Improved Cleaning Methods for Planetary Protection Bioburden Reduction
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bioburden reduction planetary protection↗
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Improved Cleaning Methods for Planetary Protection Bioburden Reduction
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bioburden reduction planetary protection↗
Mars Technology Program: Planetary Protection Technology Development
This slide presentation reviews the development of Planetary Protection Technology in the Mars Technology Program. The goal of the program is to develop technologies that will enable NASA to build, launch, and operate a mission that has subsystems with different Planetary Protection (PP) classifications, specifically for operating a Category IVb-equivalent subsystem from a Category IVa platform. The IVa category of planetary protection requires bioburden reduction (i.e., no sterilization is required) The IVb category in addition to IVa requirements: (i.e., terminal sterilization of spacecraft is required). The differences between the categories are further reviewed.
Mars Technology Program↗
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↗
Planetary Protection Concerns During Pre-Launch Radioisotope Power System Final Integration Activities
The Advanced Stirling Radioisotope Generator (ASRG) is a next-generation radioisotope-based power system that is currently being developed as an alternative to the Multi-Mission Radioisotope Thermoelectric Generator (MMRTG). Power sources such as these may be needed for proposed missions to solar system planets and bodies that have challenging Planetary Protection (PP) requirements (e.g. Mars, Europa, Enceladus) that may support NASA s search for life, remnants of past life, and the precursors of life. One concern is that the heat from the ASRG could potentially create a region in which liquid water may occur. As advised by the NASA Planetary Protection Officer, when deploying an ASRG to Mars, the current COSPAR/NASA PP policy should be followed for Category IVc mission. Thus, sterilization processing of the ASRG to achieve bioburden reduction would be essential to meet the Planetary Protection requirements. Due to thermal constraints and associated low temperature limits of elements of the ASRG, vapor hydrogen peroxide (VHP) was suggested as a candidate alternative sterilization process to complement dry heat microbial reduction (DHMR) for the assembled ASRG. The following proposed sterilization plan for the ASRG anticipates a mission Category IVc level of cleanliness. This plan provides a scenario in which VHP is used as the final sterilization process. Keywords: Advanced Stirling Radioisotope Generator (ASRG), Planetary Protection (PP), Vapor hydrogen peroxide (VHP) sterilization.
Chen, Fei↗
Ultrashort Pulse Lasers for Surface Sterilization
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bioburden reduction↗
Organic Biomarker-Based Assays to Evaluate Total Bioburden and Organic Compounds on Space Flight Hardware
Meeting planetary protection (PP) requirements for space flight hardware may involve bioburden reduction by dry heat microbial reduction (DHMR). The NASA standard assay to demonstrate the reduction of organisms involves the swabbing of surfaces, heat shock of the extracted samples, plating of the samples on Trypticase Soy Agar (TSA), and counting colony forming units after an incubation period. The standard assay uses enumeration of heat tolerant spore-formers as a proxy for total bioburden and is generally expected to provide a lower limit. We suggest that a better estimate of the total bioburden could be obtained through sampling and analysis of organic biomarkers. As biological organisms are fundamentally organic in chemistry (i.e. carbon containing materials) it is important to characterize the biomarker compounds that are released from organisms that 1) exist on flight hardware before microbial reduction and 2) left behind from the killed organisms following microbial reduction.
Locke, Darren R.↗
Europa Planetary Protection for Juno Jupiter Orbiter
NASA's Juno mission launched in 2011 and will explore the Jupiter system starting in 2016. Juno's suite of instruments is designed to investigate the atmosphere, gravitational fields, magnetic fields, and auroral regions. Its low perijove polar orbit will allow it to explore portions of the Jovian environment never before visited. While the Juno mission is not orbiting or flying close to Europa or the other Galilean satellites, planetary protection requirements for avoiding the contamination of Europa have been taken into account in the Juno mission design.The science mission is designed to conclude with a deorbit burn that disposes of the spacecraft in Jupiter's atmosphere. Compliance with planetary protection requirements is verified through a set of analyses including analysis of initial bioburden, analysis of the effect of bioburden reduction due to the space and Jovian radiation environments, probabilistic risk assessment of successful deorbit, Monte-Carlo orbit propagation, and bioburden reduction in the event of impact with an icy body.
