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At least 55 records · Page 3

Microorganisms in the Stratosphere (MIST): In-flight Sterilization with UVC Leds

The stratosphere (10 km to 50 km above sea level) is a unique place on Earth for astrobiological studies of microbes in extreme environments due to the combination of harsh conditions (high ultraviolet radiation, low pressure, desiccation, and low temperatures). Microorganisms in the Stratosphere (MIST) will attempt to characterize the diversity of microbes at these altitudes using a balloon collection device on a meteorological weather balloon. A major challenge of such an aerobiology study is the potential for ground contamination that makes it difficult to distinguish between collected microbes and contaminants. One solution is to use germicidal ultraviolet light emitting diodes (UV LEDs) to sterilize the collection strip. To use this solution, an optimal spatial arrangement of the lights had to be determined to ensure the greatest chance of complete sterilization within the 30 to 60 minute time of balloon ascent. A novel, 3D-printed test stand was developed to experimentally determine viable Bacillus pumilus SAFR-032 spore reduction after exposure to ultraviolet radiation at various times, angles, and distances. Taken together, the experimental simulations suggested that the UV LEDs on the MIST flight hardware should be active for at least 15 minutes and mounted within 4 cm of the illuminated surface at any angle to achieve optimal sterilization. These findings will aid in the production of the balloon collection device to ensure pristine stratospheric microbial samples are collected. Flight hardware capable of in-flight self-sterilization will enable future life detection missions to minimize both forward contamination and false positives.

Exobiology↗

Spacecraft Sterilization Using Non-Equilibrium Atmospheric Pressure Plasma

As a solution to chemically and thermally destructive sterilization methods currently used for spacecraft, non-equilibrium atmospheric pressure plasmas are used to treat surfaces inoculated with Bacillus subtilis and Deinococcus radiodurans. Evidence of significant morphological changes and reduction in viability due to plasma exposure will be presented, including a 4-log reduction of B. subtilis after 2 minutes of dielectric barrier discharge treatment.

disinfection↗

Inductively-Coupled RF Powered O2 Plasma as a Sterilization Source

Low-temperature or cold plasmas have been shown to be effective for the sterilization of sensitive medical devices and electronic equipment. Low-temperature plasma sterilization procedures possess certain advantages over other protocols such as ethylene oxide, gamma radiation, and heat due to the use of inexpensive reagents, the insignificant environmental impacts and the low energy requirements. In addition, plasmas may also be more efficacious in the removal of robust microorganisms due to their higher chemical reactivity. Together, these attributes render cold plasma sterilization as ideal for the surface decontamination requirements for NASA Planetary Protection. Hence, the work described in this study involves the construction, characterization, and application of an inductively-coupled, RF powered oxygen (O2) plasma.

