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Lessons Learned From the Construction of a Portable Cleanroom for NASA OSIRIS-REx Mission Deintegration

NASA Johnson Space Center (JSC) Infrastructure and Astromaterials Acquisition & Curation Office completed construction and commissioning of the OSIRIS-REx (OREx) Deintegration portable cleanroom at the Utah Test and Training Range (UTTR). The new portable cleanroom was designed to receive the OREx sample return capsule from the landing point on the range to an ISO7 environment. Scientists used the portable clean-room to deintegrate the sample canister from the sample return capsule. Once separated, the sample canister was put in a container under nitrogen purge for transportation to B31 at the Johnson Space Center for astromaterial sample extraction, preliminary analysis, and long-term curation. The portable cleanroom was built by a subcontractor at their facility and then deconstructed to be transported to the remote location at UTTR. Since construction was completed in a remote location all tools and materials had to be transported from contractor site in Dallas, TX. The cleanroom was constructed within an existing facility, which provided conditioned air, electric power, and protection from the elements. Careful coordination was required between the host facility, cleanroom contractor, mission scientists, and JSC facilities and curation personnel. An existing anteroom at JSC was transported to UTTR and added to the portable cleanroom after there was concern about contamination without one for personnel entry/exit. The scientific study of organics is critical for the mission, so a stringent contamination control plan was implemented for low organics. Given these mission requirements the cleanroom construction materials were carefully selected to not hinder the scientific search for amino acids and the study of organics in the samples. The same cleanroom contractor that built the long-term astromaterial curation cleanroom back at JSC Houston, TX was selected to build the portable cleanroom and instructed to use the same materials. The cleanroom had double doors to open and allow the sample return capsule to fit into the cleanroom on its stand and be transferred to a clean stand already in the cleanroom. The portable cleanroom successfully completed its mission and the sample canister was safely deintegrated and transported to JSC under nitrogen purge.

astromaterials curation

Portable Cleanroom for NASA OSIRIS-REx Mission Deintegration

NASA Johnson Space Center (JSC) Infrastructure and Astromaterials Acquisition & Curation Office completed construction and commissioning of the OSIRIS-REx (OREx) Deintegration portable cleanroom at the Utah Test and Training Range (UTTR). The new portable cleanroom was designed to receive the OREx sample return capsule from the landing point on the range to an ISO7 environment. Scientists used the portable clean-room to deintegrate the sample canister from the sample return capsule. Once separated, the sample canister was put in a container under nitrogen purge for transportation to B31 at the Johnson Space Center for astromaterial sample extraction, preliminary analysis, and long-term curation. The portable cleanroom was built by a subcontractor at their facility and then deconstructed to be transported to the remote location at UTTR. Since construction was completed in a remote location all tools and materials had to be transported from contractor site in Dallas, TX. The cleanroom was constructed within an existing facility, which provided conditioned air, electric power, and protection from the elements. Careful coordination was required between the host facility, cleanroom contractor, mission scientists, and JSC facilities and curation personnel. An existing anteroom at JSC was transported to UTTR and added to the portable cleanroom after there was concern about contamination without one for personnel entry/exit. The scientific study of organics is critical for the mission, so a stringent contamination control plan was implemented for low organics. Given these mission requirements the cleanroom construction materials were carefully selected to not hinder the scientific search for amino acids and the study of organics in the samples. The same cleanroom contractor that built the long-term astromaterial curation cleanroom back at JSC Houston, TX was selected to build the portable cleanroom and instructed to use the same materials. The cleanroom had double doors to open and allow the sample return capsule to fit into the cleanroom on its stand and be transferred to a clean stand already in the cleanroom. The portable cleanroom successfully completed its mission and the sample canister was safely deintegrated and transported to JSC under nitrogen purge.

