Results for “sterilization” — Page 75

NASA NTRSDate not supplied

Hazards of Lunar Surface Exploration: Determining the Immunogenicity/Allergenicity of Lunar Dust

Although infrequent, there have been Apollo program reports of lunar dust (LD) exposure leading to notable upper respiratory symptoms in select crewmembers. Possible mechanisms include particulate irritation, oxidization and release of noxious gas, or legitimate adaptive immune-mediated response. Although sterile non-protein matter would not be expected to be an allergen, one Apollo flight surgeon reported increasing symptoms upon repeated exposure with associated eosinophilia, indicative of allergy (*Acta Astronautica. 2008 63 (7–10): 980–987). Many ISS crews display a pattern of persistent immune system dysregulation and latent virus reactivation (NPJ Microgravity. 2015 Sep 3; 1:15013; NPJ Microgravity. 2017 Apr 12; 3:11). Some ISS crews manifest atypical respiratory and/or dermatitis symptoms which could have an allergic pathogenesis (J Allergy Clin. Immunol. Pract. 2016 Jul-Aug; 4(4):759-762.e8). It is logical to anticipate crew immune dysregulation would worsen during prolonged deep space missions. Planetary surface hazards will only complicate crew health risks. This study with investigate if LD exposure will elicit an IgE mediated allergic response either to the LD itself or concomitant antigen exposure during spaceflight. Allergic reactivity could adversely increase clinical and operational impacts for long-duration lunar astronauts and affect countermeasure requirements for surface vehicles. Specific aims for this study are to answer two questions: (1) Does in vitro LD exposure result in increased histamine from human peripheral blood basophils? (2) Can LD impact the capacity of CD4+ T helper and/or CD19+ B-cell mediated IgE production? To address these questions, after the proposal and selection by NASA, our laboratory has separately requested and been approved for receipt of actual LD samples from the Apollo 16 mission. These samples will be used during the study to complete the proposed set of in vitro cell culture experiments (short and long term), using human peripheral blood mononuclear cells (PBMC) and basophils from both atopic and non-atopic individuals. Cells will be co-cultured with cellular mitogens, common recall antigens (Der p1), fine ground silica quartz (as a possible allergenic component of LD), or LD, to study whether LD exposure for varying time intervals will alter the generation of selective immune responses associated with clinical allergic reactions. Measured outputs include supernatant-derived IgE, tryptase, histamine, and selected cytokine levels. Cellular activation will be monitored by assessing activation markers via flow cytometry. EM/x-ray analysis will be used to determine cellular interactions with dust particles. A series of validation experiments was initiated in FY22 once the delivery of LD was received. Based on initial experimental findings, we are optimizing the culture conditions, LD concentrations, and refining our other protocol stimuli.