Juno↗
Susceptibility of extremophiles to far-UVC light for bioburden reduction in spacecraft assembly facilities
The prevention and reduction of microbial species entering and leaving Earth's biosphere is a critical aspect of planetary protection research. While various decontamination methods exist and are currently utilized for planetary protection purposes, the use of far-UVC light (200–230 nm) as a means for microbial reduction remains underexplored. Unlike conventional germicidal ultraviolet at 254 nm, which can pose a health risk to humans even with small exposure doses, far-UVC light poses minimal health hazard making it a suitable candidate for implementation in occupied areas of spacecraft assembly facilities. This study investigates the efficacy of far-UVC 222-nm light to inactivate bacteria using microbial species which are relevant to planetary protection either in vegetative cell or spore form. All the tested vegetative cells demonstrated susceptibility to 222-nm exposure, although susceptibility varied among the tested species. Notably, Deinococcus radiodurans, a species highly tolerant to extreme environmental conditions, exhibited the most resistance to far-UVC exposure with a dose of 112 mJ/cm 2 required for a 1-log reduction in survival. While spore susceptibility was similar across the species tested, Bacillus pumilus spores were the most resistant of the tested spores when analyzed with a bi-exponential cell killing model (D 90 of 6.8 mJ/cm 2 ). Overall, these results demonstrate the efficacy of far-UVC light for reducing microbial bioburden to help ensure the success and safety of future space exploration missions.
Ultraviolet light↗
The Evolution of Planetary Protection Implementation on Mars Landed Missions
NASA has developed requirements dedicated to the prevention of forward and backward contamination during space exploration. Historically, international agreements provided guidelines to prevent contamination of the Moon and other celestial bodies, as well as the Earth (e.g., sample return missions). The UN Outer Space Treaty was established in 1967 and the Committee on Space Research (COSPAR) maintains a planetary protection policy complying with Article IX of this treaty. By avoiding forward contamination, the integrity of scientific exploration is preserved. Planetary Protection mission requirements are levied on missions to control contamination. These requirements are dependent on the science of the mission and on the celestial bodies encountered or targeted along the way. Consequently, categories are assigned to missions, and specific implementation plans are developed to meet the planetary protection requirements. NASA missions have evolved over time with increasingly more demanding scientific objectives and more complex flight systems to achieve those objectives and, thus, planetary protection methods and processes used for implementation have become much more intricate, complicated, and challenging. Here, we will portray the evolution of planetary protection implementation at JPL in several important areas throughout the course of NASA sponsored robotic Mars lander or rover missions, starting from Mars Pathfinder through the beginning of Mars 2020. Highlighted in the discussion will be process changes in planetary protection requirements development and flow down. Development and implementation of new and improved methods used in the reduction of spacecraft bioburden will be discussed as well as approaches and challenges that come along with setting up remote laboratories to perform bioassays. The consequences and forward planning of delays on missions will be highlighted as well as lessons learned on the impact of communication and training in achieving planetary protection requirements. The evolution of methods used for the detection of microbial bioburden on spacecraft hardware will be considered. These methods use standard microbiology as well as the adaptation of advances in biotechnology, molecular biology, and bioinformatics. Technical approaches developed for the prevention of contamination and recontamination of hardware during Assembly, Test, and Launch Operations will be discussed.
Kazarians, Gayane A.↗
Ours or Mars? Understanding Microbial Bioloading on the InSight Mars Lander
A review of planetary protection approaches to the reduction of "harmful contamination" in the form of endospores for the InSIght Mars Lander.
bioburden↗
Microbial Certification of the MER spacecraft
Spacecraft such as the Mars Exploration Rovers (MER) must meet acceptable microbial population levels prior to launch. Sensitive parts and materials prevent any single sterilization method from being used as a final step on the assembled spacecraft.
spore bioburden Mars Exploration Rover dry hear mi↗
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↗
Establishing and Monitoring an Aseptic Workspace for Building the MOMA Mass Spectrometer
Mars Organic Molecule Analyzer (MOMA) is an instrument suite on the European Space Agency (ESA) ExoMars 2020 Rover, and the Mass Spectrometer (MOMA-MS) is being built at Goddard Space Flight Center (GSFC). MOMA-MS is a life-detection instrument and thus falls in the most stringent category of Planetary Protection (PP) biological cleanliness requirements. Less than 0.03 spore/m2 are allowed in the instrument sample path. In order to meet these PP requirements, MOMA-MS must be built and maintained in a low bioburden environment. The MOMA-MS project at GSFC maintains three clean rooms with varying levels of bioburden control. The Aseptic Assembly Clean room has the highest level of control, applying three different bioburden reducing methods: 70% Isopropyl Alcohol (IPA), 7.5% Hydrogen Peroxide, and Ultra-Violet C (UVC) light. The three methods are used in rotation and each kills microorganisms by a different mechanism, reducing the likelihood of microorganisms developing resistance to all three. The Integration and Mars Chamber Clean rooms use less biocidal cleaning, with the option to deploy extra techniques as necessary. To support the monitoring of clean rooms and verification that MOMA-MS hardware meets PP requirements, a new Planetary Protection lab was established that currently has the capabilities of standard growth assays for spore or vegetative bacteria, rapid bioburden analysis that detects Adenosine Triphosphate (ATP), plus autoclave and Dry Heat microbial Reduction (DHMR) verification. The clean rooms are monitored for vegetative microorganisms and by rapid ATP assay, and a clear difference in bioburden is observed between the aseptic and other clean room.
