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A Lab with a View: Findings from NASA's Aircraft Bioaerosol Collector

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

bioaerosols↗

Planetary Protection Technologies for Planetary Science Instruments, Spacecraft, and Missions: Report of the NASA Planetary Protection Technology Definition Team (PPTDT)

Planetary bodies like Mars, Europa, and Enceladus pose the question, "How to study them without contaminating them and destroying future prospects to detect life, if it is there?" The natural trade-off, of course, is that the cleaner your spacecraft, the more you can explore such a body without risk of contaminating it. As chartered by NASA Headquarters, the Planetary Protection Technology Definition Team (PPTDT) was asked to provide a report covering six different areas related to the engineering and technology challenges of implementing planetary protection requirements on solar system exploration missions, including: Assessment of technical and engineering challenges to applying available microbial-reduction methods, including recontamination prevention, to spacecraft hardware and instruments, to meet current NASA requirements on preventing the forward contamination of potentially habitable worlds by future spacecraft missions (orbiters, atmospheric missions, landers, penetrators, and drills); Identification of spacecraft and instrument materials known to be compatible with existing planetary protection protocols; Planetary protection protocols/processes available or which appear promising, and areas ripe for technological development; The technical and engineering challenges in ensuring that spacecraft hardware and instruments can meet organic cleanliness requirements needed to ensure high confidence in differentiating Earth contamination from extraterrestrial signals to avoid false negative as well as false positive results; Approaches for mitigating the identified challenges that would allow instruments to be flown successfully at the required levels of cleanliness and microbial reduction, beginning with identification of commonly used materials and spacecraft hardware that are compatible (or particularly vulnerable) to planetary protection protocols; Engineering, technology, and scientific research and development that could be funded by NASA to provide future capabilities to field scientific instruments and spacecraft on missions that require either subsystem or system-level microbial reduction and recontamination prevention.

John D. Rummel↗

NASA Science Technology Development Programs for Ocean Worlds Exploration

The exploration of ocean worlds such as Europa and Enceladus supports NASA’s goal to search for life and potentially habitable regions elsewhere in the universe, and further promises to help us understand the origins, evolution, and limits of life on Earth. Over the past several years, NASA’s Planetary Science Division has funded several technology development programs to enable future surface and subsurface missions to ocean worlds. These programs include Instrument Concepts for Europa Exploration (ICEE), Concepts for Ocean Worlds Life Detection Technology (COLDTech), Scientific Exploration Subsurface Access Mechanism for Europa (SESAME), Applied Information Systems Research: Autonomous Robotics Research for Ocean Worlds (AISR:ARROW), and Astrodynamics in Support of Icy Worlds Missions. Tasks selected under these programs include the development of scientific instruments including seismometers, imagers, spectrometers, and organic analyzers, and platform technologies including drills, melt probes, through-ice communications, radiation-hard electronics, and autonomy for surface operations. This paper describes the objectives of each of these programs and provides a summary of the work that has been completed or is underway in each.

technology↗

Innovative Thermal Management & Control to Surmount Challenges of Exploring Ocean Worlds on Europa and Enceladus

