NASA Planetary Protection Research Articles

Genomic characterization and radiation tolerance of Naganishia kalamii sp. nov. and Cystobasidium onofrii sp. nov. from Mars 2020 mission assembly facilities

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

The Isolation and Characterization of Rare Mycobiome Associated With Spacecraft Assembly Cleanrooms.

Ensuring biological cleanliness while assembling and launching spacecraft is critical for robotic exploration of the solar system. To date, when preventing forward contamination of other celestial bodies, NASA Planetary Protection policies have focused on endospore-forming bacteria while fungi were neglected. In this study, for the first time the mycobiome of two spacecraft assembly facilities at Jet Propulsion Laboratory (JPL) and Kennedy Space Center (KSC) was assessed using both cultivation and sequencing techniques. To facilitate enumeration of viable fungal populations and downstream molecular analyses, collected samples were first treated with chloramphenicol for 24 h and then with propidium monoazide (PMA). Among cultivable fungi, 28 distinct species were observed, 16 at JPL and 16 at KSC facilities, while 13 isolates were potentially novel species. Only four isolated species Aureobasidium melanogenum, Penicillium fuscoglaucum, Penicillium decumbens, and Zalaria obscura were present in both cleanroom facilities, which suggests that mycobiomes differ significantly between distant locations. To better visualize the biogeography of all isolated strains the network analysis was undertaken and confirmed higher abundance of Malassezia globosa and Cyberlindnera jadinii. When amplicon sequencing was performed, JPL-SAF and KSC-PHSF showed differing mycobiomes. Metagenomic fungal reads were dominated by Ascomycota (91%) and Basidiomycota (7.15%). Similar to amplicon sequencing, the number of fungal reads changed following antibiotic treatment in both cleanrooms; however, the opposite trends were observed. Alas, treatment with the antibiotic did not allow for definitive ascribing changes observed in fungal populations between treated and untreated samples in both cleanrooms. Rather, these substantial differences in fungal abundance might be attributed to several factors, including the geographical location, climate and the in-house cleaning procedures used to maintain the cleanrooms. This study is a first step in characterizing cultivable and viable fungal populations in cleanrooms to assess fungal potential as biocontaminants during interplanetary explorations. The outcomes of this and future studies could be implemented in other cleanrooms that require to reduce microbial burden, like intensive care units, operating rooms, or cleanrooms in the semiconducting and pharmaceutical industries.

Performance of Multiple Metagenomics Pipelines in Understanding Microbial Diversity of a Low-Biomass Spacecraft Assembly Facility.

NASA planetary protection (PP) requires an assessment of the biological contamination of the potential microbial burden on spacecraft destined to explore planetary bodies that may harbor signs of life, like Mars and Europa. To help meet these goals, the performance of multiple metagenomic pipelines were compared and assessed for their ability to detect microbial diversity of a low-biomass clean room environment used to build spacecraft destined to these planetary bodies. Four vendors were chosen to implement their own metagenomic analysis pipeline on the shotgun sequences retrieved from environmental surfaces in the relevant environments at NASA’s Jet Propulsion Laboratory. None of the vendors showed the same microbial profile patterns when analyzing same raw dataset since each vendor used different pipelines, which begs the question of the validity of a single pipeline to be recommended for future NASA missions. All four vendors detected species of interest, including spore-forming and extremotolerant bacteria, that have the potential to hitch-hike on spacecraft and contaminate the planetary bodies explored. Some vendors demonstrated through functional analysis of the metagenomes that the molecular mechanisms for spore-formation and extremotolerance were represented in the data. However, relative abundances of these microorganisms varied drastically between vendor analyses, questioning the ability of these pipelines to quantify the number of PP-relevant microorganisms on a spacecraft surface. Metagenomics offers tantalizing access to the genetic and functional potential of a microbial community that may offer NASA a viable method for microbial burden assays for planetary protection purposes. However, future development of technologies such as streamlining the processing of shotgun metagenome sequence data, long read sequencing, and all-inclusive larger curated and annotated microbial genome databases will be required to validate and translate relative abundances into an actionable assessment of PP-related microbes of interest. Additionally, the future development of machine learning and artificial intelligence techniques could help enhance the quality of these metagenomic analyses by providing more accurate identification of the genetic and functional potential of a microbial community.