Audrie A. Colorado↗

NASA NTRSDate not supplied

Planetary Protection at Marshall Space Flight Center

Introduction: NASA Marshall Space Flight Center (MSFC) is historically known for its role in propulsion. While this is still the mainstay of MSFC’s expertise, many unique capabilities exist at MSFC which pertain to Planetary Protection (PP), including 1) identifying PP threats, and 2) developing novel methods to neutralize those threats. Furthermore, because these capabilities exist among diverse groups at MSFC, this work promotes collaboration both within and outside MSFC to expand and develop PP studies related to a full spectrum of NASA research, design, manufacture, and test interests. This abstract describes the PP research ongoing at MSFC and describes how it contributes to NASA’s overall PP objectives. Microbial Identification in Cleanrooms: One of the greatest threats to successful implementation of PP requirements is recontamination post bioburden reduction. One method to prevent recontamination is to keep the spacecraft in clean environments (i.e. cleanrooms) as much as possible during assembly and integration. However, cleanrooms are not without their own sources of contamination, which is why NASA is interested in monitoring the cleanliness of cleanrooms and characterizing the microbial species present. Such information allows a greater understanding of the resistance of these microbes to cleaning methods, as well as the risk of their contaminating the targeted planetary body of a given mission. MSFC has multiple cleanrooms of various ISO cleanliness levels onsite. We sampled the air and surfaces of three of these rooms, isolated microbes, and then sequenced the 16S rRNA gene or ITS region of the 18S rRNA gene for bacterial and fungal isolates, respectively. This has resulted in a microbial library which currently includes nearly 100 isolates. Microbial Enumeration of Spacecraft Materials: Currently, there are only a couple bioburden reduction methods approved by NASA, and often the harshness of these methods presents additional concerns or risks related to material properties. The goal of this research is to assess the microbial content of solid rocket motor (SRM) materials potentially used for lander missions. This work aims to more accurately define the risk of planetary contamination by providing empirical data associated with commonly used SRM raw materials. In this study, we pulverized nonmetallic SRM materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many SRM nonmetallic materials do not harbor detectable bioburden, though a range existed depending on the material. The results from this work provide quantitative data to potentially reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes. Space Environmental Effects on Microbial Survival: One potential area of microbial reduction is the space environment. Understanding the survivability of hardy microbes in space-like conditions is a crucial first step in answering how space may reduce bioburden and if it can be relied upon for adherence to PP requirements. This work studied the effects of ultraviolet (UV) and ionizing radiation on survival of Bacillus atrophaeus spores. Microbes were dried on relevant polymeric materials then exposed to space environmental stressors. Coupons were submerged in water, diluted, and plated to determine survival compared with controls. We found that both UV and ionizing radiation were capable of reducing viability by nearly 99%, but there were still survivors, some with changed morphology indicating resistance mechanisms within certain cells. Manufacturing credit: Finally, given the above-mentioned limitations of the NASA-approved bioburden reduction methods, there is interest in understanding if manufacturing processes may provide enough bioburden reduction without additional PP-specific bakeouts. For instance, some material additives may be antimicrobial. Given this, we investigated the effects of several commonly used rubber additives on the growth of B. atrophaeus spores. We found that some of the materials inhibited growth of the spores, possibly supporting the use of these additives on missions with PP constraints. Future work into manufacturing credit for bioburden reduction includes inoculation of green insulation with B. atrophaeus spores, followed by a typical cure. Thermal profiles will be verified for appropriate temperature and durations to meet PP requirements, and cured samples will be analyzed using a cryogenic grinder to determine survivability of spores.

Chelsi D. Cassilly↗

NASA NTRSDate not supplied

Evaluation of Long Term Microbial Regrowth in Slosh Water Tanks From the International Space Station

The NASA Launch Services Program (LSP) maintained the SPHERES-Slosh experiment aboard the International Space Station (ISS) between 2013 and 2019. The purpose of the Slosh experiment was to examine how liquids move inside fuel tanks in a microgravity environment. These tanks were similar to water storage tanks planned for use aboard future space systems, where large dormant periods between crew-use will provide similar conditions for biological growth or chemical leaching. The water within the SLOSH tanks remained undisturbed for over five years after testing concluded, providing a unique sample for stored water under microgravity conditions without prior protocols for microbial control such as sterilization or addition of biocides. The Slosh storage tanks were returned to Kennedy Space Center (KSC) aboard SpaceX CRS-18 mission in November 2019. Upon return of the tanks, the water within each tank was analyzed to determine how the water chemistry and biology changed during its tenure in microgravity. The data obtained and described within this publication provided a basis and reasoning for planning water storage and purification treatment methods aboard ISS, Gateway, and future space habitats. Results demonstrated that low microbial concentrations were present within the water, as expected since no biocide treatment was employed, yet no extensive biofilm formation was observed after 5 years even in the presence of microbial food sources such as the polycarbonate structure and food color additives. This experimentation demonstrates that future biofilm studies should be performed on this type of experimental setup with proper controls aboard ISS to examine microbial regrowth to improve microbial control within space water systems.

