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In a previous experiment, we demonstrated the capability of flow cytometry as a potential life detection technology for icy moons using exogenous fluorescent stains (Wallace et al., 2023). In this companion experiment, we demonstrated the capability of flow cytometry to detect life using intrinsically fluorescent biomolecules in addition to exogenous stains. We used a method similar to our previous work to positively identify six classes of intrinsically fluorescent biomolecules: flavins, carotenoids, chlorophyll, tryptophan, NAD+, and NAD(P)H. We demonstrated the effectiveness of this method with six known organisms and known abiotic material and showed that the cytometer is easily able to distinguish the known organisms and the known abiotic material by using the intrinsic fluorescence of these six biomolecules. To simulate a life detection experiment on an icy moon lander, we used six natural samples with unknown biotic and abiotic content. We showed that flow cytometry can identify all six intrinsically fluorescent biomolecules and can separate the biotic material from the known abiotic material on scatter plots. The use of intrinsically fluorescent biomolecules in addition to exogenous stains will potentially cast a wider net for life detection on icy moons using flow cytometry.

Flow cytometry is a potential technology for in situ life detection on icy moons (such as Enceladus and Europa) and on the polar ice caps of Mars. We developed a method for using flow cytometry to positively identify four classes of biomarkers using exogenous fluorescent stains: nucleic acids, proteins, carbohydrates, and lipids. We demonstrated the effectiveness of exogenous stains with six known organisms and known abiotic material and showed that the cytometer is easily able to distinguish between the known organisms and the known abiotic material using the exogenous stains. To simulate a life-detection experiment on an icy world lander, we used six natural samples with unknown biotic and abiotic content. We showed that flow cytometry can identify all four biomarkers using the exogenous stains and can separate the biotic material from the known abiotic material on scatter plots. Exogenous staining techniques would likely be used in conjunction with intrinsic fluorescence, clustering, and sorting for a more complete and capable life-detection instrument on an icy moon lander.

To support NASA's Mars 2020 mission, bioassays were performed to ensure the biological cleanliness of the spacecraft, instruments, and hardware assembly areas. Bioassays began in May 2014, as the first components were assembled, and continued until their launch in July 2020. Over this 6-year period, 1811 bioassay sampling sessions were conducted. To understand the nature of microbiological presence on and around the spacecraft, an archive of organisms resulting from the bioassays was assembled. This archive included 4232 microbial specimens preserved as frozen stocks. To date, more than 3489 microbial isolates have been tested by MALDI-TOF mass spectrometry analysis. Identifications were based on high confidence level matches to known microorganisms in the reference spectra database where 39 distinct genera were identified. Gram-positive bacteria were isolated almost exclusively. Most, but not all, were spore-forming genera. The most prevalent genera isolated in order of frequency were Bacillus, Priestia, Paenibacillus, Staphylococcus, Micrococcus, and Streptomyces . Within the largely represented Bacillus- like genera, the five most prevalent species were cereus, licheniformis, horneckiae, subtilis, and safensis.

Aims: Inactivation processes using heat are widely used for disinfection and sterilization. Dry heat sterilization of spacecraft equipment has been the preferred microbial inactivation method as part of interplanetary travel protection strategies. An antimicrobial model, based on temperature and exposure time based on experimental data, was developed to provide reliable sterilization processes to be used for interplanetary applications., Methods and Results: Bacillus atrophaeus spores, traditionally used to challenge dry heat sterilization processes, were tested over a range of temperatures in comparison with spores of Bacillus canaveralius that have been shown to have a higher heat resistance profile. D-value and Z-values were determined and used to develop a mathematical model for parametric sterilization applications. The impact of the presence of a contaminating soil, representative of Mars dust, was also tested to verify the practical application of the model to reduce the risk of microbial contamination in such environments., Conclusion: The sterilization model developed can be used as an intrinsic part of risk reduction strategies for interplanetary protection., Significance and Impact: Forward and backward planetary protection strategies to reduce the risks of microbial contamination during interplanetary exploration and research is an important consideration. The development of a modern sterilization model, with consideration of microorganisms identified with higher levels of heat resistance than traditionally deployed in terrestrial applications, allows for the consideration of optimal inactivation processes to define minimum criteria for engineering design. The ability to inactivate living microorganisms, as well as to degrade biomolecules, provides a reliable method to reduce the risk of known and potentially unknown contaminants in future applications., (© 2022 Society for Applied Microbiology.)

The National Aeronautics and Space Administration (NASA) and the European Space Agency (ESA) are studying how to improve the safety of future planetary science sample return missions that would bring back materials to Earth. Backward planetary protection requirements have been identified as a critical technology development focus in order to reduce the possibility of harm to Earth's biosphere from such returned materials. In order to meet these challenges, NASA has identified the need for an appropriate suite of biological indicators (BIs) that would be used to develop, test, and ultimately validate sample return mission sterilization systems. Traditionally, BIs are defined as test systems composed of viable microorganisms that are inactivated when necessary conditions are met during sterilization procedures, providing a level of confidence in the process. BIs used traditionally at NASA have been driven by past mission requirements, mainly focused on spore-formers. However, spore-based BIs are insufficient as the only analog for a nominal case in sample return missions. NASA has directed sample return missions from habitable worlds to manage "potential extraterrestrial life and bioactive molecules" which requires investigation of a range of potential BIs. Thus, it is important to develop a mitigation strategy that addresses various known forms of biology, from complex organisms to biomolecular assemblies (including self-perpetuating non-nucleic acid containing structures). The current effort seeks to establish a BI that would address a stable biomolecule capable of replication. Additional engineering areas that may benefit from this information include applications of brazing, sealing, and impact heating, and atmospheric entry heating. Yeast aggregating proteins exhibit aggregation behavior similar to mammalian prion protein and have been successfully employed by researchers to understand fundamental prion properties such as aggregation and self-propagation. Despite also being termed "prions," yeast proteins are not hazardous to humans and can be used as a cost effective and safer alternative to mammalian prions. We have shown that inactivation by dry heat is feasible for the prion formed by the yeast Sup35NM protein, although at higher temperature than for bacterial spores., Competing Interests: ES is employed by the company Honeybee Robotics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Seto, Hirsch, Schubert, Chandramowlishwaran and Chernoff.)