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.

Planetary protection technologies for planetary science instruments, spacecraft, and missions: Report of the NASA Planetary Protection Technology Definition Team (PPTDT)

Planetary bodies like Mars, Europa, and Enceladus pose the question, “How to study them without contaminating them and destroying future prospects to detect life, if it is there?” The natural trade-off, of course, is that the cleaner your spacecraft, the more you can explore such a body without risk of contaminating it. As chartered by NASA Headquarters, the Planetary Protection Technology Definition Team (PPTDT) was asked to provide a report covering six different areas related to the engineering and technology challenges of implementing planetary protection requirements on solar system exploration missions:•Assessment of technical and engineering challenges to applying available microbial-reduction methods, including recontamination prevention, to spacecraft hardware and instruments, to meet current NASA requirements on preventing the forward contamination of potentially habitable worlds by future spacecraft missions (orbiters, atmospheric missions, landers, penetrators, and drills);•Identification of spacecraft and instrument materials known to be compatible with existing planetary protection protocols;•Planetary protection protocols/processes available or which appear promising, and areas ripe for technological development;•The technical and engineering challenges in ensuring that spacecraft hardware and instruments can meet organic cleanliness requirements needed to ensure high confidence in differentiating Earth contamination from extraterrestrial signals to avoid false negative as well as false positive results;•Approaches for mitigating the identified challenges that would allow instruments to be flown successfully at the required levels of cleanliness and microbial reduction, beginning with identification of commonly used materials and spacecraft hardware that are compatible (or particularly vulnerable) to planetary protection protocols; and•Engineering, technology, and scientific research and development that could be funded by NASA to provide future capabilities to field scientific instruments and spacecraft on missions that require either subsystem or system-level microbial reduction and recontamination prevention.

NASA planetary protection officer suggests loosening limits on exploring Mars for life

NASA planetary protection officer suggests loosening limits on exploring Mars for life

Reaching 1 m Deep on Mars: The Icebreaker Drill

The future exploration of Mars will require access to the subsurface, along with acquisition of samples for scientific analysis and ground-truthing of water ice and mineral reserves for in situ resource utilization. The Icebreaker drill is an integral part of the Icebreaker mission concept to search for life in ice-rich regions on Mars. Since the mission targets Mars Special Regions as defined by the Committee on Space Research (COSPAR), the drill has to meet the appropriate cleanliness standards as requested by NASA's Planetary Protection Office. In addition, the Icebreaker mission carries life-detection instruments; and in turn, the drill and sample delivery system have to meet stringent contamination requirements to prevent false positives. This paper reports on the development and testing of the Icebreaker drill, a 1 m class rotary-percussive drill and triple redundant sample delivery system. The drill acquires subsurface samples in short, approximately 10 cm bites, which makes the sampling system robust and prevents thawing and phase changes in the target materials. Autonomous drilling, sample acquisition, and sample transfer have been successfully demonstrated in Mars analog environments in the Arctic and the Antarctic Dry Valleys, as well as in a Mars environmental chamber. In all environments, the drill has been shown to perform at the "1-1-100-100" level; that is, it drilled to 1 m depth in approximately 1 hour with less than 100 N weight on bit and approximately 100 W of power. The drilled substrate varied and included pure ice, ice-rich regolith with and without rocks and with and without 2% perchlorate, and whole rocks. The drill is currently at a Technology Readiness Level (TRL) of 5. The next-generation Icebreaker drill weighs 10 kg, which is representative of the flightlike model at TRL 5/6.

The statistical treatment implemented to obtain the planetary protection bioburdens for the Mars Science Laboratory mission

NASA Planetary Protection Policy requires that Category IV missions such as those going to the surface of Mars include detailed assessment and documentation of the bioburden on the spacecraft at launch. In the prior missions to Mars, the approaches used to estimate the bioburden could easily be conservative without penalizing the project because spacecraft elements such as the descent and landing stages had relatively small surface areas and volumes. With the advent of a large spacecraft such as Mars Science Laboratory (MSL), it became necessary for a modified—still conservative but more pragmatic—statistical treatment be used to obtain the standard deviations and the bioburden densities at about the 99.9% confidence limits. This article describes both the Gaussian and Poisson statistics that were implemented to analyze the bioburden data from the MSL spacecraft prior to launch. The standard deviations were weighted by the areas sampled with each swab or wipe. Some typical cases are given and discussed.