Probes to penetrate the thick ice shells of our solar system’s Ocean Worlds have been studied for nearly 20 years, since scientific evidence strongly suggested a subsurface ocean on the Jupiter moon called Europa. There is keen scientific interest in exploring four significant themes on such proposed missions: 1) Geodynamics, 2) Geochemistry, 3) Habitability, and 4) Life Detection. The ice shells of Ocean Worlds are predicted to be up to 40 km thick; they exhibit extreme thermal environments, with ice temperatures from 100 K to 270 K, and extreme pressure environments from vacuum to 53 MPa. Jet Propulsion Laboratory has conducted a broad-look investigation of proposed mission concepts to Europa to identify the significant technology and operational challenges of Europa ice-penetration. The thermal-mechanical system (TMS) of an ice penetration probe (IPP) designed to access the ocean of an icy moon using radioisotope thermoelectric generators for heat and power faces technological hurdles exacerbated by severe thermal and volume constraints. This study identified thermal management and control (TMC) challenges that are strongly linked to: ice penetration start-up, mobility and navigation in the ice, communications while in the ice sheet, and detecting and avoiding in-ice hazards. The major objectives of the TMC system are: 1) Absorb internal thermal energy from the IPP radioisotope power source, 2) Maintain liquid water conditions around the IPP at all times, 3) Manage and control thermal flows from probe nose to tail, and 4) Provide pressure containment for all internal probe components. This work discusses the baseline TMC system architecture and design developed to accomplish these objectives, and survive and transit the extreme ice thicknesses in pursuit of Icy/Ocean Worlds science goals. The proposed TMC system consisting of an internal pumped two-phase fluid loop “thermal bus” for thermal energy capture, variable conductance heat pipe system for passively adaptive thermal energy transport around the probe, and water jetting system for ice cutting is described and discussed. Critical testing performed to date is described.

Lee, Kuan-Lin↗

Venus Flagship Mission Concept: A Decadal Survey Study

More than any other known planet, Venus is essential to our understanding of the evolution and habitability of Earth-size planets throughout the galaxy. We address two critical questions for planetary science: 1) How, if at all, did Venus evolve through a habitable phase? 2) What circumstances affect how volatiles shape habitable worlds? More than any other group, volatile elements have a strong influence on the evolutionary paths of rocky bodies and are critical to understanding solar system evolution. It is clear that Venus experienced a different volatile element history from the Earth and provides the only accessible example of one end-state of habitable Earth-size planets. Venus will allow us to identify the mechanisms that operate together to produce and maintain habitable worlds like our own.The (VFM) concept architecture relies on five collaborative platforms: an Orbiter, Lander, variable-altitude Aerobot and two Small Satellites (SmallSats) delivered via a single launch on a Falcon 9 heavy expendable. The platforms would use multiple instruments to measure the exosphere, atmosphere and surface at multiple scales with high precision and over time. VFM would provide the first measurements of mineralogy and geochemistry of tessera terrain to examine rocks considered to be among the most likely to have formed in a habitable climate regime. Landed, descent, aerial and orbital platforms would work synergistically to measure the chemical composition of the atmosphere including the Aerobot operating for 60 days in the Venus clouds. Loss mechanisms would be constrained by the SmallSats in two key orbits. The baseline payload for VFM includes instruments to make the first measurements of seismicity and remanent magnetism, the first long-lived (60 day) surface platform and the first life detection instrument at Venus to interrogate what could be an inhabited world.The VFM concept directly addresses each of the three Venus Exploration Analysis Group (VEXAG) goals as well as several of the strategic objectives of the 2020 NASA Science Plan, Planetary Science Division, Heliophysics and Astrophysics. The simultaneous, synergistic measurements of the solid body, surface, atmosphere and space environment provided by the VFM would allow us to target the most accessible Earth-size planet in our galaxy, and gain a profound new understanding of the evolution of our solar system and habitable worlds.

Segura, Marcia↗

NASA Science Technology Development Programs for Ocean Worlds Exploration

The exploration of icy satellites such as Europa and Enceladus supports NASA’s goal to search for life and potentially habitable regions elsewhere in the universe, and further promises to help us understand the origins, evolution, and limits of life on Earth. Over the past several years, NASA’s Planetary Science Division has funded several technology development programs to enable future surface and subsurface missions to ocean worlds. These programs include Instrument Concepts for Europa Exploration (ICEE), Concepts for Ocean Worlds Life Detection Technology (COLDTech), Scientific Exploration Subsurface Access Mechanism for Europa (SESAME), Applied Information Systems Research: Autonomous Robotics Research for Ocean Worlds (AISR:ARROW), and Astrodynamics in Support of Icy Worlds Missions. Tasks selected under these programs include the development of scientific instruments including seismometers, imagers, spectrometers, and organic analyzers, and also platform technologies including drills, melt probes, through-ice communications, radiation-hard electronics, and autonomy for surface operations. This paper describes the objectives of each of these programs and provides a summary of the work that has been completed or is underway in each.

