strategies that mitigate volatile exchange until analysis can be completed. Executive Summary Any samples returned from Mars would be placed under quarantine at a Sample Receiving Facility (SRF) until it can be determined that they are safe to release to other laboratories for further study. The process of determining whether samples are safe for release, which may involve detailed analysis and/or sterilization, is expected to take several months. However, the process of breaking the sample tube seal and extracting the headspace gas would perturb local equilibrium conditions between gas and rock and set in motion irreversible processes that proceed as a function of time. Unless these processes are understood, planned for, and/or monitored during the quarantine period, scientific information expected from further analysis may be lost forever. Specialist members of the Mars Sample Return Planning Group Phase 2 (MSPG-2), referred to here as the Time-Sensitive Focus Group, have identified four processes that underpin the time-sensitivity of Mars returned sample science: (1) degradation of organic material of potential biological origin, (2) modification of sample headspace gas composition, (3) mineral-volatile exchange, and (4) oxidation/reduction of redox-sensitive materials (Figure 2). Consideration of the timescales and the degree to which these processes jeopardize scientific investigations of returned samples supports the conclusion that an SRF must have the capability to characterize: (1) sample tube headspace gas composition, (2) organic material of potential biological origin, (3) volatiles bound to or within minerals, and (4) minerals or other solids that host volatiles (Table 4). Most of the investigations classified as time-sensitive in this report are also sensitive to sterilization by either heat treatment and/or gamma irradiation (Velbel et al., 2022). Therefore, these investigations must be completed inside biocontainment and on timescales that minimize the irrecoverable loss of scientific information (i.e., several months or less; Section 5). To that end, the Time-Sensitive Focus Group has outlined a number of specific recommendations for sample preparation and instrumentation in order to complete these investigations as efficiently as possible within an SRF (Table 5). Constraints on the characteristic timescales that define time-sensitivity for different processes can range from relatively coarse to uncertain (Section 4). Thus, future work should focus on: (1) quantifying the timescales of volatile exchange for variably lithified core material physically and mineralogically similar to samples expected to be returned from Mars, and (2) identifying and developing stabilization strategies or temporary storage strategies that mitigate volatile exchange until analysis can be completed. List of Findings FINDING T-1: Aqueous phases, and oxidants liberated by exposure of the sample to aqueous phases, mediate and accelerate the degradation of critically important but sensitive organic compounds such as DNA. FINDING T-2: Warming samples increases reaction rates and destroys compounds making biological studies much more time-sensitive. MAJOR FINDING T-3: Given the potential for rapid degradation of biomolecules, (especially in the presence of aqueous phases and/or reactive O-containing compounds) Sample Safety Assessment Protocol (SSAP) and parallel biological analysis are time sensitive and must be carried out as soon as possible. FINDING T-4: If molecules or whole cells from either extant or extinct organisms have persisted under present-day martian conditions in the samples, then it follows that preserving sample aliquots under those same conditions (i.e., 6 mbar total pressure in a dominantly CO2 atmosphere and at an average temperature of -80°C) in a small isolation chamber is likely to allow for their continued persistence. FINDING T-5: Volatile compounds (e.g., HCN and formaldehyde) have been lost from Solar System materials stored under standard curation conditions. FINDING T-6: Reactive O-containing species have been identified in situ at the martian surface and so may be present in rock or regolith samples returned from Mars. These species rapidly degrade organic molecules and react more rapidly as temperature and humidity increase. FINDING T-7: Because the sample tubes would not be closed with perfect seals and because, after arrival on Earth, there will be a large pressure gradient across that seal such that the probability of contamination of the tube interiors by terrestrial gases increases with time, the as-received sample tubes are considered a poor choice for long-term gas sample storage. This is an important element of time sensitivity. MAJOR FINDING T-8: To determine how volatiles may have been exchanged with headspace gas during transit to Earth, the composition of martian atmosphere (in a separately sealed reservoir and/or extracted from the witness tubes), sample headspace gas composition, temperature/time history of the samples, and mineral composition (including mineral-bound volatiles) must all be quantified. When the sample tube seal is breached, mineral-bound volatile loss to the curation atmosphere jeopardizes robust determination of volatile exchange history between mineral and headspace. FINDING T-9: Previous experiments with mineral powders show that sulfate minerals are susceptible to H2O loss over timescales of hours to days. In addition to volatile loss, these processes are accompanied by mineralogical transformation. Thus, investigations targeting these minerals should be considered time-sensitive. FINDING T-10: Sulfate minerals may be stabilized by storage under fixed relative-humidity conditions, but only if the identity of the sulfate phase(s) is known a priori. In addition, other methods such as freezing may also stabilize these minerals against volatile loss. FINDING T-11: Hydrous perchlorate salts are likely to undergo phase transitions and volatile exchange with