Recommendation on Orbiting Sample Cleanliness.

The National Aeronautics and Space Administration-European Space Agency (NASA-ESA) Mars Sample Return (MSR) campaign involves the collection of samples on Mars by the Perseverance (Mars 2020) rover and their return to Earth. To accomplish this, the Orbiting Sample container (OS) will be sent to Mars to accommodate the collected samples then launched from Mars and returned to Earth, where the samples will be removed for examination in the Sample Return Facility (SRF). Crucial to this entire sequence will be establishment of the required level of cleanliness inside the OS. In February 2021, the NASA Headquarters' Mars Sample Return Program and Office of Planetary Protection assembled an MSR OS Tiger Team (OSTT) to discuss the appropriate cleanliness level options of the interior of the OS. The team's remit was primarily focused on evaluating the trade-offs between Planetary Protection cleanliness levels 4a and 4b. These cleanliness levels are determined by the Committee on Space Research (COSPAR) planetary protection regulations, where 4a requires <300 bacterial spores/m2 and <3 x 105 bacterial spores on the spacecraft (in this case, the interior of the OS) and 4b mandates the more stringent requirement of <30 bacterial spores on the spacecraft. This report documents the consensus opinion submitted by the OSTT that recommended the interior of the OS be cleaned to a 4a requirement with any feasible added effort toward 4b. This report provides, as well, the rationale for that decision.

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.

Final Report of the Mars Sample Return Science Planning Group 2 (MSPG2).

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 would 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 could, 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.

Time-Sensitive Aspects of Mars Sample Return (MSR) Science.

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 <<1 ppm O2 are likely to stabilize redox-sensitive minerals over timescales of several years. MAJOR FINDING T-19: MSR investigations targeting organic macromolecular or cellular material, mineral-bound volatile compounds, redox sensitive minerals, and/or hydrous carbonate minerals can become compromised at the timescale of weeks (after opening the sample tube), and scientific information may be completely lost within a time timescale of a few months. Because current considerations indicate that completion of SSAP, sample sterilization, and distribution to investigator laboratories cannot be completed in this time, these investigations must be completed within the Sample Receiving Facility as soon as possible.

Preliminary Planning for Mars Sample Return (MSR) Curation Activities in a Sample Receiving Facility (SRF).

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 et al., 2019) to make sure that the instrumentation suggested is sufficient to cover these key science planning questions (Table 1; Section S-6). It was determined that for Pre-Basic Characterization, two instruments are required, a Magnetometer (see Section S-1.1) and an X-ray Computed Tomography scanner (XCT see Section S-1.2). For Basic Characterization, there are four instruments that are considered necessary, which are analytical balance(s) (see Section S-2.1), binocular microscopes (see Section S-2.2), and multispectral imaging and hyperspectral scanning systems (see Section S-2.3). Then in Preliminary Examination, there is a set of instruments that should be available for generating more detailed information for the sample catalog. These are a Variable Pressure-Field Emission Scanning Electron Microscope (VP-FE-SEM see Section S-3.1), Confocal Raman spectrometer (see Section S-3.2), Deep UV Fluorescence (see Section S-3.3), a Fourier Transform Infrared Spectrometer (see Section S-3.4), a Micro X-ray Diffractometer (see Section S-3.5), X-ray Fluorescence Spectrometer (see Section S-3.6), and Petrographic and Stereo Microscope (see Section S-3.7). All instruments are summarized in Table 1. Finally, our Curation Focus Group has outlined several specific findings for sample curation within the SRF to complete the sample catalog prior to sample distribution and made several recommendations for future work (summarized in Section 8.1) to build upon the efforts that generated this report. List of Findings MAJOR FINDING C-1: The initial sample characterization in the Sample Receiving Facility of the MSR samples can be broken down into three stages for simplicity as follows: Pre-Basic Characterization (Pre-BC), Basic Characterization (BC), and Preliminary Examination (PE). While the whole collection would be assessed through Pre-BC and BC, only subsets of samples would be used during the PE phase. FINDING C-2: Immediately after Earth landing, the spacecraft would be recovered and placed in a container designed to control and stabilize its physical conditions. The optimum temperature (Toptimum) of the sample tubes during transport to the Sample Receiving Facility (SRF) should be the same as the operating temperature of the SRF to avoid unnecessary temperature shock. FINDING C-3: The Sample Receiving Facility (SRF) should operate at room temperature (∼15-25°C), and the samples should be held at this temperature through all steps of initial sample characterization, with the option for cold storage of subsamples available in the SRF when needed. MAJOR FINDING C-4: Measurements on all the sample tubes before they are opened are essential to conduct as the samples could be compromised upon opening of the tubes. This step is called Pre-Basic Characterization (Pre-BC). These are measurements that would inform how the tubes are opened, processed, and subsampled during Basic Characterization (BC). MAJOR FINDING C-5: Careful collection and storage of the serendipitous dust on the outside of the sample tubes is a critical step in the curation process in the Sample Receiving Facility. The dust collected is a valuable resource to the scientific community. MAJOR FINDING C-6: Careful collection and storage of the unaltered and unfractionated headspace gas collected from the sample tubes is a critical step in the curation process in the Sample Receiving Facility. The gas collected is a valuable resource to the scientific community. FINDING C-7: To minimize the interaction of Earth atmospheric gases and gases that are in the sealed sample tubes, once the dust is removed from the exterior of the sample tubes, they should be placed into individual sample tube isolation chambers (STIC) as quickly as possible. FINDING C-8: There are compelling reasons to perform penetrative 3D imaging prior to opening the sample tubes. A laboratory-based X-ray Computed Tomography scanner is the best technique to use and the least damaging to organics of the penetrative imaging options considered. MAJOR FINDING C-9: Measurements on all the samples once the sample tubes are opened within the pristine isolators are essential to make initial macroscopic observations such as weighing, photographing, and optical observations. The first step to this stage is removal and collection of the headspace gas, which then starts the clock for time-sensitive measurements. This step is called Basic Characterization (BC). FINDING C-10: To avoid cross contamination between samples, it is recommended that, for processing through the isolators, the samples are organized into groups that have like properties. Given what we know about the geology of Jezero Crater, a reasonable starting assumption is five such groups. MAJOR FINDING C-11: Assuming that sample processing rates are reasonable and the samples are organized into five sets for cross contamination avoidance purposes, at least twelve pristine isolators are required to perform Basic Characterization on the MSR samples. This total would increase by two for each additional distinct processing environment. MAJOR FINDING C-12: More advanced measurements on subsamples, beyond those included in BC, are essential for the allocation of material to the scientific community for investigation, including some measurements that can make irreversible changes to the samples. These types of measurements take place during Preliminary Examination (PE). FINDING C-13: The output of the initial sample characterization, and a key function of the curation activities within the Sample Receiving Facility, is to produce a sample catalog that would provide relevant information on the samples' physical and mineralogical/chemical characteristics (derived from the Pre-Basic Characterization, Basic Characterization, and Preliminary Examination investigations), sample safety assessments, time-sensitive studies, and information derived from mission operations to enable allocation of the most appropriate materials to the scientific community. FINDING C-14: A staffing model for curation activities, including technical support and informatics/ documentation support, should be developed (as part of ongoing Sample Receiving Facility development) to ensure that the Sample Receiving Facility is staffed appropriately to support sample curation activities. FINDING C-15: To reduce the risk of catastrophic loss of samples curated in a single facility up to, and including, decadal timescales, the sample collection should be split-once it is possible to do so-and housed in more than one location for the purpose of maximizing the long-term safety of the collection.