science instruments↗

Enumeration and Fluorescence In Situ Hybridization of Microbial Bioburden on Cleanroom Surfaces

Introduction: Microorganisms are everywhere on Earth, even in the cleanest of places. Spacecraft assembly cleanrooms can harbor low levels of living and dead microbial cells (e.g., [1,2]), and cleanroom bioburden can also include organic molecules from industrial sources and in situ biomass. Life detection missions require careful attention to avoid contaminants that can be easily convoluted with analytical targets. We are evaluating epifluorescent microscopy and fluorescence in situ hybridization (FISH) as methods to complement organic contamination detection techniques. Epifluorescent cell counting offers an accurate and cost-effective way to quantify low levels of surface biomass. FISH could allow for the identification of residual organisms, and can be targeted to detect active populations of specific organisms such as bacteria known to resist cleaning procedures. This effort is part of a larger study that is concentrated on characterizing the surface and airborne molecular organic contamination background in Johnson Space Center (JSC) Astromaterials curation laboratories and Goddard Space Flight Center (GSFC) spacecraft assembly rooms, and understanding contaminants in the context of cleaning procedures and residual bioburden. Methods: Samples were collected by swabbing surfaces in ISO 5 and ISO 7 equivalent cleanrooms at JSC. Swabs for FISH were fixed in 4% paraformaldehyde (PFA) for 3 hours and then stored in 1:1 ethanol:PBS, while swabs for cell counting were stored in 4% PFA until analysis to avoid any cell loss during centrifugation that could impact quantification of very low biomass samples. Cell counting was performed with SYBR Gold as in [3], but adapted for very low biomass. FISH was performed as in [4], using DAPI as a counterstain for all DNA-containing cells. Negative controls included wells with no probe applied, to test for natural fluorescence, as well as the nonsense probe NONEUB (reverse complement of EUB338) to evaluate non-specific probe binding. Results and Discussion: Cleanroom surfaces had 102-103 cells cm-2. The extremely low biomass of these samples was challenging for enumeration, and required careful and routine use of “field” and laboratory blanks. FISH was performed with the general archaeal and bacterial probes ARCH915 and EUB338 (EUBMIX, [4]), probe GAMBET ([4]), and PSE227, which targets the genus Pseudomonas [5]). The latter two probes were selected because Pseudomonas spp. and other Gammaproteobacteria have not been isolated from cleanroom surfaces but do appear frequently in rRNA gene libraries from these surfaces. While some active bacteria were identified (Fig. 1c), most cells detectable by DAPI did not have a strong or any fluorescent signal (e.g., Fig. 1d), indicating that the vast majority of cells are dead or inactive. This suggests that cleaning protocols are effective at inactivating microbial contaminants, but that dead or inactive cells can remain on surfaces. Cells were often clumped in a weakly autofluorescent matrix, possibly biofilm material (Fig. 1c,d). We also observed other particulate material that was collected by the swabs, including apparent textile fibers (Fig. 1b). Our results are consistent with other studies that show that the bioburden present in clean rooms includes active, dormant, and dead cells. We will discuss how FISH and epifluorescent cell counting could be applied in planetary protection protocols, including the advantages and disadvantages of FISH and cell counting for routine use, as well as different possible applications for more specialized FISH procedures. References: [1] Moissl-Eichinger et al. (2015) Sci Rep, 5, 9156 [2] Hendrickson et al. (2021) Microbiome, 9, 238 [3] Jones et al. (2017) Appl Environ Microbiol, 83, e00909-17 [4] Jones et al. (2015) Appl Environ Microbiol, 81, 1242-1250. [5] Watt et al. (2006) Environ Microbiol, 8, 871-884

