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NASA NTRS2019

A Lab with a View: Findings from NASA's Aircraft Bioaerosol Collector

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

bioaerosols↗
NASA NTRS2008

Effect of shadowing on survival of bacteria under conditions simulating the Martian atmosphere and UV radiation

Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (~5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.

Venkateswaran, Kasthuri↗
NASA NTRS2021

Characterization of Spacesuit Associated Microbial Communities and their Implications for NASA Missions

Crewed National Aeronautics and Space Administration (NASA) missions to other solar system bodies are currently being planned. One high-profile scientific focus during such expeditions would be life detection, specifically the discovery of past or present microbial life, if they exist. However, both humans and associated objects typically carry a high microbial burden. Thus, it is essential to distinguish between microbes brought with the expedition and those present on the exploring planets. Modern spacesuits are unique, customized spacecraft which provide protection, mobility and life support to crew during spacewalks, yet they vent continuously, and the mobility of microbes through spacesuits has not been studied. To evaluate the microbial colonization of spacesuits, NASA used an Extravehicular Activity swab kit to examine viable microbial populations of 48 samples from spacesuits using both traditional microbiological methods and molecular sequencing methods. The cultivable microbial population ranged from below the detection limit to 9 x 102 colony forming units per 25 cm2 of sample and also significantly varied by the location. The cultivable microbial diversity was dominated by members of Bacillus, Arthrobacter, and Ascomycota. However, 16S rRNA-based viable bacterial burden ranged from 105 to 106 copies per 25 cm2 of sample. Shotgun metagenome sequencing revealed the presence of a diverse microbial population on the spacesuit surfaces, including Curtobacterium and Methylobacterium from across all sets of spacesuits in high abundance. Among bacterial species identified, higher abundance of Cutibacterium acnes, Methylobacterium oryzae, and M. phyllosphaerae reads were documented. The results of this study provide evidence that identical microbial strains may live on the wrist joint, inner gauntlet, and outer gauntlet of spacesuits. This raises the possibility, but does not confirm, that viable microbes are able to migrate between the interior and exterior of spacesuits. Overall, these data provide the first estimate of microbial dispersion from internal to external spacesuit surfaces, which will help future mission planners develop effective planetary protection strategies.

spacesuit↗
NASA NTRSDate not supplied

EVA Swab Kit: Tools and Techniques for Collecting Aseptic Samples from Crewed Space Missions