Luke B. Roberson↗

NASA NTRSDate not supplied

ISS External Microorganisms: A Planetary Protection Payload Ready-for-Flight

Before NASA or COSPAR is able to set planetary protection requirements for crewed missions to locations like Mars there are a number of critical knowledge gaps that must be addressed (1). One of the most important knowledge gaps is an understanding of microbial leakage from crewed habitats and space suits. Current ECLSS (Environmental Control and Life Support System) and PLSS (Portable Life Support System) requirements do not include any provisions to control microbes that may escape along with vented or leaked gasses. The current generation of NASA space suits can leak at rates as high as 100 cm3/min. during nominal operation (2). ISS (International Space Station) intentionally vents atmospheric gases like CO2to maintain habitable conditions for the crew. Furthermore, every time an airlock is used for EVA (extravehicular activity) there is an accompanying release of internal atmosphere. Since it is not possible to sterilize a crewed mission, it is important that we understand what if any microbes are entrained in these vented and leaked products. It is also important to understand if these microbes can survive on exterior surfaces. Recent sampling of the Russian segments of ISS suggest that bacteria and fungi from inside ISS may be capable of surviving on external surfaces (3). NASA has developed an aseptic sampling tool for use during EVA and plans to collect samples from vents on ISS to build on these results. The results of this work will be used to develop planetary protection requirements for vented and leaked gasses from crewed volumes.

M S Bell↗

NASA NTRSDate not supplied

Hazards of Lunar Surface Exploration: Determining the Immunogenicity/Allergenicity of Lunar Dust

Although infrequent, there have been Apollo program reports of lunar dust (LD) exposure leading to notable upper respiratory symptoms in select crewmembers. Possible mechanisms include particulate irritation, oxidization and release of noxious gas, or legitimate adaptive immune-mediated response. Although sterile non-protein matter would not be expected to be an allergen, one Apollo flight surgeon reported increasing symptoms upon repeated exposure with associated eosinophilia, indicative of allergy (*Acta Astronautica. 2008 63 (7–10): 980–987). Many ISS crews display a pattern of persistent immune system dysregulation and latent virus reactivation (NPJ Microgravity. 2015 Sep 3; 1:15013; NPJ Microgravity. 2017 Apr 12; 3:11). Some ISS crews manifest atypical respiratory and/or dermatitis symptoms which could have an allergic pathogenesis (J Allergy Clin. Immunol. Pract. 2016 Jul-Aug; 4(4):759-762.e8). It is logical to anticipate crew immune dysregulation would worsen during prolonged deep space missions. Planetary surface hazards will only complicate crew health risks. This study with investigate if LD exposure will elicit an IgE mediated allergic response either to the LD itself or concomitant antigen exposure during spaceflight. Allergic reactivity could adversely increase clinical and operational impacts for long-duration lunar astronauts and affect countermeasure requirements for surface vehicles. Specific aims for this study are to answer two questions: (1) Does in vitro LD exposure result in increased histamine from human peripheral blood basophils? (2) Can LD impact the capacity of CD4+ T helper and/or CD19+ B-cell mediated IgE production? To address these questions, after the proposal and selection by NASA, our laboratory has separately requested and been approved for receipt of actual LD samples from the Apollo 16 mission. These samples will be used during the study to complete the proposed set of in vitro cell culture experiments (short and long term), using human peripheral blood mononuclear cells (PBMC) and basophils from both atopic and non-atopic individuals. Cells will be co-cultured with cellular mitogens, common recall antigens (Der p1), fine ground silica quartz (as a possible allergenic component of LD), or LD, to study whether LD exposure for varying time intervals will alter the generation of selective immune responses associated with clinical allergic reactions. Measured outputs include supernatant-derived IgE, tryptase, histamine, and selected cytokine levels. Cellular activation will be monitored by assessing activation markers via flow cytometry. EM/x-ray analysis will be used to determine cellular interactions with dust particles. A series of validation experiments was initiated in FY22 once the delivery of LD was received. Based on initial experimental findings, we are optimizing the culture conditions, LD concentrations, and refining our other protocol stimuli.

immunology↗

NASA NTRSDate not supplied

Recommendations for Developing Space Suit Integrated Food Systems and Delivering Nutrition Before, During, and After Lunar EVA