C J Huff↗

Continuous Habitable Zones: Pairing a GCM and Bayesian Framework to Predict Habitable Zone Evolution

In the near-future, new space telescopes like JWST, LUVOIR, and HabEx will begin attempting to explore the properties of atmospheres of potentially habitable planets. This will require a significant amount of time and resources for even a single planet, which makes it essential to prioritize observations by those most-likely to have detectable life. Here we present a statistical method to estimate the probabilities that specific exoplanets have been continuously in the habitable zone of their host stars for more than 2 billion years, the approximate time it took life on Earth to significantly increase the oxygen content of the atmosphere. We introduce the use of statistics of an ensemble of 3D planetary general circulation models to estimate these probabilities, replacing prior 1D model estimates.

habitable planets↗

Pairing a GCM and Bayesian Framework to Predict Habitable Zone Evolution

In the near-future, new space telescopes like JWST, LUVOIR, and HabEx will begin attempting to explore the properties of atmospheres of potentially habitable planets. This will require a significant amount of time and resources for even a single planet, which makes it essential to prioritize observations by those most-likely to have detectable life. Here we present a statistical method to estimate the probabilities that specific exoplanets have been continuously in the habitable zone of their host stars for more than 2 billion years, the approximate time it took life on Earth to significantly increase the oxygen content of the atmosphere. We introduce the use of statistics of an ensemble of 3D planetary general circulation models to estimate these probabilities, replacing prior 1D model estimates.

habitable planets↗

Analog Environments for Venus Aerosol Instrument and Mission Concept Testing

The most common hypothesis discussed for life on Venus is a habitat in the dense, persistent clouds, which are primarily sulfuric acid with a secondary water component. Although Earth lacks a complete Venus analog environment, several partial analogs exist which can inform our understanding of the requirements for cloud habitability as well as serving as potential testbeds for future Venus instruments or missions. Earth’s tropospheric clouds have larger particles, more water, and shorter lifetimes than Venus’s; however, they have a partial overlap in pressure and temperature, and are our only example of an airborne habitat.They can inform how particle residence time and microbial generation time constrain habitable atmospheric regions. On the biological side, this includes available nutrients and energy, environmental stressors such as radiation, osmotic potential and acidity, and time spent in protective but inactive forms (endospores, cysts, akinetes); on the physical side, gravity, air density and viscosity, thermal lofting, gravity waves, scavenging due to precipitation, and other airflow dynamics, as well as effective particle radius and electrical charge. They can also enable sampling strategy tests for airborne life detection, ranging from10 2 to10 8 viable cells/mL (10 -3 to 10 -9 cells/particle). Conversely, Earth's stratospheric sulfate layer lacks an active microbial presence, and is at a colder and less dense altitude but has partially analogous values in terms of H2SO4 and water concentration, particle size, and number density. These aerosols are accessible to aircraft and high-altitude balloons. Even more accessible potential analogs include sea spray, which overlaps with the smaller (haze) particle sizes and densities at Venus and contains trace organic constituents; marine fog, which at higher densities can match Venus’s larger (Mode 2) particle properties and contains a range of trace compounds including ammonia, phosphates, sulfates, and nitrates; and spray from acidic hydrothermal systems, which can cover a wide variety of sizes, densities, and chemical compositions. These analogs could improve the development of Venus cloud sampling instruments or sondes, including field tests of material compatibility, particle capture efficiency, and analysis sensitivity.