Introduction: When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed spacecraft meet planetary protection requirements—and to protect our science from human contamination—we'll need to assess and verify whether micro-organisms may be leaking/venting from our spacesuits. This requires collecting samples under Extravehicular Activity (EVA) conditions. Detailed, systematic research on forward contamination from robotic spacecraft has been steadily progressing since the Viking missions, but systematic studies of contamination from space suits has not been conducted in many years. The modern EMU (Extravehicular Mobility Unit) suit used by NASA is designed to leak at rates as high as 100 cc/min. Before humans land on Mars there is a critical need to understand the types and quantities of microbes that could be introduced via space suits. The Human Forward Contamination Assessment team at NASA’s Johnson Space Center (JSC) has developed a prototype EVA swab tool [1,2,3,4] designed for use in space to sample cleaned and uncleaned space suits to determine the present day microbial load and eventually the rate of leakage. The ability to assess microbial leakage early in advanced space suit and life support system design cycles will help avoid costly hardware redesign later. Test Objectives: The primary objective of EMU testing was to characterize the type of micro-organisms typically found on or near selected suit pressure joints under suit differential pressure conditions. Most human-borne microbes can fit through a 0.5 to 1.0 µm gap. Knowing which joints are more likely to leak will inform hardware design decisions. Knowing which types of micro-organisms may leak from EVA suits provides a basis for subsequent studies to characterize the viability of those organisms under destination conditions, as well as how far they might spread through natural or human-influenced processes. That data, in turn, will inform exploration mission operations and hardware design. The secondary objective of testing was to evaluate the interface between a fully suited test subject and the EVA swab tool at vacuum. Bulky EVA suits can restrict movement and limit visibility through the helmet visor. Fully suited testing is important for identifying tool design issues prior to flight. At exploration destinations, such as Mars, suited crew may be required to periodically sample their suits as part of an environmental monitoring protocol. Suit Microbial Sampling Results: This report details results of microbial swabs collected from current flight suit configurations worn by crew members assigned to upcoming ISS expedition missions as well as swabs collected from prototype suits intended for use on the Orion spacecraft. These tests were intended to characterize the types of contaminants found on flight suits under current, typical handling conditions. No attempt was made to change suit handling procedures, provide additional sterilization, or to limit typical potential contaminant sources. Using culture based techniques, we cultivated 235 CFU (colony forming units) comprised of 26 bacterial species and one fungal species on the outside of the suits. The fungal species and 14 of the bacterial species were unique to the suit surfaces and were not detected in any of the background samples collected within the chambers. We sequenced 755,434 ribosomal fragments on all of the suit surfaces from swab samples. 557,016 of these sequences represent DNA that survived at least 4 hours at vacuum. These sequences formed 2,464 OTU's (Operational Taxonomic Units, 97% similarity) showing low diversity in the samples. The most abundant sequences that survived vacuum belong to the genera Staphyloccocus, Ralstona, Bacillus and Rhodobacter all of which are common to the human microbiome. [5] See Danko et al., (2021) for more complete details of these first analyses. Further analysis of EVA suit materials with respect to the efficacy of various cleaning protocols and engineered containment solutions is planned to inform suit design for NASA’s Artemis Moon to Mars program crew testing. Swab Tool Function Results: The kit was demonstrated for fit and function in suited subject vacuum tests to determine how well the tool worked as an aseptic microbial sampling device as well as to identify any design elements that could be upgraded for EVA task specific improvement. It was found that sample acquisition efficacy could be enhanced by redesign of the sample canister to end-effector interface. Several modifications of the sample caddy assemblies to optimize EVA safety and functionality were also identified. Consequently, fabrication of the redesigned sample canister to end-effector assembly interfaces and and the sample caddy assemblies are required. Fabrication of sixteen flight sample canister assemblies (8 per each of two EVA Swab Kits) and two sample caddy assemblies are in process to be followed by hardware testing and certification to produce two flight-certified EVA Swab Kits for transport to ISS no earlier than summer of 2022. Sampling Strategy: The International Space Station is an ideal testbed for systematic studies of contamination from crewed vehicles since it has been continuously occupied for 20 years and exposed to non-terrestrial conditions. We will sample the exterior of the ISS during EVA using a purpose-built swab tool capable of maintaining sterility while undergoing temperature changes from -151 to +121°C under hard vacuum. Prior to each EVA, the project team will work with ISS mission managers to identify precise sampling locations, which will vary by EVA based on the translation paths and worksites scheduled for that particular EVA. Ideally, translation path handrails and areas near ECLSS (Environmental Control and Life Support System) external vent openings on a spacecraft would be assessed. There are currently more than a dozen ECLSS external vents on the ISS. Some are connected to systems that vent waste products, while others are intended to equalize cabin pressure. As EVA opportunity allows, microbial samples from any of these external vents would provide a valuable data point, though some will be more useful than others. Four criteria have been identified to help prioritize sampling sites near vents: • EVA Accessibility: To minimize cost, it is desired to piggy-back onto a planned EVA. Therefore, the sampling location must be readily accessible by an EVA crew • Type of Vented Products: Vent products that have been in direct contact with crew, such as cabin air, are more likely to contain microorganisms than vent products associated with isolated systems, such as experiment module combustion products. • Mass of Vented Products: Higher-flow vents are more likely to contain detectible levels of microbial contaminants than lower-flow vents. • Local Environment: Sample locations with relatively benign local conditions, such as warm surfaces shielded from direct ultraviolet (UV) radiation exposure, may be more likely to support microbial growth than locations with harsher local environmental conditions. Because EVA accessibility is the most important criteria, the proposal team worked with an astronaut and flight controllers using the Dynamic Onboard Ubiquitous Graphics (DOUG) tool. The DOUG virtual environment allows an operator to “fly” around the current ISS vehicle configuration to assess EVA translation paths, attach points, and keep-out zones. While analysis on station or rapid return to Earth would be preferable, samples collected from the exterior of the ISS have already been exposed to temperature variations between -157 and +121 °C as well as hard vacuum. Therefore, they should be fairly stable and robust. We hypothesize that samples collected from the ISS exterior could be stored for up to 6 months at -80°C without degradation. Sample canisters will be returned to Earth while frozen at -80°C for analysis, and sterilized canisters can be re-flown back to ISS to support additional sampling opportunities Relevance to NASA Exploration Objectives: These data will allow us to identify new or improved methods, technologies, and procedures for spacecraft sterilization and leakage mitigation to minimize the amount of contamination introduced to the environment by human explorers. This work is funded by NASA research grant: NNH18ZDA001N-PPR References: [1] Bell, M.S. et al. (2015) LPS XLVI, Abst. #1832 [2] Rucker et al. (2018) 42nd COSPAR (PPP.3) [3] Bell, M.S. et al. (2019) Mars Extant Life Conference, Abst. #5096.[4] Bell, M.S. et al., (2020) 43rd COSPAR (BO.2).[5] Danko D, et.al.,(2021)Front.Microbiol.12:608478.