ambient surroundings in hours to days under temperature and relative humidity ranges typical of laboratory environments. However, the exact timescale over which these processes occur is likely a function of grain size, lithification, and/or cementation. FINDING T-12: Nanocrystalline or X-ray amorphous materials are typically stabilized by high proportions of surface adsorbed H2O. Because this surface adsorbed H2O is weakly bound compared to bulk materials, nanocrystalline materials are likely to undergo irreversible ripening reactions in response to volatile loss, which in turn results in decreases in specific surface area and increases in crystallinity. These reactions are expected to occur over the timescale of weeks to months under curation conditions. Therefore, the crystallinity and specific surface area of nanocrystalline materials should be characterized and monitored within a few months of opening the sample tubes. These are considered time-sensitive measurements that must be made as soon as possible. FINDING T-13: Volcanic and impact glasses, as well as opal-CT, are metastable in air and susceptible to alteration and volatile exchange with other solid phases and ambient headspace. However, available constraints indicate that these reactions are expected to proceed slowly under typical laboratory conditions (i.e., several years) and so analyses targeting these materials are not considered time sensitive. FINDING T-14: Surface adsorbed and interlayer-bound H2O in clay minerals is susceptible to exchange with ambient surroundings at timescales of hours to days, although the timescale may be modified depending on the degree of lithification or cementation. Even though structural properties of clay minerals remain unaffected during this process (with the exception of the interlayer spacing), investigations targeting H2O or other volatiles bound on or within clay minerals should be considered time sensitive upon opening the sample tube. FINDING T-15: Hydrated Mg-carbonates are susceptible to volatile loss and recrystallization and transformation over timespans of months or longer, though this timescale may be modified by the degree of lithification and cementation. Investigations targeting hydrated carbonate minerals (either the volatiles they host or their bulk mineralogical properties) should be considered time sensitive upon opening the sample tube. MAJOR FINDING T-16: Current understanding of mineral-volatile exchange rates and processes is largely derived from monomineralic experiments and systems with high surface area; lithified sedimentary rocks (accounting for some, but not all, of the samples in the cache) will behave differently in this regard and are likely to be associated with longer time constants controlled in part by grain boundary diffusion. Although insufficient information is available to quantify this at the present time, the timescale of mineral-volatile exchange in lithified samples is likely to overlap with the sample processing and curation workflow (i.e., 1-10 months; Table 4). This underscores the need to prioritize measurements targeting mineral-hosted volatiles within biocontainment. FINDING T-17: The liberation of reactive O-species through sample treatment or processing involving H2O (e.g., rinsing, solvent extraction, particle size separation in aqueous solution, or other chemical extraction or preparation protocols) is likely to result in oxidation of some component of redox-sensitive materials in a matter of hours. The presence of reactive O-species should be examined before sample processing steps that seek to preserve or target redox-sensitive minerals. Electron paramagnetic resonance spectroscopy (EPR) is one example of an effective analytical method capable of detecting and characterizing the presence of reactive O-species. FINDING T-18: Environments that maintain anoxia under inert gas containing <

International audience; The Mars Sample Return (MSR) Campaign must meet a series of scientific and technical achievements to be successful. While the respective engineering responsibilities to retrieve the samples have been formalized through a Memorandum of Understanding between ESA and NASA, the roles and responsibilities of the scientific elements have yet to be fully defined. In April 2020, ESA and NASA jointly chartered the MSR Science Planning Group 2 (MSPG2) to build upon previous planning efforts in defining 1) an end-to-end MSR Science Program and 2) needed functionalities and design requirements for an MSR Sample Receiving Facility (SRF). The challenges for the first samples brought from another planet include not only maintaining and providing samples in pristine condition for study, but also maintaining biological containment until the samples meet sample safety criteria for distribution outside of biocontainment. The MSPG2 produced six reports outlining 66 findings. Abbreviated versions of the five additional high-level MSPG2 summary findings are: Summary-1. A long-term NASA/ESA MSR Science Program, along with the necessary funding and human resources, will be required to accomplish the end-to-end scientific objectives of MSR. Summary-2. MSR curation will need to be done concurrently with Biosafety Level-4 containment. This would lead to complex first-of-a-kind curation implementations and require further technology development. Summary-3. Most aspects of MSR sample science can, and should, be performed on samples deemed safe in laboratories outside of the SRF. However, other aspects of MSR sample science are both time-sensitive and sterilization-sensitive and would need to be carried out in the SRF. Summary-4. To meet the unique science, curation, and planetary protection needs of MSR, substantial analytical and sample management capabilities would be required in an SRF. Summary-5. Because of the long lead-time for SRF design, construction, and certification, it is important that preparations begin immediately, even if there is delay in the return of samples.