INTRODUCTION Artemis missions will include a higher tempo and frequency of extravehicular activities (EVAs) than any previous space program. Because of the physical demands expected from the crew, future space suit designs are required to incorporate nutritional support to the astronauts during lunar surface EVAs lasting longer than 4 hours. The purpose of this project was to provide recommendations to aid the development of an in-suit system that can adequately, safely, and acceptably deliver nutrition to a crewmember while confined to a space suit during EVA. METHODS Physiological, logistical, and engineering aspects of potential in-suit nutrition approaches were assessed through literature reviews, assessments of commercial off the shelf (COTS) foods, suit volumetric modeling, and feedback from subject matter experts and crewmembers. Key driving factors in the development of in-suit nutrition requirements included how much and what type of nutrition should be included, what food formulations are appropriate and safe, what are inherent limitations of space suits, what are the potential risks to the crewmember in the suit, and what practices and preferences from astronauts should be considered. Design references were conceptualized and assessed for strengths and limitations as potential in-suit nutrition systems for surface EVA. RESULTS Acute exogenous energy demands vary greatly depending on activity intensity and duration, and partial energy replenishment (i.e., 60–80 kcal∙hr-1 of EVA, or 460–680 kcal for EVAs lasting up to 8 hours) during activities could improve performance, safety, and recovery. COTS foods capable of providing these energy requirements exist; however, no COTS foods have been identified that pass NASA flight standards for microbiological safety and stability. In-suit nutrition delivery design references that were considered included in-suit concepts for a prefilled drink bag, a hydratable drink bag, and a solid food stick. In addition, a helmet feed port concept was considered for use with drink bags external to the suit. Volumetric models of the in-suit drink bag concepts, based on xEMU dimensions, indicate challenges of fitting formulations > 200 ml (equating to approximately 200 kcal). Astronaut feedback on the four concepts indicated that despite some individual preferences for inclusion of solid foods and helmet port designs, the prefilled drink bag concept was the most preferred. A prefilled drink bag can only be used if food safety and stability can be ensured, possibly requiring advancements in food delivery hardware. CONCLUSION The ability to meet the increased need for nutrition during surface EVAs through provision of nutrients in the suited configuration would benefit overall crew health, performance, and morale, and thus increase the likelihood of mission success. It is recommended that in-suit nutrition capabilities provide at least 400–600 kcal within the suit during EVAs lasting > 4 hours and that suit designs include a dedicated volume for food grade nutrition systems. The developed food system should either allow for 1) installation of prefilled (sealed sterile) liquid nutrition in the suit and provide a mechanism to break the seal at the time that consumption is desired or 2) demonstrate that the unsealed food product shelf life allows for safe consumption after at least 12 hours of EVA.

space suit↗

NASA NTRSDate not supplied

Nanopore Sequencing-Based Microbial Air Profiling Method for Crewed Spacecraft

Microbial monitoring of the International Space Station (ISS) atmosphere is vital to maintaining the health of the spacecraft and crew. Key to NASA’s microbial risk assessment is the identity of contaminating microorganisms in the environment. Historically, this has been achieved through impaction-based air sampling followed by culture. Identification of the microorganisms present requires sample return to Earth and lab-based analyses. While this culture-based approach has served to provide alerts to anomalies and overall confidence in the controls in place, it is not suitable for exploration missions with no sample return. Recently, significant advancements in molecular-based microbial monitoring via nanopore sequencing have been implemented onboard the ISS. Building on this work, multiple commercially available air samplers, compatible with downstream molecular analysis, were evaluated for use in the spaceflight environment. Through this assessment, the Coriolis Compact (Bertin Technologies), which uses cyclonic technology to collect bioaerosols onto the surface of a sterile cone, was selected for a larger-scale comparison to the current culture-based monitoring method. Using the Coriolis Compact, 1000 L of air was collected from the breakroom of an office building and a fitness center. The buffer used to dissociate the microbial cells from the surface of the cone was split between the NASA Microbiology Laboratory’s standard culture and Sanger sequencing-based method and the culture-independent nanopore sequencing method. The bacteria identified through culture were present in the nanopore data, with Micrococcus, Staphylococcus, and Moraxella being the most common cultured isolates, which is expected based on the media and growth conditions. Not surprisingly, the nanopore data yielded much higher diversity and paralleled that of previous atmospheric microbiome studies of human-occupied built environments. As compared to the culture-based data where the breakroom and fitness center data sets cluster in proximity, the nanopore data depicts the contrast of these atmospheric microbiomes. Moreover, the nanopore data were sufficient to meet NASA’s risk assessment needs and noted the culturable isolates routinely observed. This nanopore-based atmospheric microbial profiling method will enable near real-time environmental monitoring of crewed spacecraft as future missions extend beyond low-Earth orbit.