astromaterials curation

Amateur Cleanrooms: Costs May Not Offset Benefits

Contamination and Coatings Branch During my career at NASA, I have encountered a variety of cleanroom systems. With many projects, cost pressures and lack of adequate facilities have forced the managers to resort to amateur cleanrooms to manufacture spacecraft and instruments. These rooms are usually spaces that have been used for other purposes that are converted to cleanroom, usually without the assistance of a contamination control specialist. Often, scientists and engineers are successful in converting an area for experimental use. However, when the area is used for production, countless difficulties are encountered. This paper will document some of the disasters that I have personally witnessed and offer some guidelines for contamination professionals to follow if you are called upon to assist in the development of new cleanrooms. Cleanroom come in all shapes and sizes from special purpose mini-environments (such as flow benches) to large, expansive production facilities. These areas may require a variety of unit operations to be carried out within a short range of each other. The design of the cleanroom should account for compatibilities of these operations to protect the product and personnel. The level of cleanliness has traditionally been associated with the method of ventilation. However, just because and =ea has HEPA filters and greater that 20 air changes per hour does not mean that it is a cleanroom. Airflow is extremely complex; the only way to properly design a cleanroom is through the use of a computer based model. In the aerospace industry, few engineered cleanrooms are modeled. Modeling has been perceived as expensive; however, modern programs and fast computers are changing perception. It is the lack of appreciation for how air flow and location within a cleanroom affects the product that causes most of the problems I have experienced. Currently, the rules defining the best air flow design practices are based on simplistic historical data that are often wrong. The performance of a cleanroom is defined by a set of complex interactions between the airflow, sources of contamination and heat, position of the air terminals and exhausts as well as the objects occupying the space in question. These subtleties are almost never appreciated in the setup of amateur cleanrooms (and sadly, in some engineered cleanroom as well). Experience with the room, measurement of air flows in the room, and black light inspections can be used to