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

The Mars Sample Return Planning Group 2 (MSPG2) was tasked with identifying the steps that encompass all the curation activities that would happen within the MSR Sample Receiving Facility (SRF) and any anticipated curation-related requirements. An area of specific interest is the necessary analytical instrumentation. The SRF would be a Biosafety Level-4 facility where the returned MSR flight hardware would be opened, the sample tubes accessed, and the martian sample material extracted from the tubes. Characterization of the essential attributes of each sample would be required to provide enough information to prepare a sample catalog used in guiding the preparation of sample-related proposals by the world's research community and informing decisions by the sample allocation committee. The sample catalog would be populated with data and information generated during all phases of activity, including data derived concurrent with Mars 2020 sample-collecting rover activity, sample transport to Earth, and initial sample characterization within the SRF. We conclude that initial sample characterization can best be planned as a set of three sequential phases, which we have called Pre-Basic Characterization (Pre-BC), Basic Characterization (BC), and Preliminary Examination (PE), each of which requires a certain amount of instrumentation. Data on specific samples and subsamples obtained during sample safety assessments and time-sensitive scientific investigations would also be added to the catalog. There are several areas where future work would be beneficial to prepare for the receipt of samples, which would include the design of a sample tube isolation chamber and a strategy for opening the sample tubes and removing dust from the tube exteriors. Executive Summary All material collected from Mars (gases, dust, rock, regolith) would need to be carefully handled, stored, and analyzed following Earth return to minimize the alteration or contamination that could occur on Earth and maximize the scientific information that can be attained from the samples now and into the future. A Sample Receiving Facility (SRF) is where the Earth Entry System (EES) would be opened and the sample tubes opened and processed after they land on Earth. Samples should be accessible for research in biocontainment for time-sensitive studies and eventually, when deemed safe for release after sterilization or biohazard assessment, should be transferred out of biocontainment for allocation to scientific investigators in outside laboratories. There are two main mechanisms for allocation of samples outside the SRF: 1) Wait until the implementation of the Sample Safety Assessment Protocol (Planetary Protection) results concludes that the samples are non-hazardous, 2) Render splits of the samples non-hazardous by means of sterilization. To make these samples accessible, a series of observations and analytical measurements need to be completed to produce a sample catalog for the scientific community. Specialist members of the Mars Sample Return Planning Group Phase 2 (MSPG2), referred to here as the Curation Focus Group, have identified four curation goals that encompass all of the activities within the SRF: 1.Documentation of the state of the sample tubes and their contents prior to opening, 2.Inventory and tracking of the mass of each sample, 3.Preliminary assessment of lithology and any macroscopic forms of heterogeneity (on all the samples, non-invasive, in pristine isolators), 4.Sufficient characterization of the essential attributes of each sample to prepare a sample catalog and respond to requests by the sample allocation committee (partial samples, invasive, outside of pristine isolators). The sample catalog will provide data for the scientific community to make informed requests for samples for scientific investigations and for the approval of allocations of appropriate samples to satisfy these requests. The sample catalog would be populated with data and information generated during all phases of activity, including data derived from the landed Mars 2020 mission, during sample collection and transport to Earth, and reception within the Sample Receiving Facility. Data on specific samples and subsamples would also be generated during curation activities carried out within the Sample Receiving Facility and during sample safety assessments, time-sensitive studies, and a series of initial sample characterization steps we refer to as Pre-Basic Characterization (Pre-BC), Basic Characterization (BC), and Preliminary Examination (PE) phases. A significant portion of the Curation Focus Group's efforts was to determine which instrumentation would be required to produce a sample catalog for the scientific community and how and when certain instrumentation should be used. The goal is to provide enough information for the PIs to request material for their studies but to avoid facilitating studies that target scientific research that is better left to peer-reviewed competitive processes. We reviewed the proposed scientific objectives of the International MSR Objectives and Samples Team (iMOST) (Beaty