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Planetary Protection, Parts, and Perception: The Ranger Missions as the Origin Story of the Myth of Planetary Protection-Related Mission Failures
There has been a long-standing perspective in the planetary engineering community that the use of planetary protection sterilization techniques such as dry heat microbial reduction (DHMR) have led to significant mission failures in the past. This perception has led to a hesitation to craft mission designs that incorporate sterilization beyond the component-level. Via interviews with long-standing members of the planetary engineering community across the Agency, we have identified the root historical missions that have driven this perspective. The source, or Origin Story, which has been passed down to subsequent generations of engineers, is the failures tied to the early series of the Ranger missions. In this presentation, we examine the details behind the Ranger failures, the majority of which are non-planetary protection sterilization-related, and bring to light the myth of this perception.
Using a Blender to Assess the Microbial Density of Encapsulated Organisms
There are specific NASA requirements for source-specific encapsulated microbial density for encapsulated organisms in non-metallic materials. Projects such as the Mars Science Laboratory (MSL) that use large volumes of non-metallic materials of planetary protection concern pose a challenge to their bioburden budget. An optimized and adapted destructive hardware technology employing a commercial blender was developed to assess the embedded bioburden of thermal paint for the MSL project. The main objective of this optimization was to blend the painted foil pieces in the smallest sizes possible without excessive heating. The small size increased the surface area of the paint and enabled the release of the maximum number of encapsulated microbes. During a trial run, a piece of foil was placed into a blender for 10 minutes. The outside of the blender was very hot to the touch. Thus, the grinding was reduced to five 2-minute periods with 2-minute cooling periods between cycles. However, almost 20% of the foil fraction was larger (>2 mm). Thus, the largest fractions were then put into the blender and reground, resulting in a 71% increase in particles less than 1 mm in size, and a 76% decrease in particles greater than 2 mm in size. Because a repeatable process had been developed, a painted sample was processed with over 80% of the particles being <2 mm. It was not perceived that the properties (i.e. weight and rubber-like nature) of the painted/foil pieces would allow for a finer size distribution. With these constraints, each section would be ground for a total of 10 minutes with five cycles of a 2-minute pulse followed by a 2-minute pause. It was observed on several occasions that a larger blade affected the recovery of seeded spores by approximately half an order of magnitude. In the standard approach, each piece of painted foil was aseptically removed from the bag and placed onto a sterile tray where they were sized, cut, and cleaned. Each section was then weighed and placed into a sterile Waring Laboratory Blender. Samples were processed on low speed. The ground-up samples were then transferred to a 500-mL bottle using a sterile 1-in. (.2.5-cm) trim brush. To each of the bottles sterile planetary protection rinse solution was added and a modified NASA Standard Assay (NASA HBK 6022) was performed. Both vegetative and spore plates were analyzed.
Ultrashort Pulsed Laser Treatment: A Novel Sterilization Method for Planetary Protection
Introduction: On missions seeking signs of potential life elsewhere in the solar system, we have an obligation not to bring our own Earthly life with us. Reducing the bioburden (number of living microbes) on spacecraft is therefore required by the COSPAR Policy on Planetary Protection to target bodies that are of interest for understanding the origins of life (such as Mars). The method of spacecraft sterilization predominantly used by NASA is Heat Microbial Reduction, which is typically incompatible with heat-sensitive components such as optics and electronics, and is expensive and time-consuming. Here, we present preliminary results on a novel method for spacecraft hardware sterilization: high-intensity ultrashort (femtosecond) pulsed laser illumination. Femtosecond lasers use extremely high photon fluxes (10^29 photons/sec*cm^2, ~0.03 J/cm^2) in extremely short pulses, which can inactivate even stress-tolerant microbial spores with minimal damage to the spacecraft surface. This rapid sterilization technique could be carried out in situ in a spacecraft assembly clean room using high-speed surface scanning, saving critical time and resources. It also can potentially remove or reduce debris from inactivated cells and spores. Methods: To develop optimal laser processing parameters for inactivating planetary protection-relevant organisms on metal surfaces, we inoculated 1-cm^2 mirror-polished aluminum coupons with Bacillus subtilis spores (2x10^5 spores/coupon) and tested the effect of several parameters, including pulse count and fluence. Sterilization effectiveness was measured by recovering spores using a PVA (polyvinyl acetate) peel and conducting serial dilution and plating for colony-forming units (CFUs). Results: Our results show that pulse count and fluence both affect sterilization effectiveness and that within a certain pulse count range, increasing fluence increases effectiveness. We have demonstrated the ability to reduce viable microbial counts by at least 10^-4. Future work will include testing higher abundances and different species of microorganisms, effectiveness on complex surfaces, compatibility with sensitive surfaces, and quantification of cell debris removal. Femtosecond pulsed laser illumination has the potential to provide dramatic savings in both cost and schedule over current methods of bioburden reduction to prevent forward contamination. It may also have the potential for use in surface sterilization of returned samples to prevent back contamination.