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).

COSPAR Sample Safety Assessment Framework (SSAF).

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Sulfur isotope enrichment during maintenance metabolism in the thermophilic sulfate-reducing bacterium Desulfotomaculum putei.

Values of Delta(34)S (=delta(34)S(HS)-delta(34)S(SO(4)), where delta(34)S(HS) and delta(34)S(SO(4)) indicate the differences in the isotopic compositions of the HS(-) and SO(4)(2-) in the eluent, respectively) for many modern marine sediments are in the range of -55 to -75 per thousand, much greater than the -2 to -46 per thousand epsilon(34)S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or "retentostat." The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10(-16) to 10(-18) mol of SO(4) cell(-1) h(-1) toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the delta(34)S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Delta(34)S of -20.9 per thousand, than the -9.7 per thousand observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.

Microbial communities in subpermafrost saline fracture water at the Lupin Au mine, Nunavut, Canada.

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). 14C, 3H, and noble gas isotope analyses suggest that the Na-Ca-Cl, suboxic, fracture water represents a mixture of geologically ancient brine, approximately25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were approximately10(3) cells mL(-1). Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH4 isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH4 on Mars.

Unanticipated Geochemical and Microbial Community Structure under Seasonal Ice Cover in a Dilute, Dimictic Arctic Lake.

Despite most lakes in the Arctic being perennially or seasonally frozen for at least 40% of the year, little is known about microbial communities and nutrient cycling under ice cover. We assessed the vertical microbial community distribution and geochemical composition in early spring under ice in a seasonally ice-covered lake in southwest Greenland using amplicon-based sequencing that targeted 16S rRNA genes and using a combination of field and laboratory aqueous geochemical methods. Microbial communities changed consistently with changes in geochemistry. Composition of the abundant members responded strongly to redox conditions, shifting downward from a predominantly heterotrophic aerobic community in the suboxic waters to a heterotrophic anaerobic community in the anoxic waters. Operational taxonomic units (OTUs) of Sporichthyaceae, Comamonadaceae, and the SAR11 Clade had higher relative abundances above the oxycline and OTUs within the genus Methylobacter, the phylum Lentisphaerae, and purple sulfur bacteria (PSB) below the oxycline. Notably, a 13-fold increase in sulfide at the oxycline was reflected in an increase and change in community composition of potential sulfur oxidizers. Purple non-sulfur bacteria were present above the oxycline and green sulfur bacteria and PSB coexisted below the oxycline, however, PSB were most abundant. For the first time we show the importance of PSB as potential sulfur oxidizers in an Arctic dimictic lake.