Mary Suzanne Bell↗
NASA NTRS2020

Prokaryotic and Fungal Characterization of the Facilities Used to Assemble, Test, and Launch the OSIRIS-REx Spacecraft

To characterize the ATLO (Assembly, Test, and Launch Operations) environment of the OSIRIS-REx spacecraft, we analyzed 17 aluminum witness foils and two blanks for bacterial, archaeal, fungal, and arthropod DNA. Under NASA’s Planetary Protection guidelines, OSIRIS-REx is a Category II outbound, Category V unrestricted sample return mission. As a result, it has no bioburden restrictions. However, the mission does have strict organic contamination requirements to achieve its primary objective of returning pristine carbonaceous asteroid regolith to Earth. Its target, near-Earth asteroid (101955) Bennu, is likely to contain organic compounds that are biologically available. Therefore, it is useful to understand what organisms were present during ATLO as part of the larger contamination knowledge effort—even though it is unlikely that any of the organisms will survive the multi-year deep space journey. Even though these samples of opportunity were not collected or preserved for DNA analysis, we successfully amplified bacterial and archaeal DNA (16S rRNA gene) from 16 of the 17 witness foils containing as few as 7 ± 3 cells per sample. Fungal DNA (ITS1) was detected in 12 of the 17 witness foils. Despite observing arthropods in some of the ATLO facilities, arthropod DNA (COI gene) was not detected. We observed 1,009 bacterial and archaeal sOTUs (sub-operational taxonomic units, 100% unique) and 167 fungal sOTUs across all of our samples (25–84 sOTUs per sample). The most abundant bacterial sOTU belonged to the genus Bacillus. This sOTU was present in blanks and may represent contamination during sample handling or storage. The sample collected from inside the fairing just prior to launch contained several unique bacterial and fungal sOTUs that describe previously uncharacterized potential for contamination during the final phase of ATLO. Additionally, fungal richness (number of sOTUs) negatively correlates with the number of carbon-bearing particles detected on samples. The total number of fungal sequences positively correlates with total amino acid concentration. These results demonstrate that it is possible to use samples of opportunity to characterize the microbiology of low-biomass environments while also revealing the limitations imposed by sample collection and preservation methods not specifically designed with biology in mind.