Sharma, S. P.↗

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗

Establishing and Monitoring an Aseptic Workspace

When are aseptic operations necessary? In order to meet certain bioburden requirements, some components must undergo dry heat microbial reduction (DHMR) or other sterilizing procedures. If sensitive surfaces must be re-exposed after DHMR, this could compromise the bioburden levels. Recontaminating sterilized surfaces could be costly both in time by requiring repeated DHMR and risk to the hardware, which may not be compatible with repeated high temperature bakes. In order to prevent recontamination of the sensitive surfaces, an aseptic environment and sterile technique must be employed. Aseptic environments mean working in a space with almost no detectable bioburden in the air or on surfaces. Ideally, DHMR happens as late as possible to avoid requiring aseptic operations, as it can be considered a high-risk operations. Preparing the cleanroom for aseptic operations Establishing an ISO (International Organization for Standardization) class 5 space to minimize airborne particles. Maintain low bioburden in the cleanroom by using biocidal cleaners. Using multiple biocidal techniques decreases the likelihood of selecting for resistant microorganisms. 70% Isopropyl Alcohol (IPA) denatures the proteins in a microorganism (note: 70% IPA is better at killing microorganisms than 100% IPA) 7% hydrogen peroxide: damages DNA and proteins through oxygen radical damage. Ultraviolet-C (UV-C) lamps: causes crosslinking in DNA which prevents replication. Monitor cleanroom regularly for bioburden trending: Standard bioassay: Swab or wipe samples of cleanroom surfaces processed for colony forming unity (viable or spore selected); Rapid bioassay: Adenosine triphosphate (ATP) or Limulus amebocyte lysate (LAL) for a bioburden snapshot. High levels can signal an immediate re-cleaning before standard bioassay samples are taken. Airborne monitoring: Active (pulling air through a filter) or passive (particle fallout) for bioburden. Verify bioburden levels just before aseptic operation. Test hardware and cleanroom surfaces and air 3 days before the planned aseptic operation. Rapid bioburden just before aseptic operation to ensure room was not re-contaminated. Preparing personnel and tools: Personnel training. Everyone in the cleanroom: Standard cleanroom certification Everyone on the team: 1 day Planetary Protection overview. Aseptic operators only: Half-day aseptic operations training. Covers sterile garmenting/gloves, Sterile handling with a focus on contact transfer risk, tool/GSE preparation, and two-operator system for opening sterilized tools/components. Tool sterilization: All tools to be used during an aseptic operation need to be identified. Compatible tools are sterilized by DHMR or Autoclave. Double wrapped so that the exterior bag can be handled by a non-sterile operator, and the sterile. Tools that are not compatible with high heat do not come in contact with sensitive surfaces: either substitutes are found, or tools are isolated by wrapping in sterile foil. During an aseptic operation. Pre-task to make sure everyone understands the operations, who is handling what, and when the most critical surfaces will be exposed. Monitoring during the operation. Bioburden: active and passive airborne bioburden sampling, glove-tip dabs onto a plate after completion of operation (3 days for results). Particles: real time particle counter constantly running, with alarm for exceeding ISO 5 conditions.

aseptic processing↗

Biological Contamination of Mars: Issues and Recommendations

The ad hoc Task Group on Planetary Protection formed by the Space Studies Board (SSB) of the National Research Council focused on making recommendations concerning the protection of Mars from forward contamination (i.e., Earth to Mars) during upcoming missions by both the United States and the former Soviet Union. In so doing, it distinguished between missions whose goals include reconnaissance and measurement and those that specifically include experiments to detect life. The task group also discussed what additional knowledge will be needed in order to assure that future recommendations regarding contamination of Earth from Mars might be made with a higher degree of certainty than is now possible. Following a short introduction to the rationale underlying planetary exploration (Chapter 1) is a brief summary of approved and contemplated missions to Mars (Chapter 2). Chapter 3 briefly reviews the state of knowledge in several areas pertinent to the problem of planetary protection, in the limits of life on Earth and the abilities of known terrestrial organisms to withstand extreme environment conditions, as well as new approaches to detecting life forms. Chapter 5 includes a review and comments (made in light of current knowledge)- on the recommendations made in 'Recommendations on Quarantine Policy for Mars, Jupiter, Saturn, Uranus, Neptune, and Titan'. Updates to the recommendations made in 1978 are also given in Chapter 5. Chapter 6 gives additional recommendations concerning collection of essential data, spacecraft sterilization and bioburden assessment, and future research, as well as legal and social issues and NASA's overall planetary protection program.

Source record↗

Aseptic Handling of the MOMA Mass Spectrometer After Dry Heat Microbial Reduction

Mars Organic Molecule Analyzer Mass Spectrometer (MOMA-MS) is an instrument in the larger MOMA instrument suite for the European Space Agency (ESA) ExoMars 2020 Rover. As a life-detection instrument on a Mars landing mission, MOMA-MS has very stringent Planetary Protection (PP) bioburden requirements. Within the MOMA instrument suite, the hardware surfaces of the sample path must be cleaned to a level of 0.03 spore/sq m. To meet this requirement, a process called Dry Heat Microbial Reduction (DHMR) is used to decrease the number of viable spores by 4 orders of magnitude. Before DHMR, the hardware is handled using standard cleanroom practices, while after DHMR, all sample path surfaces must be handled aseptically when exposed. Aseptic handling of the sample path involves a number of strategies and protocols including working only in an aseptic ISO class 5 work space, limiting the amount of time of exposure, using sterile garmenting with sterile gloves, and using sterile tools. Before work begins, the aseptic workspace will be tested for bioburden and particle fallout, and all tools that will contact sample path surfaces must be sterilized. During the exposure activity, sterile garments will be worn, sterile tools will be handled in a 2 person set up so that the operator touches only the sterile tool and not the exterior surfaces of the sterile pouch, and the environment will be monitored with active and passive fallout for bioburden and particle levels. Any breach in the planetary protection cleanliness can necessitate repeating DHMR, which not only has significant cost and schedule implications, it also become a risk to hardware that is not rated for repeated long exposures to high temperatures.