Ramsey, W. Lawrence

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

2023 Cleanroom Monitoring for OSIRIS-REx

OSIRIS-REx is an asteroid sample return mission that delivered asteroid regolith from Bennu to Earth on 9/24/2023 [1]. Cleanroom monitoring of OSIRIS-REx Curation facilities has been ongoing since 2021 when construction of the OSIRIS-REx lab at Johnson Space Center (JSC) was completed [2]. The monitoring continues inside of the OSIRIS-REx lab in Houston. Monitoring was also conducted on a temporary cleanroom constructed inside a hangar at the Utah Test and Training Range (UTTR) to initially receive the Sample Return Capsule (SRC) upon its landing there. The following reports on cleanroom monitoring that has taken place in the months leading up to as well as after OSIRIS-REx return. Monitoring techniques used include particle counts, deployment of Si wafer witness plates (Balazs, Inc.) and Al foil witness plates, gas sampling (Balazs, Inc.) and microbiological monitoring. We collected particle counts at both the lab at JSC as well as the temporary cleanroom at UTTR. Particle counts were taken monthly from six locations in the lab at JSC. Particle counts were taken from seven locations at UTTR in July, August, and September. The UTTR cleanroom particle counts were taken daily in the five days leading to OSIRIS-REx return as well as consistently the day of return (every 2 minutes and 15 seconds 7:21 am- 6:27 pm) from one location inside the cleanroom. The particle counts at UTTR consistently stayed well within the ranges of its required ISO 7 equivalent classification (352,000 maximum particles/ m3 ≥ 0.5 μm). Most analysis showed that the particle counts were far below ISO 7 requirements (highest measured count was 61,484 particles/ m3 ≥ 0.5 um). but the elevated instances correlated to exterior hangar doors being open. Particle counts were reduced when keeping exterior hangar doors closed. The particle counts at JSC have stayed consistently within their ISO 5 equivalent classification (3,520 maximum particles/ m3 ≥ 0.5 um). In most instances, the counts are much better except in one instance where the count measured 13,381 particles/ m3 ≥ 0.5 um. The high spike in particle counts in the JSC lab correlated with the air handlers being shut down temporarily the previous day. The particle counts taken in the same location quickly reduced and measured 0 when taken a month later with the air handlers on. We deployed 8-inch semiconductor polished Si wafer witness plates for 24-hour periods inside of the OSIRIS-REx Curation lab at JSC (in July, August, September, and November 2023) and the cleanroom at UTTR (July and September 2023) to test for possible organic and inorganic contaminants. We also deployed these wafers inside of the OSIRIS-REx Touch-and-Go sample Acquisition Mechanism (TAGSAM) glovebox (where Bennu sample processing occurs) and desiccator (Bennu sample storage) at JSC in August 2023. These samples were all sent to Balazs for analysis via Thermal Desorption Gas Chromatography Mass Spectroscopy (TD-GC-MS) to quantify organic compounds and vapor phase decomposition inductively coupled plasma mass spectrometry (VPD-ICP-MS) to quantify inorganic contaminants. We collected air samples using an adsorbent tube connected to a pump for six hours (100 mL/minute) inside of both the JSC lab and the UTTR cleanroom. These samples were taken at JSC in July, August, September, November, and December and at UTTR in July and September. Additionally, a sample was taken just outside of the TAGSAM glovebox airlock door in July. These samples were sent to Balazs for analysis of volatile organics in air. Overall, the OSIRIS-REx lab at JSC and cleanroom at UTTR yielded very low organic and inorganic contaminants (similar results to Genesis lab at JSC which is ISO 4 equivalent) except for boron, which is attributed to the borosilicate glass in the fan filter units. We collected monthly surface and air samples inside the JSC lab to monitor potential microbial contamination. We report the recovery rate for each sampling event, which is defined as the number of samples exhibiting bacterial or fungal growth divided by the total number of samples collected. Since the lab was commissioned in 2021 the median recovery rate is 29%. This recovery rate is consistent with other ISO 5 equivalent labs used to curate astromaterials collections. Increases in recovery rate correspond to construction and/or extra activity in the lab. However, increases in recovery rate do not reliably correlate to increases in particle counts, which highlights the need for a dedicated microbial monitoring program for biologically sensitive collections. We sampled the temporary clean room at UTTR in July, August, and September of 2023 to monitor potential microbial contamination. The recovery rate decreased from 80% in August to 29% in September. However, in all instances, the diversity of bacteria and fungi was higher in the UTTR cleanroom than in the JSC lab. We routinely collected more than twenty different organisms from the UTTR cleanroom. In the JSC lab the median diversity is 2 organisms. Based on these results we hypothesize that microbes from UTTR could be transported back to JSC with the Bennu samples. To mitigate this risk, in September of 2023, we implemented additional cleaning procedures in the JSC lab to reduce the bioburden on surfaces that could come into direct contact with hardware used to process Bennu samples. Since adopting these additional cleaning measures, the median recovery rate has decreased to 14%. We did not observe an increase in fungal or bacterial diversity in the lab in the October sampling. We will continue to monitor this trend closely for the next several months. Supported by NASA under Award NNH09ZDA007O and Contract NNM10AA11C.

Rachel Comstock Funk

Low-Pressure Generator Makes Cleanrooms Cleaner

Scientists at NASA's Kennedy Space Center work in cleanrooms: laboratories with high degrees of cleanliness provided by strict control of particles such as dust, lint, or human skin. They are contaminant-free facilities, where the air is repeatedly filtered, and surfaces are smooth to prevent particles from getting lodged. Technicians working in these environments wear specially designed cleanroom "bunny suits" and booties over their street clothes, as well as gloves and face masks to avoid any contamination that may be imparted from the outside world. Even normal paper is not allowed in cleanrooms, only cleanroom low-particulate paper. These are sensitive environments where precision work, like the production of silicon chips or hard disk drives, is performed. Often in cleanrooms, positive air pressure is used to force particles outside of the isolated area. The air pressure in the Kennedy cleanrooms is monitored using high-accuracy, low-differential pressure transducers that require periodic calibration. Calibration of the transducers is a tricky business. In prior years, the analysis was performed by sending the transducers to the Kennedy Standards Laboratory, where a very expensive cross-floated, labor- intensive, dead-weight test was conducted. In the early 1990s, scientists at Kennedy determined to develop a technique and find equipment to perform qualification testing on new low-differential pressure transducers in an accurate, cost-effective manner onsite, without requiring an environmentally controlled room. They decided to use the highly accurate, cost-effective Setra Model C264 as the test transducer. For qualification testing of the Setra, though, a portable, lower-cost calibrator was needed that could control the differential pressure to a high degree of resolution and transfer the accuracy of the Standards Laboratory testing to the qualification testing. The researchers decided that, to generate the low-differential pressure setpoints needed for qualification testing, very small gas volume changes could be made against the test article, and a corresponding pressure change would be detected by a pressure standard. This allowed the researchers to recreate cleanroom air pressure settings without the use of a cleanroom. Thus was born the low-differential pressure generator. In 1993, a prototype was developed using a pair of PVC tanks, a volume controller, and a 1-pound-per-square-inch pressure standard. By 1995, the prototype was perfected into the unit that is still used today.

