Clean room microbiome complexity impacts planetary protection bioburden

Background The Spacecraft Assembly Facility (SAF) at the NASA’s Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. Results Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. Conclusion This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays.

Mars: new insights and unresolved questions

Mars exploration motivates the search for extraterrestrial life, the development of space technologies, and the design of human missions and habitations. Here, we seek new insights and pose unresolved questions relating to the natural history of Mars, habitability, robotic and human exploration, planetary protection, and the impacts on human society. Key observations and findings include: – high escape rates of early Mars' atmosphere, including loss of water, impact present-day habitability; – putative fossils on Mars will likely be ambiguous biomarkers for life; – microbial contamination resulting from human habitation is unavoidable; and – based on Mars' current planetary protection category, robotic payload(s) should characterize the local martian environment for any life-forms prior to human habitation. Some of the outstanding questions are: – which interpretation of the hemispheric dichotomy of the planet is correct; – to what degree did deep-penetrating faults transport subsurface liquids to Mars' surface; – in what abundance are carbonates formed by atmospheric processes; – what properties of martian meteorites could be used to constrain their source locations; – the origin(s) of organic macromolecules; – was/is Mars inhabited; – how can missions designed to uncover microbial activity in the subsurface eliminate potential false positives caused by microbial contaminants from Earth; – how can we ensure that humans and microbes form a stable and benign biosphere; and – should humans relate to putative extraterrestrial life from a biocentric viewpoint (preservation of all biology), or anthropocentric viewpoint of expanding habitation of space? Studies of Mars' evolution can shed light on the habitability of extrasolar planets. In addition, Mars exploration can drive future policy developments and confirm (or put into question) the feasibility and/or extent of human habitability of space.

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.

International environmental law and environmentally harmful space activities: learning from the past for a more sustainable future

Purpose The purpose of this paper is to present and analyse part of the relevant legal instruments currently available for regulating environmentally harmful space activities. Design/methodology/approach This paper opted for a functional research method combined with a comparative methodology. To make the argument, this paper relies on the contextual analysis of primary and secondary sources of law, instrument of soft law and the relevant background material (e.g. journal articles, textbooks, law reform and policy papers). Findings The central section will focus on the principles of international environmental law to outline their utility in the contemporary context. Finally, the conclusive part will point out the several ways in which the use of analogies can shape the outer space regime, especially concerning how those principles that are developed to safeguard the Earth, can also be extended for the protection of the space ecosystem. Originality/value Environmental hazards are rapidly increasing and the current international law and policy on planetary protection are inadequate to meet the challenges of the near future. There is no possibility of an environment-friendly and sustainable future if not strictly connecting it with a comprehensive and transparent acknowledgement of the human mistakes made on Earth. There are valuable lessons to be learned from our past, and it is under this perspective that the trend of polluting the outer space can be reverted. This paper fulfils an identified need to study the correlation between principles of international environmental law, space law and the current situation in the outer space.

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

BackgroundCrewed 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, and the mobility of microbes through spacesuits has not been studied.ResultsTo 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 × 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.ConclusionThe 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 microbial contaminants on the outside of the suits could contaminate planetary science operations unless additional measures are taken. Overall, these data provide the first estimate of microbial distribution associated with spacesuit surfaces, which will help future mission planners develop effective planetary protection strategies.

PanCam: the ‘science eyes’ of the Rosalind Franklin (ExoMars 2022) rover

<p>The scientific objectives of the ExoMars Rosalind Franklin rover [1] are designed to answer several key questions in the search for life on Mars. In particular, the unique subsurface drill will address some of these questions for the first time, such as the possible existence and stability of sub-surface organics. PanCam [2] will establish the surface geological and morphological context for the mission, working in collaboration with other context instruments. Here, we describe the PanCam scientific objectives in geology, atmospheric science and 3D vision. We discuss the design of PanCam, which includes a stereo pair of Wide Angle Cameras (WACs), each of which has an 11 position filter wheel, and a High Resolution Camera (HRC) for high resolution investigations of rock texture at a distance. The cameras and electronics are housed in an optical bench that provides the mechanical interface to the rover mast and a planetary protection barrier.  The electronic interface is via the PanCam Interface Unit (PIU), and power conditioning is via a DC-DC converter. PanCam also includes a calibration target mounted on the rover deck for radiometric calibration, fiducial markers for geometric calibration and a rover inspection mirror. Recent simulations [3] show the view from PanCam, the ‘science eyes’ of the Rosalind Franklin rover.</p> <p><strong>References:</strong></p> <p>[1] Vago, J.L., F. Westall, A.J. Coates, et al., Habitability on Early Mars and the Search for Biosignatures with the ExoMars Rover, <em>Astrobiology</em>, 17(6-7), 471-510, doi:10.1089/ast.2016.1533, Jul 2017.</p> <p>[2] Coates, A.J., R. Jaumann, A.D. Griffiths, et al., The PanCam instrument for the ExoMars rover, <em>Astrobiology</em>, 17 (6-7), 511-541, doi: 10.1089/ast.2016.1548, Jul 2017.</p> <p>[3] Miles, H.C., M.D. Gunn and A.J. Coates, Seeing through the ‘Science Eyes’ of the ExoMars Rover, IEEE Computer Graphics & Applications, Applications Department, 40, 71-81, doi: 10.1109/MCG.2020.2970796, Mar-Apr 2020.</p>