Life Detection↗

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↗

How to create an artificial magnetosphere for Mars

If humanity is ever to consider substantial, long-term colonization of Mars, the resources needed are going to be extensive. For a long-term human presence on Mars to be established, serious thought would need to be given to terraforming the planet. One major requirement for such terraforming is having the protection of a planetary magnetic field - which Mars currently does not have. Here, the Earth's magnetosphere helps protect the planet from the potential sterilizing effects of cosmic rays and also helps retain the atmosphere, which would otherwise by stripped by large solar storms as they pass over the planet. Mars does have small patches of remnant surface magnetic field, but these are localized in the southern hemisphere and are not of sufficient size or magnitude to protect the planet or a colony.

79 ASTRONOMY AND ASTROPHYSICS↗

Aseptic Operations for Post DHMR Processing of MOMA Mass Spectrometer

Mars Organic Molecule Analyzer - Mass Spectrometer (MOMA-MS) is an instrument in the MOMA instrument suite for the European Space Agency (ESA) ExoMars 2020 Rover. The rover is Planetary Protection Mission Category IVb, the first IVb mission since the Viking missions in the 1970s. Within the sample path of the MOMA instrument suite, the hardware surfaces of the must be sanitized to a level of less than 0.03 spore/m sq. To meet this requirement, the MS sample path was subjected to Dry Heat Microbial Reduction (DHMR) to decrease the number of viable spores by 4 orders of magnitude from a measured 88 spores/m sq to 0.009 spores/m sq. Before DHMR, the hardware is handled using standard cleanroom practices. After DHMR, planetary protection filters protect the sample path for most of integration, but when sample path exposure is required, aseptic operations are instituted and exposure times are kept to an absolute minimum. The surface area of exposure is also taken into account to determine safe exposure times. Before work begins, the ISO class 5 aseptic workspace is cleaned and tested for surface and airborne bioburden, and all tools that will contact or be used near sample path surfaces are sterilized. During the exposure activity, sterile garments are worn, sterile gloves are changed as often as necessary, and the environment is monitored with active and passive fallout for bioburden and real time airborne particle counts. Sterile tools are handled by a two person team so that the operator touches only the tool and not the exterior surfaces of the sterilization pouch, and a sterile operating field is established as a safe place to organize tools or parts during the aseptic operations. In cases where aseptic operations are not feasible, localized DHMR is used after exposure. Any breach in the planetary protection cleanliness can necessitate repeating instrument level DHMR, which not only has significant cost and schedule implications, it also become a risk to hardware that is not rated for repeated long exposures to high temperatures.

Spacecraft Design↗

Treatment of Solid Rocket Motors that Complies with Established Protocols to Ensure Planetary Protection

This presentation discusses recent work being conducted by the National Aeronautics and Space Administration (NASA) at Marshall Space Flight Center (MSFC) to evaluate various methods that could be employed to provide for planetary protection of those solar system bodies that are candidates for extraterrestrial life, thus preventing contamination of such bodies. MSFC is presently involved in the development phase of the Europa Lander De-Orbital Stage (DOS) braking motor. In order to prevent bio-contamination of this Jovian satellite, three paths are currently being considered. The first is (1) Bio-Reduction of those microscopic organisms in or on the vehicle (in this case a solid rocket motor (SRM)) that might otherwise be transported during the mission. Possible methods being investigated include heat sterilization, application or incorporation of biocide materials, and irradiation. While each method can be made to work, effects on the SRM's components (propellant, liner, insulation, etc.) could well prove deleterious. A second path would be use of (2) Bio-Barrier material(s). So long as such barrier(s) can maintain their integrity, planetary protection should be afforded. Under the harsh conditions encountered during extended spaceflight (vacuum, temperature extremes, radiation), however, such barrier(s) could well experience a breach. Finally, a third path would be to perform (3) Pyrotechnic Sterilization of the SRM during its end-of-mission phase. Multiple pyrotechnic units would be triggered to ensure activation of such an event and provide for a final sterilization before vehicle impact. In light of Europa's stringent bio-reduction targets, the final and best choice to minimize risk will probably be some combination of the above.