Analog↗

NASA Science Technology Development Programs for Ocean Worlds Exploration

The exploration of ocean worlds such as Europa and Enceladus supports NASA’s goal to search for life and potentially habitable regions elsewhere in the universe, and further promises to help us understand the origins, evolution, and limits of life on Earth. Over the past several years, NASA’s Planetary Science Division has funded several technology development programs to enable future surface and subsurface missions to ocean worlds. These programs include Instrument Concepts for Europa Exploration (ICEE), Concepts for Ocean Worlds Life Detection Technology (COLDTech), Scientific Exploration Subsurface Access Mechanism for Europa (SESAME), Applied Information Systems Research: Autonomous Robotics Research for Ocean Worlds (AISR:ARROW), and Astrodynamics in Support of Icy Worlds Missions. Tasks selected under these programs include the development of scientific instruments including seismometers, imagers, spectrometers, and organic analyzers, and platform technologies including drills, melt probes, through-ice communications, radiation-hard electronics, and autonomy for surface operations. This paper describes the objectives of each of these programs and provides a summary of the work that has been completed or is underway in each.

Nayar, Hari D↗

Microbial Bioburden on NASA Cleanroom Surfaces: Cell Counting and Fluorescence in situ Hybridization

Spacecraft assembly cleanrooms can harbor a low number of microbial cells that could contaminate hardware designed for life detection missions (known as “bioburden”). We are evaluating epifluorescence microscopy and fluorescence in situ hybridization (FISH) as rapid, accurate, and cost-effective methods to complement other contamination detection techniques. This study is part of a larger effort to characterize background organic contamination and the efficacy of cleaning procedures in Johnson Space Center (JSC) Astromaterials Curation Laboratories and Goddard Space Flight Center (GSFC) spacecraft assembly rooms. The low level of biomass required careful use of blanks during each stage of sample collection and preparation. FISH was performed using general archaeal and bacterial probes, as well as some group-specific probes. While some active bacteria were identified, a large majority of them were dead or inactive, and thus were difficult to identify by standard FISH. Our results are consistent with other studies that show that the bioburden present in clean rooms includes active, dormant, and dead cells.

C J Huff↗

Analysis of Bioburden Associated with Nonmetallic Spacecraft Materials

Planetary Protection (PP) requirements help to ensure the integrity of life-detection science conducted on solar system bodies, particularly those with liquid water. Such requirements are often met by a combination of cleaning and verification steps to reduce microbial contamination (i.e. bioburden) to an acceptable limit. There are only two cleaning methods approved by the National and Space Administration (NASA), vapor hydrogen peroxide (VHP) and heat microbial reduction (HMR), the latter of which is the most commonly employed. However, HMR can be harsh and produce its own concerns about material integrity, like aging or increasing hardness. When bioburden reduction methods cannot be employed, the project must rely on assumed NASA specification values of bioburden which can cause compliance issues with strict PP bioburden limitations. This work aimed to more accurately define the risk of contamination of a target body by providing empirical data associated with raw materials commonly used in solid rocket motors for lander missions. In this study, we pulverized nonmetallic materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many nonmetallic materials do not harbor large bioburden when compared with the NASA specification values, though a range existed depending on the material, treatment, and handling. The results from this work provide quantitative data to help reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes.