Contamination Control Engineering↗
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.
Planetary Protection; Lasers; Sterilization; Bacil↗
The Validation of Vapor Phase Hydrogen Peroxide Microbial Reduction for Planetary Protection and a Proposed Vacuum Process Specification
The Jet Propulsion Laboratory, in conjunction with the NASA Planetary Protection Officer, has selected the vapor phase hydrogen peroxide sterilization process for continued development as a NASA approved sterilization technique for spacecraft subsystems and systems. The goal is to include this technique, with an appropriate specification, in NPR 8020.12C as a low temperature complementary technique to the dry heat sterilization process.To meet microbial reduction requirements for all Mars in-situ life detection and sample return missions, various planetary spacecraft subsystems will have to be exposed to a qualified sterilization process. This process could be the elevated temperature dry heat sterilization process (~115 C for 40 hours) which was used to sterilize the Viking lander spacecraft. However, with utilization of such elements as highly sophisticated electronics and sensors in modern spacecraft, this process presents significant materials challenges and is thus an undesirable bioburden reduction method to design engineers. The objective of this work is to introduce vapor hydrogen peroxide (VHP) as an alternative to dry heat microbial reduction to meet planetary protection requirements.The VHP process is widely used by the medical industry to sterilize surgical instruments and biomedical devices, but high doses of VHP may degrade the performance of flight hardware, or compromise material properties. Our goal for this study was to determine the minimum VHP process conditions to achieve microbial reduction levels acceptable for planetary protection.
planetary protection↗
Analysis of Bioreduction Strategies for a Solid Rocket Motor on an Interplanetary Mission to Europa
The Europa Lander De-Orbit stage braking motor must comply with the Planetary Protection requirements established for a category IV mission. In its mission to Europa, a motor that hasn’t gone through bioreduction environments will carry microbial spores and other biosignature molecules that might jeopardize a mission of astrobiological concern as well as future missions to come. A motor with solid rocket propellant represents exclusive challenges associated to the calculation of high numbers of an encapsulated bioburden hidden behind a nozzle plug. Existing techniques for bioburden reduction are analysed in perspective to motor assembly facilities and the series of events that are involved in manufacturing a solid rocket motor. These techniques include antimicrobial effects of chemical components already present within the motor and bioreduction due to assembly and operational environments induced in the motor. Analysis of the manufacturing process, adhered bioburden and recent investigations into the effectiveness of microbiological techniques in finding inherent antimicrobial properties have generated a step by step outline of Planetary Protection for lander mission associated solid rocket motors.
Velez-Justiniano, Yo-Ann↗
Development and Implementation of Aseptic Operations for the MOMA-Mass Spectrometer
The ExoMars 2020 Rover is a life detection mission, and is classified as Planetary Protection (PP) Mission Category IVb, the first IVb mission since the Viking missions. Mars Organic Molecule Analyzer Mass Spectrometer (MOMA-MS) is a life detection instrument for the rover. To meet the stringent bioburden requirement of 0.03 spore/m2, the MS is subjected to Dry Heat Microbial Reduction (DHMR) to decrease the bioburden from a measured 88 spores/m2 to 0.009 spores/m2. After DHMR, exposure of the sample path must be kept to an absolute minimum and requires aseptic operations. Aseptic operations include determining the safe exposure time based on the surface area of exposure and particle fallout expected in the aseptic ISO class 5 workspace, preparing an aseptic ISO class 5 workspace, and using sterile garments and tools. During the exposure activity 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 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 PP cleanliness can necessitate repeating instrument level DHMR, which not only has significant cost and schedule implications, but also is a risk to hardware that is not rated for repeated long exposures to high temperatures.
Aseptic processing↗