Aaron B. Regberg↗
NASA NTRSDate not supplied

Planetary Protection at Marshall Space Flight Center

Introduction: NASA Marshall Space Flight Center (MSFC) is historically known for its role in propulsion. While this is still the mainstay of MSFC’s expertise, many unique capabilities exist at MSFC which pertain to Planetary Protection (PP), including 1) identifying PP threats, and 2) developing novel methods to neutralize those threats. Furthermore, because these capabilities exist among diverse groups at MSFC, this work promotes collaboration both within and outside MSFC to expand and develop PP studies related to a full spectrum of NASA research, design, manufacture, and test interests. This abstract describes the PP research ongoing at MSFC and describes how it contributes to NASA’s overall PP objectives. Microbial Identification in Cleanrooms: One of the greatest threats to successful implementation of PP requirements is recontamination post bioburden reduction. One method to prevent recontamination is to keep the spacecraft in clean environments (i.e. cleanrooms) as much as possible during assembly and integration. However, cleanrooms are not without their own sources of contamination, which is why NASA is interested in monitoring the cleanliness of cleanrooms and characterizing the microbial species present. Such information allows a greater understanding of the resistance of these microbes to cleaning methods, as well as the risk of their contaminating the targeted planetary body of a given mission. MSFC has multiple cleanrooms of various ISO cleanliness levels onsite. We sampled the air and surfaces of three of these rooms, isolated microbes, and then sequenced the 16S rRNA gene or ITS region of the 18S rRNA gene for bacterial and fungal isolates, respectively. This has resulted in a microbial library which currently includes nearly 100 isolates. Microbial Enumeration of Spacecraft Materials: Currently, there are only a couple bioburden reduction methods approved by NASA, and often the harshness of these methods presents additional concerns or risks related to material properties. The goal of this research is to assess the microbial content of solid rocket motor (SRM) materials potentially used for lander missions. This work aims to more accurately define the risk of planetary contamination by providing empirical data associated with commonly used SRM raw materials. In this study, we pulverized nonmetallic SRM materials using a cryogenic grinder, then analyzed the resulting substrate for microbial colony forming units (CFU). We found that many SRM nonmetallic materials do not harbor detectable bioburden, though a range existed depending on the material. The results from this work provide quantitative data to potentially reduce concerns of contamination, while also providing a foundation for follow up studies into additional sterilization methods and molecular identification of contaminating microbes. Space Environmental Effects on Microbial Survival: One potential area of microbial reduction is the space environment. Understanding the survivability of hardy microbes in space-like conditions is a crucial first step in answering how space may reduce bioburden and if it can be relied upon for adherence to PP requirements. This work studied the effects of ultraviolet (UV) and ionizing radiation on survival of Bacillus atrophaeus spores. Microbes were dried on relevant polymeric materials then exposed to space environmental stressors. Coupons were submerged in water, diluted, and plated to determine survival compared with controls. We found that both UV and ionizing radiation were capable of reducing viability by nearly 99%, but there were still survivors, some with changed morphology indicating resistance mechanisms within certain cells. Manufacturing credit: Finally, given the above-mentioned limitations of the NASA-approved bioburden reduction methods, there is interest in understanding if manufacturing processes may provide enough bioburden reduction without additional PP-specific bakeouts. For instance, some material additives may be antimicrobial. Given this, we investigated the effects of several commonly used rubber additives on the growth of B. atrophaeus spores. We found that some of the materials inhibited growth of the spores, possibly supporting the use of these additives on missions with PP constraints. Future work into manufacturing credit for bioburden reduction includes inoculation of green insulation with B. atrophaeus spores, followed by a typical cure. Thermal profiles will be verified for appropriate temperature and durations to meet PP requirements, and cured samples will be analyzed using a cryogenic grinder to determine survivability of spores.

Chelsi D. Cassilly↗
NASA NTRSDate not supplied

Microbial Methods for Testing a Novel Spacecraft Bioburden Reduction Method

In order to circumvent the contamination of microorganisms in both extraterrestrial and terrestrial environments, spacecraft hardware must often be sterilized. Current sterilization practices are both time-consuming and expensive. Techniques such as dry heat sterilization and scattered/localized electric field enhancements are incompatible with a variety of hardware materials as well as complex surface shapes. Our group is testing a novel femtosecond pulsed laser for the rapid sterilization of spacecraft hardware using high photon fluxes, which is both an efficient and effective technique to ensure a reduced microbial burden aboard spacecraft. This method is likely to be effective because microorganisms have not encountered high photon fluxes in their evolutionary history and thus contain no protective mechanisms against them. To assess the effectiveness of pulsed laser sterilization, we used a combination of scanning electron microscopy and elemental analysis for the examination of damaged spores on metal coupons, as well as microbiology methods for spore assays. The surfaces of metal coupons inoculated with Bacillus subtilis were illuminated with femtosecond pulses at various wavelengths below the damage threshold for the aluminum. After laser treatment, metal coupon samples were sonicated to dislodge spores, serially diluted, and finally plated and incubated. Results show that sterilization efficiency is favorable in some conditions. The NASA standards for spore assays are a preliminary demonstration in establishing the trend in ability to kill spores. The results show potential for the dual pulse illumination method as a sterilization technique for planetary protection applications.