NASA's Planetary Protection Officer

NASA's Planetary Protection Officer

Setting a Standard: TheLimulusAmebocyte Lysate Assay and the Assessment of Microbial Contamination on Spacecraft Surfaces

Historically, colony-forming units as determined by plate cultures have been the standard unit for microbiological analysis of environmental samples, medical diagnostics, and products for human use. However, the time and materials required make plate cultures expensive and potentially hazardous in the closed environments of future NASA missions aboard the International Space Station and missions to other Solar System targets. The Limulus Amebocyte Lysate (LAL) assay is an established method for ensuring the sterility and cleanliness of samples in the meat-packing and pharmaceutical industries. Each of these industries has verified numerical requirements for the correct interpretation of results from this assay. The LAL assay is a rapid, point-of-use, verified assay that has already been approved by NASA Planetary Protection as an alternate, molecular method for the examination of outbound spacecraft. We hypothesize that standards for molecular techniques, similar to those used by the pharmaceutical and meat-packing industries, need to be set by space agencies to ensure accurate data interpretation and subsequent decision making. In support of this idea, we present research that has been conducted to relate the LAL assay to plate cultures, and we recommend values obtained from these investigations that could assist in interpretation and analysis of data obtained from the LAL assay.

Vapor hydrogen peroxide as alternative to dry heat microbial reduction

The Jet Propulsion Laboratory (JPL), in conjunction with the NASA Planetary Protection Officer, has selected vapor phase hydrogen peroxide (VHP) sterilization process for continued development as a NASA approved sterilization technique for spacecraft subsystems and systems. The goal was to include this technique, with an appropriate specification, in NASA Procedural Requirements 8020.12 as a low-temperature complementary technique to the dry heat sterilization process. The VHP process is widely used by the medical industry to sterilize surgical instruments and biomedical devices, but high doses of VHP may degrade the performance of flight hardware, or compromise material compatibility. The goal for this study was to determine the minimum VHP process conditions for planetary protection acceptable microbial reduction levels. Experiments were conducted by the STERIS Corporation, under contract to JPL, to evaluate the effectiveness of vapor hydrogen peroxide for the inactivation of the standard spore challenge, Geobacillus stearothermophilus. VHP process parameters were determined that provide significant reductions in spore viability while allowing survival of sufficient spores for statistically significant enumeration. In addition to the obvious process parameters of interest: hydrogen peroxide concentration, number of injection cycles, and exposure duration, the investigation also considered the possible effect on lethality of environmental parameters: temperature, absolute humidity, and material substrate. This study delineated a range of test sterilizer process conditions: VHP concentration, process duration, a process temperature range for which the worst case D-value may be imposed, a process humidity range for which the worst case D-value may be imposed, and the dependence on selected spacecraft material substrates. The derivation of D-values from the lethality data permitted conservative planetary protection recommendations.

Archaeal diversity analysis of spacecraft assembly clean rooms

One of the main tasks of NASA's planetary protection program is to prevent the forward contamination of extraterrestrial environments with Earth life, and in turn preserve other planets and the integrity of future life detection missions. Despite information regarding bacterial diversity in NASA's clean rooms, little is known about the presence of Archaea. Archaeal community analysis of spacecraft-associated surfaces is important, as they are considered by some to represent terrestrial life most capable of surviving on Mars. The first insights into the archaeal diversity of clean rooms where spacecraft assembled are attempted. Nucleic acid sequences clustering with uncultivable Archaea within the Eury- and Crenarchaeota were retrieved from 8 of 26 samples collected from several spacecraft assembly clean rooms. Due to their potential capability to survive and proliferate in Martian conditions, screening for Archaea on spacecraft surfaces and instruments that are associated with future life detection missions may be necessary.