Source record

Dynamic Monitoring of Cleanroom Fallout Using an Air Particle Counter

The particle fallout limitations and periodic allocations for the James Webb Space Telescope are very stringent. Standard prediction methods are complicated by non-linearity and monitoring methods that are insufficiently responsive. A method for dynamically predicting the particle fallout in a cleanroom using air particle counter data was determined by numerical correlation. This method provides a simple linear correlation to both time and air quality, which can be monitored in real time. The summation of effects provides the program better understanding of the cleanliness and assists in the planning of future activities. Definition of fallout rates within a cleanroom during assembly and integration of contamination-sensitive hardware, such as the James Webb Space Telescope, is essential for budgeting purposes. Balancing the activity levels for assembly and test with the particle accumulation rate is paramount. The current approach to predicting particle fallout in a cleanroom assumes a constant air quality based on the rated class of a cleanroom, with adjustments for projected work or exposure times. Actual cleanroom class can also depend on the number of personnel present and the type of activities. A linear correlation of air quality and normalized particle fallout was determined numerically. An air particle counter (standard cleanroom equipment) can be used to monitor the air quality on a real-time basis and determine the "class" of the cleanroom (per FED-STD-209 or ISO-14644). The correlation function provides an area coverage coefficient per class-hour of exposure. The prediction of particle accumulations provides scheduling inputs for activity levels and cleanroom class requirements.

Perry, Radford

Reduced Organic Outgassing in the NASA Osiris-Rex and Hayabusa2 Curation Facility by Careful Selection and Implementation of Cleanroom Construction Materials

In October 2021, NASA Johnson Space Center (JSC) Astromaterials Acquisition and Cu-ration Office in JSC bldg. 31 completed construction and commissioning of the OSIRIS-REx and Hayabusa2cleanroom laboratory suites along with new precision cleaning and advanced curation laboratories: •OSIRIS-REx Curation Cleanroom: ISO Class 5•Hayabusa2 Curation Cleanroom: ISO Class 5•Final Precision Cleaning Cleanroom: ISO Class 5•Advanced Precision Cleaning Cleanroom: ISO Class 6•PreClean Precision Cleaning Cleanroom: ISO Class 6•Advanced Curation Cleanroom: ISO Class 7The new curation facilities are designed for initial receiving, basic characterization, curation processing, and preliminary examination of carbonaceous asteroidal material. The facilities are also designed to enable long-term pristine sample storage to preserve the scientific integrity of each sample to enable decades of future re-search by the international science community. The scientific study of organics in general is critical for both missions. The OSIRIS-REx mission executed a stringent contamination control plan [1] where all sample hardware at time of sample acquisition would be at Level 100 A/2 per IEST-STD-CC1246D (non-volatile residue (NVR) < 500 ng/cm2). In addition, the mission imposed a requirement of <180 ng/cm2 for amino acids (and hydrazine) [1]. Given these mission requirements, long-term storage preservation requirements, and information from the Organic Contamination Baseline Study at JSC [2], the JSC Curation team decided to carefully select cleanroom construction materials that would not hinder the scientific search for amino acids and the study of organics in the samples [3].

astromaterials curation

Operating and Maintaining the JWST Cleanroom attached to JSC's Chamber A during preparations for the OTIS Cryogenic Thermal Vacuum Test