Exposing flight hardware to dry heat is a NASA-approved sterilization method for reducing microbial bioburden on spacecraft. The existing NASA specification only allows heating the flight hardware between 104°C and 125°C to reduce the number of viable microbes and bacterial spores. Also, the NASA specifications only allow a four log reduction by dry heat microbial reduction because very heat-resistant spores are presumed to exist in a diverse population (0.1%). The goal of this research was to obtain data at higher temperatures than 125°C for one of the most heat-resistant microorganisms discovered in a spacecraft assembly area. These data support expanding the NASA specifications to temperatures higher than 125°C and relaxing the four log reduction specification. Small stainless steel vessels with spores of the Bacillus strain ATCC 29669 were exposed to constant temperatures between 125°C and 200°C under both dry and ambient room humidity for set time durations. After exposures, the thermal spore exposure vessels were cooled and the remaining spores recovered and plated out. Survivor ratios, lethality rate constants, and D-values were determined at each temperature. The D-values for the spores exposed under dry humidity conditions were always found to be shorter than those under ambient humidity. The temperature dependence of the lethality rate constants was obtained by assuming that they obeyed Arrhenius behavior. The results are compared to those of B. atrophaeus ATCC 9372. In all cases, the D-values of ATCC 29669 are between 20 and 50 times longer than those of B. atrophaeus ATCC 9372.

Dry heat microbial reduction is the NASA-approved sterilization method to reduce the microbial bioburden on spaceflight hardware for missions with planetary protection requirements. The method involves heating the spaceflight hardware to temperatures between 104 degrees C and 125 degrees C for up to 50 hours, while controlling the humidity to very low values. Collection of lethality data at temperatures above 125 degrees C and with ambient (uncontrolled) humidity conditions would establish whether any microbial reduction credit can be offered to the flight project for processes that occur at temperatures greater than 125 degrees C. The goal of this research is to determine the survival rates of Bacillus atrophaeus (ATCC 9372) spores subjected to temperatures higher than 125 degrees C under both dry (controlled) and room ambient humidity (36-66% relative humidity) conditions. Spores were deposited inside thin, stainless steel thermal spore exposure vessels (TSEVs) and heated under ambient or controlled humidity conditions from 115 degrees C to 170 degrees C. After the exposures, the TSEVs were cooled rapidly, and the spores were recovered and plated. Survivor ratios, lethality rate constants, and D-values were calculated at each temperature. At 115 degrees C and 125 degrees C, the controlled humidity lethality rate constant was faster than the ambient humidity lethality rate constant. At 135 degrees C, the ambient and controlled humidity lethality rate constants were statistically identical. At 150 degrees C and 170 degrees C, the ambient humidity lethality rate constant was slightly faster than the controlled humidity lethality rate constant. These results provide evidence for possibly modifying the NASA dry heat microbial reduction specification.

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA Biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Right sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.

A subset of the Caenorhabditis elegans nematodes flown aboard Biorack on IML-1 was analyzed for the fidelity of development and the mechanics of chromosomes at meiosis. To assess meiosis, mutant worms marked at two linked or unlinked loci were inoculated as heterozygous hermaphrodites and allowed to self fertilize. Mendelian segregation ratios and recombination frequency were measured for offspring produced at 1XG or in microgravity. To assess development, worms and embryos were fixed and stained with the DNA dye, DAPI, or antibodies specific for antigens expressed in germ cells, pharyngeal and body wall muscles, and gut cells. The distribution of cytoplasmic determinants, cell nuclei counts and positions were scored to assess symmetry relations and anatomical features.

The biological effects of heavy charged particle (HZE) radiation are of particular interest to travellers and planners for long-duration space flights where exposure levels represent a potential health hazard. The unique feature of HZE radiation is the structured pattern of its energy deposition in targets. There are many consequences of this feature to biological endpoints when compared with effects of ionizing photons. Dose vs response and dose-rate kinetics may be modified, DNA and cellular repair systems may be altered in their abilities to cope with damage, and the qualitative features of damage may be unique for different ions. The nematode Caenorhabditis elegans is being used to address these and related questions associated with exposure to radiation. HZE-induced mutation, chromosome aberration, cell inactivation and altered organogenesis are discussed along with plans for radiobiological experiments in space.

The nematode C. elegans was used to measure the effectiveness of high-energy ionized particles in the induction of 3 types of genetic lesions. Recessive lethal mutations in a 40-map unit autosomal region, sterility, and X-chromosome nondisjunction or damage were investigated. Induction rates were measured as a function of linear energy transfer, LET infinity, for 9 ions of atomic number 1-57 accelerated at the BEVALAC accelerator. Linear kinetics were observed for all 3 types of lesions within the dose/fluence ranges tested and varied strongly as a function of particle LET infinity. Relative Biological Effectiveness (RBE) values of up to 4.2 were measured and action cross sections were calculated and compared to mutagenic responses in other systems.