Planetary Protection↗

Autonomous Ocean World Exploration: Advancement of a Virtual Testbed

The search for life (extinct or extant) and potentially habitable bodies in our solar system and beyond is one of the 12 priority science questions outlined in the National Acadamies’ 2022 decadal survey [5]. Extraterrestrial destinations containing liquid water present an opportunity to search for life as we know it, and in recent years an increasing number of such locations have been discovered within our solar system. Several Jovian moons—Europa, Ganymede, and Callisto [10]—and the Saturnian moons Enceladus [8] and Titan [9] are known or suspected to harbor massive subsurface oceans. Of these "ocean worlds", Europa is the focus of at least one planned NASA orbiter mission, Europa Clipper [4], and an early lander mission concept, the Europa Lander [2, 3]. Whereas most robotic missions to the Moon and Mars (e.g. orbiters, rovers, landers) to date have had ground controllers on Earth tightly involved in mission operations, missions to more distant worlds will require a high degree of onboard autonomy due to long communication lags and blackouts, harsh environments (radiation, cold), and more limited battery and hardware life. The past decade has seen great advances in both AI technologies and computing scalability and performance that offer promising solutions for spacecraft autonomy and motivate the software system and research programs described in this paper. The Ocean Worlds Autonomy Testbed for Exploration, Research, and Simulation (OceanWATERS) [1], which has been in development at the NASA Ames Research Center since 2018, is a virtual environment for testing lander autonomy solutions. It is built on the Robot Operating System (ROS), runs on consumer-grade Linux workstations, and was released as open source in 2020. OceanWATERS provides a physical and visual simulation of a prototypical lander in a Europa-like environment (Figure 1). The lander was modeled after requirements and specifications made in JPL’s Europa Lander Study of 2016 [3]. Simulated lander systems include stereo cameras and spotlights mounted on an antenna mast that pans and tilts, a 6 degrees of freedom (DoF) robotic arm with a force-torque sensor and two interchangeable end effectors, and a battery pack power system. The environment consists of multiple terrain models including a highly detailed model sourced from the FROST dataset [11], simulation of surrounding planetary bodies based on an ephemeris model, and lighting from the sun with associated surface illumination, reflectance, and shadows. Operations supported by OceanWATERS include panoramic and directed imaging of the environment and lander workspace, Cartesian and joint-level arm commanding, grinding of the terrain surface (e.g. digging a trench), and scooping of ground material (Figure 2) which can be discarded or collected as science samples in a receptacle that can be emptied (science operations themselves are not simulated). These operations are realized as ROS Actions and are complimented by a wide selection of telemetry that is continually produced by each lander subsystem. The power system model is driven by the open-source Generic Software Architecture for Prognostics (GSAP) [11] that predicts the battery’s remaining useful life and other characteristics. As a testbed for high-level autonomy, OceanWATERS provides an execution framework based on PLEXIL [12], an open-source plan specification language and execution engine developed largely at Ames. NASA's initial development of OceanWATERS, as well the Ocean Worlds Lander Autonomy Testbed (OWLAT) [6], a complimentary physical testbed developed at JPL, was the first step in a plan for realizing candidate onboard autonomy solutions for such planetary landers. In 2020 NASA solicited applications for its Autonomous Robotics Research for Ocean Worlds (ARROW) program, and in 2021 the similar Concepts for Ocean worlds Life Detection Technology (COLDTech) program. Collectively six research teams, based in universities and companies across the United States, were awarded grants to develop and demonstrate autonomy solutions on OceanWATERS and OWLAT. These 1–2-year projects have now finished or are nearing completion, and a wide variety of autonomy challenges in ocean world surface missions were addressed. Prototyped and demonstrated solutions have included autonomous discovery, response and adaptation to system faults and unexpected environmental events, world model synthesis through perception, plan synthesis using learned models, methods to optimize sample target selection and prioritize science data transmission, extension of PLEXIL for stochastic decision-making, and an integration of a model of JPL’s mission-ready COLDArm [7]. Technologies used in these projects include many forms of machine learning, causal reasoning, automated planning, Markov decision processes, formal methods, and other advanced techniques. A more detailed summary of the ARROW and COLDTech projects is given herein. OceanWATERS has had significant enhancements since its open-source release in 2020. Many of its new features were driven or shaped by feedback from the ARROW and COLDTech teams and requirements of their projects. In support of enabling autonomous adaptation to spacecraft faults (a specific capability solicited by both programs), a fault injection and detection framework was developed that supports a wide and growing range of fault types such as locked joints, image loss, and battery failures. The power system model was completed and integrated into the simulator, starting as a single-cell battery model and later upgraded to a multi-cell model with associated faults such as cell disconnection. Arm/terrain interaction was improved by adding a force-torque sensor and associated faults, and an analytic dig force model based on the Balovnev bucket force equations. Environment fidelity was increased by modeling terrain deformation resulting from digging and scooping; visual improvements were made in textures, lighting, and shadows. To facilitate interoperation with OWLAT, a unified command and telemetry interface between the testbeds was developed at the ROS level, along with a PLEXIL interface. The number of lander operations was greatly expanded (e.g. with Cartesian-based arm and antenna movement), and a framework was designed for users to build their own lander actions. A GUI for PLEXIL plan selection was created (Figure 3), and an expansive set of plans were added, such as those that illustrate patterns for fault handling. This paper provides a self-contained high-level description of OceanWATERS, focusing on more detailed coverage of the aforementioned enhancements. It provides a high-level summary of the projects undertaken by participants in the ARROW and COLDTech programs and how these efforts have helped shape OceanWATERS. Finally, potential future work and directions for the testbed are listed, as likely informed by the recent planetary science decadal survey [5].

