Ultrashort Pulse Lasers for Surface Sterilization
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bioburden reduction↗
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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↗
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.↗
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.
Kaleb McQuillan↗
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↗
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↗
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↗
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↗
An Overview of Surface Heat Microbial Reduction as a Viable Microbial Reduction Modality for Spacecraft Surfaces
In accordance with NASA Planetary Protection (PP) policy requirements, flight project hardware may be required to undergo microbial reduction processes to prevent the forward contamination of target planetary bodies with Earth organisms. Heat microbial reduction (HMR) is the most commonly employed modality used at JPL for reducing the microbial bioburden on flight hardware. In 2013, longstanding HMR specifications were abandoned, and revised specifications were developed which integrated the latest findings on bacterial spore heat resistivity. Revised decimal reduction values (D-values) for time-temperature lethality curves (110 °C to 200 °C) were developed to account for “hardy” bacterial spores that exhibit greater heat resistance than previously understood. Presented here is a comparative analysis of the revised NASA HMR specifications against empirical data compiled from recent JPL studies, and peer-reviewed, published literature. Bacillus sp. strain ATCC 29669 displayed high heat resistance, and this strain’s 4-log heat lethality curve was comparable to the revised 4-log specification. Spores of Bacillus atrophaeus ATCC 9372 displayed less heat resistance, and exhibited D-values which were less than the revised 3-log microbial reduction specifications. Extrapolations indicate that the current 6-log reduction credit applied to 350 °C for 1 hr. and 500 °C for 0.5 sec. is highly conservative. Projections indicate that a 10- to 18-log reduction of both hardy and non-hardy spores may be achievable at bakeouts of 350 °C for 1 hr. The findings reported here indicate the revised NASA HMR specifications from 110 °C to 200 °C are appropriate for achieving 4-log and 6-log reductions with hardy spore populations; however, for non-hardy spore populations, or for temperatures above 200 °C, the specifications are exceedingly conservative.
Shirey, Brian T.↗
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↗
Microbial certification of the MER spacecraft
We conclude in this paper that a combination of Dry Heat Microbrial Reduction and control measures during complex mechanical assembly processes can result in a total spore bioburden that meets requirements.
spore bioburden MER spacecraft bacteria bar code↗
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↗
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↗
Refinement of planetary protection policy for Mars missions
Under existing COSPAR policy adopted in 1984, missions to Mars (landers, probes, and some orbiters) are designated as Category IV missions. As such, the procedures for implementing planetary protection requirements could include trajectory biasing, cleanrooms, bioload reduction, sterilization of hardware, and bioshields. In 1992, a U.S. National Research Council study recommended that controls on forward contamination of Mars be tied to specific mission objectives. The report recommended that Mars landers with life detection instruments be subject to at least Viking-level sterilization procedures for bioload reduction, while spacecraft (including orbiters) without life detection instruments be subject to at least Viking-level pre-sterilization procedures for bioload reduction but need not be sterilized. In light of this, it is proposed that the current policy's Category IV and its planetary protection requirements be divided into two sub-categories as follows: Category IVa, for missions comprising landers and probes without life detection experiments, which will meet a specified bioburden limit for exposed surfaces, and Category IVb, for landers and probes with life detection experiments, which will require sterilization of landed systems. In addition, Category III orbiter mission specifications are expanded to be consistent with these recommendations.
NASA Discipline Exobiology↗
Revision to Planetary Protection Policy for Mars Missions
Under existing COSPAR policy adopted in 1984, missions to Mars (landers, probes, and some orbiters) are designated as Category IV missions. As such, the procedures for implementing planetary protection requirements could include trajectory biasing, cleanrooms, bioload reduction, sterilization of hardware, and bioshields, i. e. requirements could be similar to Viking. However, in 1992, a U. S. National Academy of Sciences study recommended that controls on forward contamination of Mars be tied to specific mission objectives. The report recommended that Mars landers with life detection instruments be subject to at least Viking-level sterilization procedures for bioload reduction, while spacecraft (including orbiters) without life detection instruments be subject to at least Viking-level pre sterilization procedures for bioload reduction but need not be sterilized. In light of this, it is proposed that the current policy's Category IV missions and their planetary protection requirements be divided into two subcategories as follows: Category IV A, for missions comprising landers and probes without life detection experiments and some orbiters, which will meet a specified bioburden limit for exposed surfaces; Category IV B, for landers and probes with life detection experiments, which will require complete system sterilization. For Category IV A missions, bioburden specifications will be proposed and implementing procedures discussed. A resolution will be proposed to modify the existing COSPAR policy to reflect these changes. Similar specifications, procedures, and resolution for Category IV B missions will be the subject of a later study.