Brandon Dunbar↗

NASA NTRS2023

Molecular Identification of Microbial Contaminants

Microorganisms can have significant impacts on the success of NASA’s missions, including the integrity of materials, the reliability of scientific results, and maintenance of crew health. Robust cleaning and sterilization protocols are currently in place in NASA facilities, but agency experts agree that microbial contamination is unavoidable and its impact on NASA’s missions and science must be minimized. Therefore, it is critical to understand: 1) what specific microorganisms are present, 2) how they may impact scientific objectives, and 3) how to select appropriate mitigation strategies. The Marshall Space Flight Center (MSFC) Planetary Protection (PP) microbiology lab historically relied solely upon enumeration of culturable microbial contamination associated with spacecraft materials or cleanrooms. However, this process is time consuming, many microbes cannot be cultured, and very few can be identified with any fidelity using NASA standard microbiological methods. The work described in this white paper includes the establishment of molecular identification capabilities at MSFC, including DNA isolation, amplification, purification, and Sanger sequencing. This capability will not only improve planetary protection efforts at MSFC (i.e. by identifying contaminating microorganisms in cleanrooms or on spacecraft) but also offers a service center-wide for the identification of contaminants that arise in other projects, processing locations, or during set up and roll out of spacecraft. This work also lays the foundation for higher throughput efforts to identify large populations of microbes across the lifetime of a project and serves as the starting point for future work into whole genome sequencing, non-culture based methods, or additional characterization studies. Ultimately, accurate identification informs appropriate mitigation strategies, increasing the chances of success for NASA’s missions and objectives.

C. D. Cassilly↗

NASA NTRSDate not supplied

NASA Capture, Containment, and Return System: Bringing Mars Samples to Earth

The Capture, Containment, and Return System (CCRS) project is NASA’s last step in bringing back Mars samples. CCRS will close a decades-long multi-mission and multi-agency effort to bring Mars surface samples back to Earth for scientific studies. CCRS will launch in 2027 on the European Earth Return Orbiter (ERO) spacecraft, which will provide communications relay for the Mars Sample Return ground missions, Perseverance rover and the Sample Retrieval Lander (SRL) (to be launched in 2028). The main mission for CCRS begins when the first-ever orbital planetary capture operation occurs with CCRS catching and securing the Orbiting Sample (OS)in low Mars orbit. From this point, the system will perform additional "firsts": it will autonomously contain the OS with heat-shrink-fit, sterilize the outside surface, and assemble the Earth entry capsule, named Earth Entry System (EES), in orbit around Mars using a gantry mechanism. At approximately 2.8 Lunar distances from Earth, or 3-days from entry into Earth’s atmosphere, CCRS will open its micrometeoroid shield and release the EES on a ballistic trajectory to Earth. The EES is designed to be a fully passive system that will enter the atmosphere and land without parachute at the Utah Test and Training Range (UTTR).