Stefanski, Philip L.↗

Mechanical Abrasion as a Low Cost Technique for Contamination-Free Sample Acquisition from a Category IVA Clean Platform

The proposed Mars Sample Transfer Chain Architecture provides Planetary Protection Officers with clean samples that are required for the eventual release from confinement of the returned Martian samples. At the same time, absolute cleanliness and sterility requirement is not placed of any part of the Lander (including the deep drill), Mars Assent Vehicle (MAV), any part of the Orbiting Sample container (OS), Rover mobility platform, any part of the Minicorer, Robotic arm (including instrument sensors), and most of the caching equipment on the Rover. The removal of the strict requirements in excess of the Category IVa cleanliness (Pathfinder clean) is expected to lead to significant cost savings. The proposed architecture assumes that crosscontamination renders all surfaces in the vicinity of the rover(s) and the lander(s) contaminated. Thus, no accessible surface of Martian rocks and soil is Earth contamination free. As a result of the latter, only subsurface samples (either rock or soil) can be and will be collected for eventual return to Earth. Uncontaminated samples can be collected from a Category IVa clean platform. Both subsurface soil and rock samples can be maintained clean if they are collected by devices that are self-contained and clean and sterile inside only. The top layer of the sample is removed in a manner that does not contaminate the collection tools. Biobarrier (e.g., aluminum foil) covering the moving parts of these devices may be used as the only self removing bio-blanket that is required. The samples never leave the collection tools. The lids are placed on these tools inside the collection device. These single use tools with the lid and the sample inside are brought to Earth in the OS. The lids have to be designed impenetrable to the Earth organisms. The latter is a well established art.

Dolgin, B.↗

Examination of B. subtilis var. niger Spore Killing by Dry Heat Methods

Dry heat microbial reduction is the only NASA approved sterilization method to reduce the microbial bioburden on space-flight hardware prior to launch. Reduction of the microbial bioburden on spacecraft is necessary to meet planetary protection requirements specific for the mission. Microbial bioburden reduction also occurs if a spacecraft enters a planetary atmosphere (e.g., Mars) and is heated due to frictional forces. Temperatures reached during atmospheric entry events (>200 C) are sufficient to damage or destroy flight hardware and also kill microbial spores that reside on the in-bound spacecraft. The goal of this research is to determine the survival rates of bacterial spores when they are subjected to conditions similar to those the spacecraft would encounter (i.e., temperature, pressure, etc.). B. subtilis var. niger spore coupons were exposed to a range of temperatures from 125 C to 200 C in a vacuum oven (at <1 Torr). After the exposures, the spores were removed by sonication, dilutions were made, and the spores were plated using the pour plate method with tryptic soy agar. After 3 days incubation at 32 C, the number of colony-forming units was counted. Lethality rate constants and D-values were calculated at each temperature. The calculated D-values were: 27 minutes (at 125 C), 13 minutes (at 135 C), and <0.1 minutes (at 150 C). The 125 C and 135 C survivor curves appeared as concavedownward curves. The 150 C survivor curve appeared as a straight-line. Due to the prolonged ramp-up time to the exposure conditions, spore killing during the ramp-up resulted in insufficient data to draw curves for exposures at 160 C, 175 C, and 200 C. Exploratory experiments using novel techniques, with short ramp times, for performing high temperature exposures were also examined. Several of these techniques, such as vacuum furnaces, thermal spore exposure vessels, and laser heating of the coupons, will be discussed.