In order to maintain cleanliness during preparations for JWST’s OTIS (Optical Telescope Element-Integrated Science Instrument Module) Cryogenic Thermal Vacuum Test, a cleanroom was built that attached directly to the 60-year-old Chamber A. The cleanroom and chamber were outfitted with independent environmental control systems each providing ISO Class 71 air cleanliness. To maintain balanced, positive pressure in both the cleanroom and chamber volumes, a special control protocol was developed and successfully implemented. Dual back-up environmental control units (one each for the chamber and cleanroom) were installed just outside the building to provide environmental control redundancy due to a single source chilled water supply and weather threats. In addition, lack of a dedicated cleanroom airlock facilitating clean ingress and egress made it necessary to perform additional cleaning and packaging, as well as augment the uncontrolled truck lock space with small clean tents for pre-cleaning. Special procedures were developed to allow ingress of extra-large support equipment required for load testing of the cleanroom crane, installation of optical equipment in Chamber A and accommodation of the OTIS shipping container. A thorough bake-out and cleaning of Chamber A was also necessary to reduce volatiles from the shroud’s black thermal paint and to reduce particle fallout. Acrylic adhesive fracture discovered during early cryo-testing represented a significant challenge that was successfully mitigated prior to OTIS testing. A dedicated team of Contamination Control (CC) Technicians was specifically trained to clean support equipment and screen materials entering the cleanroom and chamber to ensure cleanliness and vacuum compatibility.

James Webb Space Telescope

Lessons Learned From Designing and Building Low Organic Outgassing Cleanrooms for NASA OSIRIS-REx and Hayabusa2 Curation Facility

NASA Johnson Space Center Astromaterials Acquisition and Curation Office completed construction and commissioning of the OSIRIS-REx and Hayabusa2 cleanroom laboratory suites along with new precision cleaning and advanced curation laboratories consisting of ISO Class 5, 6, and 7 cleanrooms. The new curation facilities are designed for initial receiving, basic characterization, curation processing, and preliminary examination of carbonaceous asteroidal material. The facilities are also designed to enable long-term pristine sample storage to preserve the scientific integrity of each sample to enable future research by the international science community. The scientific study of organics is critical for both missions. The OSIRIS-REx mission instituted a stringent contamination control plan for low organics. Given these mission requirements and long-term storage preservation requirements, cleanroom construction materials were carefully selected to not hinder the scientific search for amino acids and the study of organics in the samples. Cleanroom candidate construction materials were researched for the HVAC system, floors, walls, ceiling, and plenum areas. The team chose several candidate materials that were further tested for their specific outgassing characteristics using ASTM E-595 per ASTM E-2312. The information gained provided the foundation for the final design selection of the cleanroom materials. While material selection is important before and during the facility design phase, the construction phase is where these choices are implemented by the prime construction contractor through final product and material submittals. These submittals are the last check point and changes are often submitted due to engineering/construction conflicts, especially when renovating an existing building. Any new material or equipment/product change must be identified and scrutinized for low particulate shedding and outgassing properties. In some cases, quick testing of new material is required, and compromises must be made in real-time. Careful selection and implementation of cleanroom materials significantly reduced organic and inorganic contamination beyond normal cleanroom baselines.

astromaterials curation

Lessons Learned From Designing and Building Low Organic Outgassing Cleanrooms for NASA OSIRIS-REx and Hayabusa2 Curation Facility

NASA Johnson Space Center Astromaterials Acquisition and Curation Office completed construction and commissioning of the OSIRIS-REx and Hayabusa2 cleanroom laboratory suites along with new precision cleaning and advanced curation laboratories consisting of ISO Class 5, 6, and 7 cleanrooms. The new curation facilities are designed for initial receiving, basic characterization, curation processing, and preliminary examination of carbonaceous asteroidal material. The facilities are also designed to enable long-term pristine sample storage to preserve the scientific integrity of each sample to enable future research by the international science community. The scientific study of organics is critical for both missions. The OSIRIS-REx mission instituted a stringent contamination control plan for low organics. Given these mission requirements and long-term storage preservation requirements, cleanroom construction materials were carefully selected to not hinder the scientific search for amino acids and the study of organics in the samples. Cleanroom candidate construction materials were researched for the HVAC system, floors, walls, ceiling, and plenum areas. The team chose several candidate materials that were further tested for their specific outgassing characteristics using ASTM E-595 per ASTM E-2312. The information gained provided the foundation for the final design selection of the cleanroom materials. While material selection is important before and during the facility design phase, the construction phase is where these choices are implemented by the prime construction contractor through final product and material submittals. These submittals are the last check point and changes are often submitted due to engineering/construction conflicts, especially when renovating an existing building. Any new material or equipment/product change must be identified and scrutinized for low particulate shedding and outgassing properties. In some cases, quick testing of new material is required, and compromises must be made in real-time. Careful selection and implementation of cleanroom materials significantly reduced organic and inorganic contamination beyond normal cleanroom baselines.

