Life Detection on Icy Moons Using Flow Cytometry and Intrinsically Fluorescent Biomolecules.

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.

Life Detection on Icy Moons Using Flow Cytometry and Exogenous Fluorescent Stains.

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.

Identification and Archive of Mars 2020 Spacecraft Microbial Isolates.

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.

Heat inactivation of stable proteinaceous particles for future sample return mission architecture.

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.

Dry heat sterilization modelling for spacecraft applications.

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.

Determination of lethality rate constants and D-values for heat-resistant Bacillus spores ATCC 29669 exposed to dry heat from 125°C to 200°C.

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.