bacillus subtilis var niger↗

EFFECT OF STERILIZATION IN SPACECRAFT DESIGN

Modern spacecraft design no longer involves only the Engineering Sciences. To get "the bugs" out of a system must now be taken literally to signify eradication of actual free-living microorganisms. A severe impact of a non-sterilized space vehicle could result in overt, reckless contamination, virtually eliminating a celestial body as a scientific base for the exobiological phases of space exploration. Recognizing the potentially damaging effects of septic launches, the National Aeronautics and Space Administration in December, 1960 authorized spacecraft sterilization and decontamination as National policy, thereby elevating the space effort onto a higher scientific plane. In the NASA directive, the basic reasons for sterilization were established, viz., "to preserve clues to the origin of life and of the universe, which may be hidden beneath the lunar strata or under the atmospheres of strange planets, to prevent inadvertent seeding of extraterrestrial surfaces by earth-life cultures, and to protect the earth from mutual contamination." In light of the foregoing, spacecraft sterilization should be regarded as the most significant single mission as well as design requirement of planetary space exploration --- especially if our objectives are primarily scientific.

Ranger project↗

Analysis of CO2 Composite Spray Cleaning System Results

In a previous study, the cleaning efficiency of a CO2 composite cleaning system for particulate removal was tested. The study covered particles from spores to fluorescent particles of different sizes as well as a variety of substrate surfaces, including aluminum, titanium, stainless steel, and nitinol. Particles were deposited using aerosol (dry) and droplet (wet) deposition. Results from the previous study show that the CO2 composite spray system is capable of cleaning to sterility for aerosol deposited spores and is capable of cleaning a minimum of a 4-log reduction for droplet deposited spores. This minimum 4-log reduction matches current Planetary Protection dry heat microbial reduction requirements. In this paper we will present new data to further correlate the cleaning efficiency with how contamination was introduced to the surface, the surface roughness, and particle size. Possible causes for such correlations will be discussed.

Chung, Shirley↗

Genotypic & Phenotypic Diversity of Microbial Isolates from the Mars Exploration Rovers

Mars-bound rovers such as the Mars Exploration Rover (MER) endure strict planetary protection implementation campaigns to assess bioburden. The objective of this study is to identify cultivable microorganisms isolated by the NASA Standard Assay from spacecraft during pre-launch and evaluate their potential to survive conditions on the Martian surface. Of approximately 350 isolates collected from the MER spacecraft archive, 171 microorganisms were reconstituted for characterization via 16S rRNA fingerprinting. Alignment of 16S sequences revealed high levels of sequence similarity to spore-forming species, overwhelmingly of the genera Bacillus (73.7%) and Paenibacillus (14.0%). Samples underwent phenotype characterization employing multiple carbon sources and ion concentrations in an automated microarray format using the Omnilog system. Working and stock cultures were prepared to address the immediate needs for day-to-day culture utilization and long-term preservation, respectively. Results from this study produced details about the microbes that contaminate surfaces of spacecraft, as well as a preliminary evaluation of a rapid biochemical ID method that also provides a phenotypic assessment of contaminants. The overall outcome of this study will benefit emerging cleaning and sterilization technologies for preventing forward contamination that could negatively impact future life detection or sample return missions.

Mars Exploration Rovers↗

Testing the Efficacy of Laser Sterilization as A Spacecraft Bioburden Reduction Alternative

As NASA continues to put forth efforts in seeking out life in the solar system with missions like Europa Clipper, Mars Sample Return, and Dragonfly, reducing the bioburden (number of living microbes) on spacecraft is required by NASA policy for the protection of potentially habitable worlds. In this project, we are testing the efficacy of a novel method for laser sterilization on stress tolerant bacterial spores (Bacillus subtilis) using a high energy femtosecond laser. To test the laser, we inoculate metal coupons with Bacillus subtilis spores. After laser treatment, we do a PVA (polyvinyl acetate) peel to recover the spores then we calculate to find the number of spores that survived the treatment. Our results show that within a certain pulse count range, increasing fluence increases sterilization effectiveness. We have demonstrated the ability to reduce viable microbial counts by at least 10-4. By studying laser sterilization, we could change the way spacecraft are sterilized by making the process more efficient and cost-effective.

Planetary protection↗