DeVincenzi, D. L.↗
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↗
Microbial Monitoring of New Cleanrooms Used to Curate Astrobiologically Relevant Asteroid Samples from Bennu and Ryugu
Introduction: NASA has constructed two new cleanrooms to house materials from the OSRIS-REx and Hayabusa2 missions to the asteroids Ryugu (162173) and Bennu (101955), respectively. In accordance with standard astromaterials curation practices, these cleanrooms will be monitored for particulate contamination and maintained to ISO 5 equivalent standards1. Since the samples in these collections are expected to contain prebiotic organic compounds that may help explain the origin of life on Earth, these labs will also be monitored for organic and biological contamination2. Samples from Ryugu arrived on Earth in December, 2020. After basic characterization in Japan, NASA received a subset of these samples at the astromaterials curation facility in Houston in December of 2021. OSIRIS-REx is expected to return samples in September, 2023. Here we present preliminary microbial monitoring results from monthly monitoring of these new labs and the connected microtomy and staging areas that support them, as they are being commissioned. We also compare these results to baseline values for other astromaterials curation labs. We will also briefly describe additional cleaning efforts employed to reduce the bioburden in these new cleanrooms. Methods: Microbial samples were collected from surfaces using a dry macrofoam swab (Puritan Brand 2518051PFRNDFD). Swabs were also opened in the lab but not touched to any surfaces to function as negative controls. Samples and controls were processed inside a class II biosafety cabinet to avoid inadvertent cross contamination. The swabs were suspended in 15 ml of PBS (Phosphate Buffered Saline) and vortexed for 20 seconds to remove cells from the swab surface. The PBS was used to inoculate Petri dishes filled with TSA (Tryptic Soy Agar), Blood Agar, or Reasoners 2 agar to check for microbial growth. Each plate was inoculated with 0.1 ml of PBS. The TSA and blood agar plates were incubated at 35˚C and the Reasoners 2 agar plates were incubated at 25˚C for seven days. Petri dishes filled with Potato dextrose agar, Saboraud dextrose agar, or Saboraud dextrose agar with 0.1 mg/ml of chloramphenicol, an antibiotic, were used to check for fungal growth. These plates were inoculated with 0.3 ml of PBS and incubated at 30˚C. The remaining PBS was frozen at -80 ˚C for DNA sequencing. After incubation, isolates were counted and reisolated for identification. Isolates were identified using the VITEK23 system or by sequencing a portion of the 16S rRNA gene for bacteria or the ribosomal internal transcribed spacer (ITS) for fungi. Sequencing was performed with an ABI 3500 Sanger sequencer. Results: During our initial sampling, six of the seven sites sampled (86%) displayed bacterial or fungal growth. Samples collected from the staging areas and microtomy labs are not included in this calculation since those areas are maintained at a lower ISO 7 equivalent cleanliness standard. A month later, only three of the seven sites (43%) displayed bacterial growth. No fungal growth was detected in the second sampling. Since new equipment had been introduced to the Hayabusa2 lab since the first round of sampling, an additional three sampling sites were included in the second round of sampling. None of these sites displayed microbial growth. These sites will be included in all future sampling efforts. Bacterial isolates have been identified from the following genera at multiple time points: Micrococcus, Staphylococcus, and Bacillus. Isolates from the genera: Microbacterium, Nocardioides, Methylocystis, and Microvirga were identified in the initial sampling, but were not present at later time points. Identification of fungal isolates is in progress. Results are summarized in Table 1. Discussion: The recovery rate or percentage of positive samples4 was initially 86%, which is higher