astromaterials curation

Operating and Maintaining the JWST Cleanroom attached to JSC's Chamber A during preparations for the OTIS Cryogenic Thermal Vacuum Test

In order to maintain cleanliness during preparations for JWST’s OTIS (Optical Telescope Element-Integrated Science Instrument Module) Cryogenic Thermal Vacuum Test, a cleanroom was built that attached directly to the 60-year-old Chamber A. The cleanroom and chamber were outfitted with independent environmental control systems each providing ISO Class 71 air cleanliness. To maintain balanced, positive pressure in both the cleanroom and chamber volumes, a special control protocol was developed and successfully implemented. Dual back-up environmental control units (one each for the chamber and cleanroom) were installed just outside the building to provide environmental control redundancy due to a single source chilled water supply and weather threats. In addition, lack of a dedicated cleanroom airlock facilitating clean ingress and egress made it necessary to perform additional cleaning and packaging, as well as augment the uncontrolled truck lock space with small clean tents for pre-cleaning. Special procedures were developed to allow ingress of extra-large support equipment required for load testing of the cleanroom crane, installation of optical equipment in Chamber A and accommodation of the OTIS shipping container. A thorough bake-out and cleaning of Chamber A was also necessary to reduce volatiles from the shroud’s black thermal paint and to reduce particle fallout. Acrylic adhesive fracture discovered during early cryo-testing represented a significant challenge that was successfully mitigated prior to OTIS testing. A dedicated team of Contamination Control (CC) Technicians was specifically trained to clean support equipment and screen materials entering the cleanroom and chamber to ensure cleanliness and vacuum compatibility.

Optics

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg

The cleanroom case study in the Software Engineering Laboratory: Project description and early analysis

This case study analyzes the application of the cleanroom software development methodology to the development of production software at the NASA/Goddard Space Flight Center. The cleanroom methodology emphasizes human discipline in program verification to produce reliable software products that are right the first time. Preliminary analysis of the cleanroom case study shows that the method can be applied successfully in the FDD environment and may increase staff productivity and product quality. Compared to typical Software Engineering Laboratory (SEL) activities, there is evidence of lower failure rates, a more complete and consistent set of inline code documentation, a different distribution of phase effort activity, and a different growth profile in terms of lines of code developed. The major goals of the study were to: (1) assess the process used in the SEL cleanroom model with respect to team structure, team activities, and effort distribution; (2) analyze the products of the SEL cleanroom model and determine the impact on measures of interest, including reliability, productivity, overall life-cycle cost, and software quality; and (3) analyze the residual products in the application of the SEL cleanroom model, such as fault distribution, error characteristics, system growth, and computer usage.

Green, Scott

Software Engineering Laboratory (SEL) cleanroom process model

The Software Engineering Laboratory (SEL) cleanroom process model is described. The term 'cleanroom' originates in the integrated circuit (IC) production process, where IC's are assembled in dust free 'clean rooms' to prevent the destructive effects of dust. When applying the clean room methodology to the development of software systems, the primary focus is on software defect prevention rather than defect removal. The model is based on data and analysis from previous cleanroom efforts within the SEL and is tailored to serve as a guideline in applying the methodology to future production software efforts. The phases that are part of the process model life cycle from the delivery of requirements to the start of acceptance testing are described. For each defined phase, a set of specific activities is discussed, and the appropriate data flow is described. Pertinent managerial issues, key similarities and differences between the SEL's cleanroom process model and the standard development approach used on SEL projects, and significant lessons learned from prior cleanroom projects are presented. It is intended that the process model described here will be further tailored as additional SEL cleanroom projects are analyzed.