Maha S Ulhaq↗
NASA NTRSDate not supplied

Spaceflight Autonomous Multigenerational Microbial Sequencer in Support of Plant-Growth Systems

The CubeSat platform has proven successful in obtaining meaningful life science information when biological payloads are incorporated. Examples include: 1) the first-ever CubeSat with a biological payload, GeneSat-1, which demonstrated decreased growth rates for flight samples of Escherichia coli in low Earth orbit (Parra et al. 2008); 2) PharmaSat, demonstrated that Saccharomyces cerevisiae in the microgravity environment exhibits a significant level of metabolic activity even at high doses of applied antifungal (Ricco et al. 2011); 3) O/OREOS, which used Bacillus subtilis(bacteria) to demonstrate for the first time that microorganisms can be loaded in a dried, dormant form and then rehydrated and grown in orbit months after launch (Nicholson et al. 2011; Ehrenfreund et al. 2014; 4) the SporeSat payload, which investigated Ceratopteris richardii(fern spores) using lab-on-a-chip devices (BioCDs) and minicentrifuges to produce artificial gravitational forces in ground studies (Park et al. 2017), with demonstration of the BioCD and minicentrifuge in space; 5) EcAMSat, the first CubeSat to be directly deployed from the ISS for an experiment assessing antibiotic resistance of E. coli in the microgravity environment (Padgen et al. 2020); 6) BioSentinel, exposed a culture of yeast to galactic cosmic radiation (GCR) and solar particle events while in heliocentric orbit to measure the rate of double-strand-break repair using DNA-repair-deficient mutants. This effort measures the metabolic parameters of yeast in a deep-space environment compared to Earth ambient conditions using a 3-color LED detection system (Ricco et al. 2020; Padgen et al. 2021). We aim to expand this list to include a Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS). SAMMS will allow for the genome level understanding of changes in growth and metabolic activity for any organism. While microbes are suitable for early studies in our proposed platform because of their small size, small and relatively less-complicated genomes, fast generation times, and relevance to life support systems; multicellular organisms can similarly be evaluated for their genetic response to the spaceflight environment. The Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS) will enable autonomous sequencing of biological samples in plant production units, cislunar orbit and on the lunar surface to examine spaceflight effects (ie. radiation, altered gravity, reduced pressures) on plant and microbial genomes.On this team a Kennedy Space Center (KSC) space crop production and water systems microbiologist/molecular biologist works with a Johnson Space Center (JSC) International Space Station (ISS) microbial sequencing expert and an Ames Research Center (ARC) CubeSat Engineering team to convert an automated Oxford Nanopore librarypreparation and sequencing method to a fluidic CubeSat payload system. The Oxford Nanopore MinION sequencing platform has proven successful in the spaceflight environment onboard the ISS (Stahl-Rommel et al. 2021). Further long-duration spaceflight and exposure to high levels of radiation will cause genotypic effects in biological organisms that may affect their function. Monitoring the adaption of a population to the spaceflight environment and any subsequent beneficial mutations will allow for the harnessing of organisms best suited for use in life support systems. This will ensure that the selected life support-essential microorganisms maintain their intended specified function over generations of culturing in the relevant spaceflight environment without becoming hazardous to crew or spacecraft systems.