Clean is not Sterile: A Planetary Science and Planetary Protection Perspective on Cleanroom Microbiology at NASA
The Astromaterials Acquisition and Curation Office at NASA is responsible for the curation of extraterrestrial samples from NASA’s past and future sample return missions. Our office curates samples from the moon, meteorites, comets, asteroids, cosmic dust and solar wind particles. All these samples are kept in cleanrooms to limit particulate and trace metal contamination, but none of these cleanrooms are specifically designed to control microbial contamination. During the early Apollo missions NASA scientists were very concerned with protecting the Earth from potential microbial contamination from the moon and with protecting the lunar samples from terrestrial microbes. NASA developed specialized equipment and clean rooms to keep these collections pristine. However, as we learned more about the lunar environment our concerns about microbial contamination lessened. Today none of the existing collections have microbial contamination requirements because they are not considered susceptible to microbial alteration under curation conditions (e.g. solar wind samples, and lunar samples) or have already been contaminated by terrestrial biology (meteorites collected in Antarctica). However, NASA’s OSIRIS-REx mission will land in 2023 with samples from a carbon rich asteroid that will be susceptible to microbial alteration. The Perseverance rover on Mars will begin to collect and cache samples that will be returned to Earth as soon as 2031. Martian samples may contain signs of extraterrestrial life and will have to be treated like the early Apollo samples. Martian samples will be isolated to protect the Earth, and must also be protected from terrestrial contamination. I will present microbial monitoring data from existing NASA cleanrooms and discuss how NASA is planning to use techniques from the pharmaceutical industry and academia to design new laboratories and equipment that will protect astromaterials and the earth from unwanted microbial contamination. I will also discuss a project to sample the external microbiome of the International Space Station. Results from this research will be used to design facilities for use on Mars that limit the amount of contamination associated with crewed missions.
Ultrashort Pulse Laser Surface Processing Techniques for Sterilization of Surfaces for Planetary Protection
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Ultrashort Pulse Laser Surface Processing Techniques for Sterilization of Metal Surfaces for Planetary Protection
To prevent forward contamination from microbes aboard spacecraft intended to search for extraterrestrial life, there is a need for effective sterilization methods. However, current techniques are both time-consuming and expensive. For example, dry heat sterilization requires removal from the assembly site and several days of treatment. Furthermore, some components such as optics and electronics are not compatible with current sterilization techniques. Here, we report the latest results in our development of a novel femtosecond laser processing technique for the rapid sterilization of spacecraft hardware. Femtosecond lasers produce extremely high photon fluxes (10^29 photons/sec*cm^2, ~0.03 J/cm^2) in extremely short pulses, which can inactivate even stress-tolerant microbial spores with minimal damage to the spacecraft surface. Aluminum coupons were inoculated with specific densities of Bacillus subtilis bacterial endospores. These coupons were treated with various laser illumination parameters. Afterward, metal coupon samples were assayed for viable spores using a polyvinyl alcohol (PVA) peel, serial dilution, and plating for colony-forming units (CFU). Results indicate that with high enough energy density and pulse counts, most bacterial spores are inactivated with minimal damage to the metal. The sterilization is dependent on both the fluence and pulse count. In addition, femtosecond pulses are more effective than longer pulses for inactivation. These experiments have consistently achieved 4-log reduction in viable spores. Sterilization has been achieved on both flat metal coupons and non-flat surfaces with microchannels, with a slight reduction in sterilization efficiency on the uneven surface. The application of air flow during laser processing was also investigated as a way to remove spores that are dislodged from the surface by the laser illumination, which would contribute to the reduction of spacecraft bioburden. With laser processing technology rapidly evolving, our results support the possibility of an extremely rapid, in-situ surface sterilization method for use in spacecraft assembly clean rooms.