Effects of rapid global warming at the Paleocene-Eocene boundary on neotropical vegetation.

Temperatures in tropical regions are estimated to have increased by 3° to 5°C, compared with Late Paleocene values, during the Paleocene-Eocene Thermal Maximum (PETM, 56.3 million years ago) event. We investigated the tropical forest response to this rapid warming by evaluating the palynological record of three stratigraphic sections in eastern Colombia and western Venezuela. We observed a rapid and distinct increase in plant diversity and origination rates, with a set of new taxa, mostly angiosperms, added to the existing stock of low-diversity Paleocene flora. There is no evidence for enhanced aridity in the northern Neotropics. The tropical rainforest was able to persist under elevated temperatures and high levels of atmospheric carbon dioxide, in contrast to speculations that tropical ecosystems were severely compromised by heat stress.

Environmental genomics reveals a single-species ecosystem deep within Earth.

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

Versatile inlet system for on-line compound-specific deltaD and delta13C gas chromatography-oxidation/reduction-isotope ratio mass spectrometry analysis of gaseous mixtures.

Compound-specific deltaD and delta13C analyses of gas mixtures are useful indicators of geochemical and environmental factors. However, the relative concentrations of individual components in gas mixtures (e.g., H2, CO2, methane, ethane, propane, i-butane, n-butane) may vary over several orders of magnitude. The determination of hydrogen and carbon compound-specific stable isotope ratios requires that the hydrogen and carbon dioxide produced from each separated component has a concentration adjusted to match the dynamic range of the stable isotope mass spectrometer. We present a custom-built gas sampling and injection system (GASIS) linked with a Delta Plus XP mass spectrometer that provides flexibility, ease of operation, and economical use of small gas samples with wide ranges of analyte concentrations. The overall on-line GC-ox/red-IRMS (Gas Chromatography - oxidation/reduction - Isotope Ratio Mass Spectrometry) system consists of (i) a customized GASIS inlet system and (ii) two alternative reactors, namely an oxidative Cu-Ni-Pt reactor at 950 degrees C for production of CO2 and a reductive graphitized Al2O3 reactor at 1420 degrees C for production of H2. In addition, the system is equipped with (iii) a liquid nitrogen spray-cooling unit for cryo-GC-focusing at -20 degrees C, and (iv) a Nafion dryer for removal of water vapor from product CO2. The three injection loops of the GASIS inlet allow flexibility in the volume of injected analyte gas (e.g., from 0.06 to 500 microL) in order to measure reproducible deltaD and delta13C values for gases at concentrations ranging from 100% down to 10 ppm. We calibrate our GC-ox/red-IRMS system with two isotopically distinct methane references gases that are combusted off-line and characterized using dual-inlet IRMS.

Long-term sustainability of a high-energy, low-diversity crustal biome.

Geochemical, microbiological, and molecular analyses of alkaline saline groundwater at 2.8 kilometers depth in Archaean metabasalt revealed a microbial biome dominated by a single phylotype affiliated with thermophilic sulfate reducers belonging to Firmicutes. These sulfate reducers were sustained by geologically produced sulfate and hydrogen at concentrations sufficient to maintain activities for millions of years with no apparent reliance on photosynthetically derived substrates.

Desulfotomaculum and Methanobacterium spp. dominate a 4- to 5-kilometer-deep fault.

Alkaline, sulfidic, 54 to 60 degrees C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO4(2-) in the presence of high pH, high concentrations of H2 and CH4, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO4(2-) reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.

34S isotope effect on sulfate ester hydrolysis: mechanistic implications.

In this communication, we report the first determination of 34S kinetic isotope effects (KIEs) for the hydrolysis of sulfate monoesters. The method involves the conversion of the inorganic sulfate, acquired at partial extent of reaction, to SO2, followed by isotope ratio determination by mass spectrometry. The KIEs determined for p-nitrophenyl sulfate and p-acetylphenyl sulfate are 1.0154 (+/-0.0002) and 1.0172 (+/-0.0003), respectively. These results, together with previous peripheral 18O KIE values, are inconsistent with an associative mechanism. The isotope effect method we report should also prove useful for studying the mechanism of other sulfuryl group transfers, including sulfatase and sulfotransferase reactions, as well as sulfate hydrolyses under other conditions.

Structure--activity relationships of the peptide deformylase inhibitor BB-3497: modification of the P2' and P3' side chains.

Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to both the P2' and P3' side chains. Enzyme inhibition and antibacterial activity data revealed that a variety of substituents are tolerated at the P2' and P3' positions of the inhibitor backbone. The data from this study highlights the potential for modification at the P2' and P3' positions to optimise the physicochemical properties.

Structure-activity relationships of the peptide deformylase inhibitor BB-3497: modification of the metal binding group.

A series of analogues of the potent peptide deformylase (PDF) inhibitor BB-3497 containing alternative metal binding groups was synthesised. Enzyme inhibition and antibacterial activity data for these compounds revealed that the bidentate hydroxamic acid and N-formyl hydroxylamine structural motifs represent the optimum chelating groups on the pseudopeptidic BB-3497 backbone.