Aubrie E Orourke↗
NASA NTRSDate not supplied

Microbial Survival in Brewed Tea

Loose-leaf bagged tea is a commonly requested crew food item that comprises up to 7% of samples in the spaceflight food system. Tea preferences are typically specific and vary between different crew members, and there is significant interest to provide preferred food selections for the physical and mental wellbeing of the crew. Loose leaf bagged teas are made from herbs and spices that frequently have high microbial loads which do not meet the microbiological standards outlined in NASA-STD-3001. The food industry on earth does not require standards for microbial load of loose-leaf teas, because tea is brewed in boiling (100C) water, which is assumed to reduce the number of microorganisms. However, hot water from the ISS PWD is typically dispensed at a temperature between 155F (68C) and 175F (79C), which is below the suggested temperature range for safe brewing (180F/80C – 212F/100C). In this study, a commonly requested chamomile tea bags were infected with Bacillus cereus, a pathogenic, spore forming bacterium known to cause foodborne illness. Brewing B. cereus infected tea bags with boiling (212F/100C) or ISS PWD (155F/68C) temperature water showed no significant reduction in the overall microbial load of the tea (p=0.535). The addition of boiling and ISS PWD temperature water also was not sufficient to kill 100% of the infected B. cereus, and in one case of ISS PWD temperature water, B. cereus was detected at levels above a previously reported infectious dose. This study showed that the microbiological risk associated with drinking brewed tea on ISS is not reduced by the brewing process.

S G Thornhill↗
NASA NTRSDate not supplied

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↗
NASA NTRS2024

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↗
NASA NTRSDate not supplied

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↗
DOE OSTI2021

Application of the Metabolic Modeling Pipeline in KBase to Categorize Reactions, Predict Essential Genes, and Predict Pathways in an Isolate Genome

The DOE Systems Biology Knowledgebase (KBase) platform offers a range of powerful tools for the reconstruction, refinement, and analysis of genome-scale metabolic models built from microbial isolate genomes. In this chapter, we describe and demonstrate these tools in action with an analysis of isoprene production in the Bacillus subtilis DSM genome. Two different methods are applied to build initial metabolic models for the DSM genome, then the models are gapfilled in three different growth conditions. Next, flux balance analysis (FBA) and flux variability analysis (FVA) techniques are applied to both study the growth of these models in minimal media and classify reactions within each model based on essentiality and functionality. The models are applied with the FBA method to predict essential genes, which are then compared to an updated list of essential genes obtained for B. subtilis 168, a very similar strain to the DSM isolate. The models are also applied to simulate Biolog growth conditions, and these results are compared with Biolog data collected for B. subtilis 168. Finally, the DSM metabolic models are applied to explore the pathways and genes responsible for producing isoprene in this strain. These studies demonstrate the accuracy and utility of models generated from the KBase pipelines, as well as exploring the tools available for analyzing these models.

Allen, Benjamin↗
DOE OSTI2020

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index

In recent decades, the taxonomy of Bacteria and Archaea , and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Microbiomes of commercially-available pine nuts and sesame seeds

Metagenomic analysis of food is becoming more routine and can provide important information pertaining to the shelf life potential and the safety of these products. However, less information is available on the microbiomes associated with low water activity foods. Pine nuts and sesame seeds, and food products which contain these ingredients, have been associated with recalls due to contamination with bacterial foodborne pathogens. The objective of this study was to identify the microbial community of pine nuts and sesame seeds using targeted 16S rRNA sequencing technology. Ten different brands of each seed type were assessed, and core microbiomes were determined. A total of 21 and 16 unique taxa with proportional abundances >1% in at least one brand were identified in the pine nuts and sesame seeds, respectively. Members of the core pine nut microbiome included the genera Alishewanella, Aminivibrio, Mycoplasma, Streptococcus, and unassigned OTUs in the families of Desulfobacteraceae and Xanthomonadaceae. For sesame seeds, the core microbiome included Aminivibrio, Chryseolina, Okibacterium, and unassigned OTUs in the family Flavobacteriaceae. The microbiomes of these seeds revealed that these products are dominated by environmental bacterial genera commonly isolated from soil, water, and plants; bacterial genera containing species known as commensal organisms were also identified. Understanding these microbiomes can aid in the risk assessment of these products by identifying food spoilage potential and community members which may co-enrich with foodborne bacterial pathogens.

59 BASIC BIOLOGICAL SCIENCES↗