than the median recovery rate for comparable ISO 5 equivalent curation labs like Stardust (33%), Hayabusa (33%), and Cosmic Dust (50%). However, after a month of operation, the recovery rate for these same sites decreased to 43%, which is similar to what we observe in comparable curation cleanrooms with no microbial control requirements. Adding in the new sampling sites further decreases the recovery rate to 30%. With the reduction in recovery rate, we also observed a decrease in microbial diversity. At the first time point, we observed at least 10 different bacterial species and at least two different fungi. This is a higher diversity than the median values for comparable ISO 5 equivalent labs (2-4 isolates per sampling event). After the second sampling, we observed at least 4 bacterial species and no fungi, which is more consistent with comparable labs. We expect the recovery rate and diversity in both labs to continue to decrease as routine operation continues. We will use ultrapure hydrogen peroxide to disinfect equipment and work areas prior to opening any sample containers. Most of the bacterial and fungal isolates were detected on samples from the cleanroom floors. This is consistent with baseline results from other curation labs. Organisms from the genera Bacillus, Staphylococcus, and Micrococcus that were repeatedly detected are common in cleanrooms and on human skin5,6. These organisms are generally thought to be introduced when people enter the cleanroom. Microbacterium, Nocardioides, and Microvirga have also previously been identified in astromaterials cleanrooms, but not as frequently as Bacillus, Staphylococcus, and Micrococcus. Methylocystis is a novel genus in the astromaterials cleanrooms, but it was identified with low accuracy (93% match in the sequenced region of the 16S rRNA gene) and further work is needed to confirm this identification. Microbacterium is a diverse genus with isolates identified from terrestrial and aquatic sediments. Some species of Microbacterium are capable of degrading complex organic compounds found in crude oil. The presence of these bacteria in the OSIRIS REx and Hayabusa2 cleanrooms should be closely monitored. Methylocystis is a genus of methanotrophic bacteria capable of oxidizing methane. If this identification proves to be correct and it is detected again, it should be closely monitored as well. Under nominal operating conditions, samples should not ever encounter the cleanroom floor or other high traffic areas. If we observe an increase in the bioburden in sensitive work areas that appears to be influenced by organism transfer from high traffic areas like the floors, we can employ additional hydrogen peroxide treatments to disinfect high traffic areas. Routine microbial monitoring of these labs will ensure that NASA’s astromaterials collections remain pristine and useful for scientific study. Table 1. Sampling Locations and Colony Counts Bacterial CFUa Fungal CFU Bacterial CFU Fungal CFU Lab - Location 11/2/2021 11/2/2021 12/13/2021 12/13/2021 H2b-Floor 4 8 1 0 H2-staging pass through 3 0 0 0 H2-microtomy pass through TNTCc 0 0 0 H2 Microscope 1 NA NA 0 0 H2 Microscope 2 NA NA 0 0 H2-Table NA NA 0 0 OREXd- microtomy pass through 0 0 6 0 OREX – Anteroom pass through 0 0 0 0 OREX – Floor 1 2 0 0 OREX Witness Foil Table 3 0 1 0 Staging-Floor 16 0 15 0 Microtomy-Floor 3 0 2 0 a: CFU = Colony Forming Unit b: H2 = Hayabusa2 Lab c: TNTC = too numerous to count d: OREX = OSIRIS-REx Lab References: 1. ISO 14644-1:2015 - Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration. 37 (2015). 2. McCubbin, F. M. et al. Space Sci Rev 215, (2019). 3. Pincus, D. H. Encyclopedia of Rapid Microbiological Methods (2005). 4. The United States Pharmacopeial Convention. USP General Chapter <1116> 17, 784–794 (2013). 5. Sheraba, N. S., Yassin, A. S. & Amin, M. BMC Research Notes 3, 278 (2010). 6. Utescher, C. L. de A., Franzolin, M. R., Trabulsi, L. R. & Gambale, V. Brazilian Journal of Microbiology 38, 710–716 (2007).
A B Regberg↗