Development of vapor phase hydrogen peroxide sterilization process
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The Use of Liquid Isopropyl Alcohol and Hydrogen Peroxide Gas Plasma to Biologically Decontaminate Spacecraft Electronics
Legitimate concern exists regarding sending spacecraft and their associated hardware to solar system bodies where they could possibly contaminate the body's surface with terrestrial microorganisms. The NASA approved guidelines for sterilization as set forth in NPG 8020.12C, which is consistent with the biological contamination control objectives of the Committee on Space Research (COSPAR), recommends subjecting the spacecraft and its associated hardware to dry heat-a dry heat regimen that could potentially employ a temperature of 110(deg)C for up to 200 hours. Such a temperature exposure could prove detrimental to the spacecraft electronics. The stimulated growth of intermetallic compounds (IMCs) in metallic interconnects and/or thermal degradation of organic materials composing much of the hardware could take place over a prolonged temperature regimen. Such detrimental phenomena would almost certainly compromise the integrity and reliability of the electronics. Investigation of sterilization procedures in the medical field suggests that hydrogen peroxide (H202) gas plasma (HPGP) technology can effectively function as an alternative to heat sterilization, especially for heat-sensitive items. Treatment with isopropyl alcohol (IPA) in liquid form prior to exposure of the hardware to HPGP should also prove beneficial. Although IPA is not a sterilant, it is frequently used as a disinfectant because of its bactericidal properties. The use of IPA in electronics cleaning is widely recognized and has been utilized for many years with no adverse affects reported. In addition, IPA is the principal ingredient of the test fluid used in ionic contamination testers to assess the amount of ionic contamination found on the surfaces of printed wiring assemblies. This paper will set forth experimental data confirming the feasibility of the IPA/H202 approach to reach acceptable microbial reduction (MR) levels of spacecraft electronic hardware. In addition, a proposed process flow in which both IPA liquid and HPGP are utilized will be presented in Section 7.0.
Designing an Ultraviolet Illumination System for Mars Sample Return’s Capture, Containment & Return System
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Synchronous Separation, Seaming, Sealing and Sterilization (S4) using Brazing for Sample Containerization and Planetary Protection
The potential return of samples back to Earth from other planetary bodies would be based on planetary protection requirements that vary depending on the type of body [1]. Potential Mars Sample Return would require the protection of our planet from backward contamination. To fulfill this requirement, it would be necessary to implement “break the chain of contact (BTC)” process, where any material reaching Earth would have to be inside a container that is sealed with an extremely high level of confidence. In order to accomplish this, it would be necessary to contain the acquired samples and destroy any potential biological materials that may have contaminated the external surface of the container, while protecting the samples for further analysis. Using brazing, a novel synchronous separation, seaming, sealing and sterilization (S4) process for sample containerization and planetary protection has been conceived and demonstrated. A prototype double-wall container with inner and outer shells and Earth clean interstitial space was used for this demonstration. For potential Mars sample return, the double wall container would be consist of two halves and prepared on Earth. The on-orbit execution would consist of inserting the sample into one of the halves and then mating to the other half and melt the braze material to perform the S4 process. The use of brazing material that melts at temperatures higher than 500OC would assure sterilization of the exposed areas due to pyrolysis since carbon bonds are broken at this temperature. The process consists of two-steps, Step-1: the double wall container halves are fabricated and brazed on Earth; and Step-2: Assembly and brazing the samples on orbit. To prevent potential jamming during the process of mating the two halves of the double-wall container and the extraction of the brazed inner container, a double cone-within-cone approach has been conceived. The results of this study are described and discussed in this manuscript.
Identifying and Inactivating Bacterial Spores
Problems associated with, and new strategies for, inactivating resistant organisms like Bacillus canaveralius (found at Kennedy Space Center during a survey of three NASA cleanrooms) have been defined. Identifying the particular component of the spore that allows its heightened resistance can guide the development of sterilization procedures that are targeted to the specific molecules responsible for resistance, while avoiding using unduly harsh methods that jeopardize equipment. The key element of spore resistance is a multilayered protein shell that encases the spore called the spore coat. The coat of the best-studied spore-forming microbe, B. subtilis, consists of at least 45 proteins, most of which are poorly characterized. Several protective roles for the coat are well characterized including resistance to desiccation, large toxic molecules, ortho-phthalaldehyde, and ultraviolet (UV) radiation. One important long-term specific goal is an improved sterilization procedure that will enable NASA to meet planetary protection requirements without a terminal heat sterilization step. This would support the implementation of planetary protection policies for life-detection missions. Typically, hospitals and government agencies use biological indicators to ensure the quality control of sterilization processes. The spores of B. canaveralius that are more resistant to osmotic stress would serve as a better biological indicator for potential survival than those in use currently.