Mars mission, Sample return, Mission design↗

NASA NTRSDate not supplied

The Containment Assurance Risk Framework of the Mars Sample Return Program

The Mars Sample Return campaign aims at bringing rock and atmospheric samples from Mars to Earth through a series of robotic missions. These missions would collect the samples being cached and deposited on Martian soil by the Perseverance rover, place them in a container, and launch them into Martian orbit for subsequent capture by an orbiter that would bring them back. Given there exists a non-zero probability that the samples contain biological material, precautions are being taken to design systems that would break the chain of contact between Mars and Earth. These include techniques such as sterilization of Martian particles, redundant containment vessels, and a robust reentry capsule capable of accurate landings without a parachute. Requirements exist that the probability of containment not assured of Martian-contaminated material into Earth’s biosphere be less than one in a million. To demonstrate compliance with this strict requirement, a statistical framework was developed to assess the likelihood of containment loss during each sample return phase and make a statement about the total combined mission probability of containment not assured. The work presented here describes this framework, which considers failure modes or fault conditions that can initiate failure sequences ultimately leading to containment not assured. Reliability estimates are generated from databases, design heritage, component specifications, or expert opinion in the form of probability density functions or point estimates and provided as inputs to the mathematical models that simulate the different failure sequences. The probabilistic outputs are then combined following the logic of several fault trees to compute the ultimate probability of containment not assured. Given the multidisciplinary nature of the problem and the different types of mathematical models used, the statistical tools needed for analysis are required to be computationally efficient. While standard Monte Carlo approaches are used for fast models, a multi-fidelity approach to rare event probabilities is proposed for expensive models. In this paradigm, inexpensive low-fidelity models are developed for computational acceleration purposes while the expensive high-fidelity model is kept in the loop to retain accuracy in the results. This work presents an example of end-to-end application of this framework highlighting the computational benefits of a multi-fidelity approach.

Giuseppe Cataldo↗

NASA NTRS2022

Design of a Low-Cost, Submersible, Digital Holographic Microscope for in Situ Microbial Imaging

The methodologies for studying marine microbiology typically consist of utilizing instrumentation within a laboratory. This typically requires extracting a sample from its place of origin prior to examination, which may be days after collection. Oftentimes, the solution is to bring the lab to the ocean, which may be costly and provide further limitations for a sterile and stable laboratory environment. Here we present a low-cost, submersible, digital holographic microscope (DHM) designed to image marine microorganisms (such as bacteria and plankton) in their natural underwater environment. Our instrument eliminates the need to transport samples and allows for instantaneous data collection of microbes in-situ. The DHM achieves sub-micron spatial resolution and is paired with artificial intelligence for the detection and tracking of specimens to reduce the overall collected data. This instrument also aims to reduce the cost of manufacturing and field expenses relative to marine microbiological research. “Off the shelf” components were selected in the design process of this instrument which allows us to achieve precise results without sacrificing data quality. The DHM itself costs under one thousand dollars and features a low-cost high-resolution camera, the Arducam MT9J001. Included in the design were five main subsystems: optical, mechanical, electrical, power, and machine learning. Our on board computer and artificial intelligence consist of a Raspberry Pi 4 (8 Gb) and Google Coral USB Tensorflow accelerator. Instrument testing has successfully proven our abilities of data acquisition for at least two hours in depths of at least forty meters below sea level. Additionally, our artificial intelligence system is currently capable of tracking up to ten areas of interest in a fraction of a second with over ninety percent confidence via the neural net driven by the tensor cores on the Google Coral. Furthermore, we have demonstrated the versatility of our instrument by mounting it on an ocean-going ROV, the BlueRov2 by BlueRobotics. Our tests on the BlueRov2 exemplified the cost-effective nature of a submersible and reusable instrument that can be implemented in moderate environments and on most vessels.

Wallace, James Kent↗

NASA NTRSDate not supplied

Overview of the Capture, Containment, and Return System (CCRS)