Planetary protection implementation on Mars Reconnaissance Orbiter mission

In August 2005 NASA launched a large orbiting science observatory, the Mars Reconnaissance Orbiter (MRO), for what is scheduled to be a 5.4-year mission. High resolution imaging of the surface is a principal goal of the mission. One consequence of this goal however is the need for a low science orbit. Unfortunately this orbit fails the required 20-year orbit life set in NASA Planetary Protection (PP) requirements [NASA. Planetary protection provisions for robotic extraterrestrial missions, NASA procedural requirements NPR 8020.12C, NASA HQ, Washington, DC, April 2005.]. So rather than sacrifice the science goals of the mission by raising the science orbit, the MRO Project chose to be the first orbiter to pursue the bio-burden reduction approach. Cleaning alone for a large orbiter like MRO is insufficient to achieve the bio-burden threshold requirement in NASA PP requirements. The burden requirement for an orbiter includes spores encapsulated in non-metallic materials and trapped in joints, as well as located on all internal and external surfaces (the total spore burden). Total burden estimates are dominated by the mated and encapsulated burden. The encapsulated burden cannot be cleaned. The total burden of a smaller orbiter (e.g., Mars Odyssey) likely could not have met the requirement by cleaning; for the large MRO it is clearly impossible. Of course, a system-level partial sterilization, with its attendant costs and system design issues, could have been employed. In the approach taken by the MRO Project, hardware which will burn up (completely vaporize or ablate) before reaching the surface or will at least attain high temperature (500 °C for 0.5 s or more) due to entry heating was exempt from burden accounting. Thus the bio-burden estimate was reduced. Lockheed Martin engineers developed a process to perform what is called breakup and burn-up (B&B) analysis. 2 Lockheed Martin Corporation. 2 The use of the B&B analysis to comply with the spore burden requirement is the main subject of this article. However, several components aboard the orbiter were predicted to fail the minimum time at temperature requirements (or could not conservatively be shown to meet the conditions). An implementation plan was generated to address the highest contributors to the bio-burden assessment that fail to meet the requirements. The spore burden for these components was estimated by direct and proxy burden assays, NASA PP specifications, and dry heat microbial reduction, as appropriate. Items on the orbiter that required rework during assembly were also individually assessed. MRO met the spore burden requirement based on the B&B analysis, the MRO Planetary Protection Implementation Plan, and verification by the NASA Planetary Protection Officer’s (PPO) independent assays. The compliance was documented in the MRO PP Pre-Launch Report. MRO was approved for flight by the NASA PPO.

Planetary protection policy for MUSES-C

MUSES-C is a Japanese sample return mission to a near-earth asteroid 1998 SF36(25143). This object is S-type and spectrally similar to L- and LL-type chondrites. Part of this mission is collaborated with NASA and Australia, and international consensus on the planetary protection policy for it was strongly required as well as the NASA policy. The mission plan of MUSES-C and the properties of the targeted asteroid were reviewed in a NASA Planetary Protection Advisory Committee meeting and a COSPAR/IAU workshop. Both bodies classified this mission as Category V with unrestricted earth return.

Implementing planetary protection requirements for sample return missions

NASA is committed to exploring space while avoiding the biological contamination of other solar system bodies and protecting the Earth against potential harm from materials returned from space. NASA's planetary protection program evaluates missions (with external advice from the US National Research Council and others) and imposes particular constraints on individual missions to achieve these objectives. In 1997 the National Research Council's Space Studies Board published the report, Mars Sample Return: Issues and Recommendations, which reported advice to NASA on Mars sample return missions, complementing their 1992 report, The Biological Contamination of Mars Issues and Recommendations. Meanwhile, NASA has requested a new Space Studies Board study to address sample returns from bodies other than Mars. This study recognizes the variety of worlds that have been opened up to NASA and its partners by small, relatively inexpensive, missions of the Discovery class, as well as the reshaping of our ideas about life in the solar system that have been occasioned by the Galileo spacecraft's discovery that an ocean under the ice on Jupiter's moon Europa might, indeed, exist. This paper will report on NASA's planned implementation of planetary protection provisions based on these recent National Research Council recommendations, and will suggest measures for incorporation in the planetary protection policy of COSPAR.