Microbial Methods for Testing a Novel Spacecraft Bioburden Reduction Method
In order to circumvent the contamination of microorganisms in both extraterrestrial and terrestrial environments, spacecraft hardware must often be sterilized. Current sterilization practices are both time-consuming and expensive. Techniques such as dry heat sterilization and scattered/localized electric field enhancements are incompatible with a variety of hardware materials as well as complex surface shapes. Our group is testing a novel femtosecond pulsed laser for the rapid sterilization of spacecraft hardware using high photon fluxes, which is both an efficient and effective technique to ensure a reduced microbial burden aboard spacecraft. This method is likely to be effective because microorganisms have not encountered high photon fluxes in their evolutionary history and thus contain no protective mechanisms against them. To assess the effectiveness of pulsed laser sterilization, we used a combination of scanning electron microscopy and elemental analysis for the examination of damaged spores on metal coupons, as well as microbiology methods for spore assays. The surfaces of metal coupons inoculated with Bacillus subtilis were illuminated with femtosecond pulses at various wavelengths below the damage threshold for the aluminum. After laser treatment, metal coupon samples were sonicated to dislodge spores, serially diluted, and finally plated and incubated. Results show that sterilization efficiency is favorable in some conditions. The NASA standards for spore assays are a preliminary demonstration in establishing the trend in ability to kill spores. The results show potential for the dual pulse illumination method as a sterilization technique for planetary protection applications.
Synchronous Separation, Seaming, Sealing and Sterilization (S4) using Brazing for Sample Containerization and Planetary Protection
The return of samples back to Earth in future missions would require protection of our planet from the risk of bringing uncontrolled biological materials back with the samples. This protection would require “breaking the chain of contact (BTC)”, where any returned material reaching Earth for further analysis would have to be sealed inside a container with extremely high confidence. Therefore, the acquired samples would need to be contained while destroying any potential biological materials that may contaminate the external surface of the container. A novel process that could be used to contain returning samples has been developed and demonstrated in a quarter scale size. The process consists of brazing using non-contact induction heating that synchronously separates, seams, seals and sterilizes (S4) the container. The use of brazing involves melting at temperatures higher than 500°C and this level of heating assures sterilization of the exposed areas since all carbon bonds (namely, organic materials) are broken at this temperature. The mechanism consists of a double wall container with inner and outer shells having Earth-clean interior surfaces. The process consists of two-steps, Step-1: the double wall container halves are fabricated and brazed (equivalent to production on Earth); and Step-2 is the S4 process and it is the equivalent to the execution on-orbit around Mars. In a potential future mission, the double wall container would be split into two halves and prepared on Earth. The potential on-orbit execution would consist of inserting the orbiting sample (OS) container into one of the halves and then mated to the other half and brazed. The latest results of this effort will be described and discussed in this manuscript.
Lessons Learned in Designing A Proposed Ultraviolet Sterilization System for Space
This paper presents a number of lessons learned while designing a proposed sterilization system for Mars Sample Return. This sterilization system is needed to inactivate any potentially hazardous Mars material on the exterior surface of the vessel containing sealed sample tubes filled with Mars rock cores, regolith and atmosphere. These returned samples would provide information on the geologic history of Mars, the evolution of its climate and the potential for ancient life. Mars Sample Return is categorized at Planetary Protection Category V Restricted Earth Return, so it is required to protect the Earth–Moon system from the biological impact of returning samples from Mars to Earth. This article reviews lessons learned in the development of a particular engineering implementation to support the protection of the Earth–Moon biosphere: the use of in situ ultraviolet LED illumination. The details of the biological efficacy of this approach or the policy-related impacts are outside of the scope of this manuscript. The lessons learned presented here include establishing design requirements for the system, the selection of a light source, optical design options, contamination control and approaches to thermal and power management.
Mechanical abrasion as a low cost technique for contamination-free sample acquisition from contaminated (IVA clean) platforms
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Silicon Carbide, Vacuum Tube Nanoelectronics: Application for Exploration Missions Requiring Category III/IV Planetary Protection
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