The Mars Sample Return (MSR) campaign is one of the most ambitious and complex planetary exploration missions currently underway. With the participation of NASA, ESA, and a large number of industry partners, MSR aims to bring Martian soil, rock, and atmospheric samples back to Earth, in order to answer key questions about Mars’ biological evolution. To accomplish this goal the campaign relies on four coordinated missions, each fulfilling a fundamental role to bring the samples to Earth. The Mars Perseverance rover, the first of the four missions, landed safely on Mars on February 18, 2021 and has already acquired candidate samples for Earth return. A selection of the samples of Martian soil and atmosphere that Perseverance has captured during its mission will be recovered, launched into Mars orbit, and transported back to Earth. The Sample Fetch Rover and Mars Ascent System, both parts of the Sample Return Lander project, perform the Mars surface missions to retrieve the collected samples and launch them into Mars orbit. NASA’s Capture, Containment, and Return System (CCRS), hosted on ESA’s Earth Return Orbiter (ERO), brings the samples back to Earth from Mars orbit. These retrieval and return missions are currently in the planning and design stages of development. The NASA-provided CCRS is the payload of the ESA ERO and is the focus of this presentation. ERO will enter Mars orbit and provide communication relay to Earth for the other MSR elements. The Sample Return Lander systems will fetch the sample tubes and integrate them into a protective vessel – the Orbiting Sample (OS) system – which is then launched into low Mars orbit. ERO will perform rendezvous maneuvers, allowing its CCRS payload to capture the OS, contain it, and perform the first automated in-space assembly of a spacecraft, the Earth Entry System (EES), while in Mars orbit. ERO will then begin its journey back to Earth, with CCRS and its assembled EES spacecraft. Three days prior to arrival, CCRS will release the EES on an Earth entry trajectory from a distance beyond the orbit of the Moon. The passive EES spacecraft will then enter Earth’s atmosphere, flying on a ballistic trajectory, followed by a terminal descent (without a parachute) and landing at the Utah Test and Training Range (UTTR). This presentation will show the current design of the CCRS system and its concept of operations. ERO and CCRS will perform several firsts in planetary exploration: (a) orbital rendezvous and capture in Mars orbit, (b) in-space sterilization and containment, (c) on-orbit spacecraft assembly at Mars, and (d) fully-passive entry, descent, and landing sequence for sample return.

Carlie H. Zumwalt↗

NASA NTRSDate not supplied

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↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

NASA NTRSDate not supplied

Mars Sample Return: Considerations for the Curation of Astromaterials from a Restricted Planet

The joint NASA/ESA Mars Sample Return (MSR) campaign is underway. The Perseverance Rover has already collected a returnable sample suite currently cached at Three Forks and the pairs to these samples are stored on the Rover with more compelling samples planned to be collected. The MSR collection would represent the most geologically diverse astromaterial collection ever returned and should provide information on topics ranging from Martian geological and biological history to Martian environmental hazards and in situ resource utilization to support potential human exploration. Although Jezero Crater and the surrounding area are not Mars special regions, the scientific opinion is that Mars as a whole is of significant interest to the process of chemical evolution and/or the origin of life. Therefore, due to possibility, however remote, that the samples could contain extraterrestrial life, MSR is classified as a Category V: Restricted Earth Return mission by the NASA Planetary Protection Office. As a result of this classification, a MSR Sample Receiving Facility (SRF) must not only provide a pristine environment to ensure samples are protected from terrestrial contamination for scientific investigations, but it must also provide high-containment to isolate the samples from Earth’s biosphere until the samples are deemed safe for release and/or sterilized.

Mars Sample Return↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

NASA NTRS2024

IVGen Mini Techport May 2024

Intravenous (IV) fluids are an important treatment modality for multiple medical conditions that have the potential to occur during Mars missions and that may lead to adverse crew health and mission outcomes without appropriate treatment. However, carrying sufficient volumes of IV fluids with adequate shelf life to support such missions is currently not feasible due to anticipated mass and volume constraints of future vehicles and the relatively short shelf-life of terrestrial IV fluids. The goal of the IntraVenous Fluid Generation for Exploration Missions, Miniaturized, (IVGen Mini) project is to develop a low mass and volume IV fluid generation device that can produce IV fluids in situ and that reduces the need to launch and store large quantities of IV fluids. It builds on the original IVGEN system developed by the Human Research Program’s Exploration Medical Capability Element that successfully demonstrated the capability to produce in-situ IV fluids aboard the ISS in 2010 using an ISS potable water source. Flight demonstration objectives for this system included purification via packed bed resin, bubble removal, sterilization, passive mixing in microgravity, and United States Pharmacopeia (USP) tested compliance. The project will culminate in a flight technical demonstration on the ISS in 2025/2026.

Courtney M Schkurko↗

NASA NTRSDate not supplied

Cleanroom Microbes Survive Drying, Vacuum, and Proton Irradiation

Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.

Chelsi Cassilly↗