Green, Scott

Preparing for Mars 2020 and Future Space Missions: Technology Advancements Driving Contamination Control Requirements and Cleanroom Protocols

Future space exploration missions will force contamination control requirements to become more strict to support increasingly sensitive instrumentation and search for life missions. Driving issues for these extremely clean requirements include increased instrument sensitivity, return sample science, and protecting ambitious mission science objectives. Preparing to meet these requirements mandates that contamination control provide new guidelines and more involved support for the cleanrooms during flight hardware assembly, including establishing better methods for setting cleanroom personnel limits to reduce particle fall out in cleanrooms. Limited literature exists for universal methods of determining cleanroom personnel limits, and what does exist includes mostly theory and assumptions on determining the limit. In this work, published method will be assessed against particle fall out data collected from the ISO 5 cleanrooms of the Mars 2020 Perseverance Rover assembly. Additional evaluations will assess contamination control required cleanroom protocols and the overall success of meeting strict cleanliness requirements of the Adaptive Caching Assembly (ACA) and sample tubes to safeguard future scientific endeavors.

Chen, Nicole

Establishing Cleaning Methods for Cleanroom and Safety Suits at Remote Sites with Available Resources

"The James Webb Space Telescope (JWST) spent considerable time in multiple clean facilities during its launch campaign at the Centre Spatial Guyanais (CSG) in French Guiana. Throughout this time, it was imperative that personnel wear approved cleanroom or safety suits when working in proximity to the JWST observatory. This proved to be challenging for two reasons: 1) A large quantity of NASA cleanroom suits needed to be shipped to CSG to account for the heavy volume of NASA and ESA personnel requiring access to the clean facilities. It became evident that shipping clean suits from the US to CSG in a timely fashion during the launch campaign would be challenging. Consequently, a backup plan needed to be established to avoid running out. 2) For safety purposes, Self-Contained Atmospheric Protection Ensemble (SCAPE) and Splash suits were required for hazardous operations. Neither type of suit was cleanroom compatible; therefore, a viable cleaning process needed to be developed to ensure both types of suits met JWST’s approved cleanliness standards. The NASA Contamination Control (CC) team partnered with CSG’s S5E cleanroom and suit processing team. The teams were able to make modifications to the S5E facility to create a NASA approved makeshift clean environment where NASA cleanroom suits and CSG Splash suits were inspected, folded, and bagged for cleanroom use once they were washed and dried. Since SCAPE suits could not be cleaned using conventional methods, a procedure was developed to clean and inspect the suits by the CC team in a temporary clean environment prior to fueling. "

James Webb Space Telescope

Establishing Cleaning Methods For Cleanroom And Safety Suits At Remote Sites With Available Resources

The James Webb Space Telescope (JWST) spent considerable time in multiple clean facilities during its launch campaign at the Centre Spatial Guyanais (CSG) in French Guiana. Throughout this time, it was imperative that personnel wear approved cleanroom or safety suits when working in proximity to the JWST observatory. This proved to be challenging for two reasons: 1) A large quantity of NASA cleanroom suits needed to be shipped to CSG to account for the heavy volume of NASA and ESA personnel requiring access to the clean facilities. It became evident that shipping clean suits from the US to CSG in a timely fashion during the launch campaign would be challenging. Consequently, a backup plan needed to be established to avoid running out. 2) For safety purposes, Self-Contained Atmospheric Protection Ensemble (SCAPE) and Splash suits were required for hazardous operations. Neither type of suit was cleanroom compatible; therefore, a viable cleaning process needed to be developed to ensure both types of suits met JWST’s approved cleanliness standards. The NASA Contamination Control (CC) team partnered with CSG’s S5E cleanroom and suit processing team. The teams were able to make modifications to the S5E facility to create a NASA approved makeshift clean environment where NASA cleanroom suits and CSG Splash suits were inspected, folded, and bagged for cleanroom use once they were washed and dried. Since SCAPE suits could not be cleaned using conventional methods, a procedure was developed to clean and inspect the suits by the CC team in a temporary clean environment prior to fueling.

James Webb Space Telescope