K Michael Dalal↗

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗

Planetary Protection at Marshall Space Flight Center

Planetary Protection (PP) at NASA aims to 1) protect solar system bodies from contamination by Earth life and 2) protect Earth from possible life forms that may be returned from other solar system bodies. It is particularly relevant for missions focused on life detection. PP specialists at MSFC act as subject matter experts to interpret PP requirements and communicate with other NASA centers and/or vendors to ensure compliance. This includes insight and oversight to help projects consider and plan PP implementation during design phases. It also includes physical implementation during build, assembly, and test operations, (i.e. microbial sampling of environments and spacecraft) to help maintain cleanliness. MSFC has also expanded to include PP research which included standing up a fully functional BSL2 microbiology laboratory.

Planetary Protection↗

Autonomous Ocean World Exploration: Advancement of a Software Testbed

The search for life signs and potentially habitable bodies in our solar system and beyond is one of NASA’s top priorities. The prime destinations for such exploration are bodies containing liquid water, such as Jupiter’s moon Europa and Saturn’s moon Enceladus. Initial missions to these “ocean worlds” will be robotic, and because of long communication lags and blackouts, harsh environments, and limited battery life, a high degree of onboard autonomy will be required. To this end NASA has developed the Ocean Worlds Autonomy Testbed for Exploration, Research, and Simulation (OceanWATERS), a software testbed for surface mission autonomy. Under grants from NASA’s Autonomous Robotics Research for Ocean Worlds (ARROW) and Concepts for Ocean worlds Life Detection Technology (COLDTech) programs, collectively 6 research teams at universities and companies across the United States have developed autonomy solutions using OceanWATERS, tackling a variety of surface mission challenges and employing state-of-the-art AI-based solutions. This paper provides a self-contained high-level description of OceanWATERS while detailing enhancements made since its 2020 release. It provides a high-level summary of the projects undertaken by participants in the ARROW and COLDTech programs and how these efforts have helped shape OceanWATERS. Finally, potential future work and directions for the testbed are listed.

K Michael Dalal↗

HAZMAT. IX. An Analysis of the UV and X-Ray Evolution of Low-mass Stars in the Era of Gaia

Low-mass stars (≤1 M⊙) are some of the best candidates for hosting planets with detectable life because of these stars' long lifetimes and relative ratios of planet to star mass and radius. An important aspect of these stars to consider is the amount of ultraviolet (UV) and X-ray radiation incident on planets in the habitable zones due to the ability of UV and X-ray radiation to alter the chemistry and evolution of planetary atmospheres. In this work, we build on the results of the HAZMAT I and HAZMAT III M-star studies to determine the intrinsic UV and X-ray flux evolution with age for M stars using Gaia parallactic distances. We then compare these results to the intrinsic fluxes of K stars adapted from HAZMAT V. We find that although the intrinsic M-star UV flux is 10–100 times lower than that of K stars, the UV fluxes in their respective habitable zone are similar. However, the habitable zone X-ray flux evolutions are slightly more distinguishable with a factor of 3–15 times larger X-ray flux for late M stars than for K stars. These results suggest that there may not be a K-dwarf advantage compared to M stars in the UV, but one may still exist in the X-ray.

stars↗