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NASA NTRSDate not supplied

Sample Processor for Life on Icy Worlds (SPLIce): Monolithic Manifold-Based System to Recover, Prepare, and Deliver Samples and Standards to Instrumentation Suites for Ocean World Life-Search Missions

A claim of life detection on one of the solar system’s icy ocean worlds would necessitate extraordinarily convincing evidence. Limited energy availability in the oceans of such bodies as Europa and Enceladus argues for microbes as most probable among possible life forms, but evidence of their existence in the surface layers of an icy moon or in a frozen plume ejected into space could take various forms, pointing to instrumentation suites as a preferred means to detect diverse molecular and morphological life indicators. Multiple disparate categories of positive detections could provide truly convincing evidence from samples that may be only a few micro-liters. SPLIce’s Foundation. Teams led by NASA’s Ames Research Center have developed and operated numerous small, live-biology and astrobiology science payloads in space over two decades. Since 2016, we have adapted and augmented their biological sample-handling systems to create compact, robust search-for-life fluidic processors designed to function after a decade or more in transit, in environments with very little gravity and lots of radiation: up to 100’s of kilorads.

Instrumentation suite↗
NASA NTRSDate not supplied

Investigating Commercial Off-The-Shelf (COTS) Glovebox and Support Components Compared to Custom Curatorial Laboratories

There is a need envisioned to investigate the application of commercial off-the-shelf (COTS) systems as tools that could be used within commercial preliminary curation as the expected space economy is supported and begins to take flight. NASA is involved with supporting and developing the space economy [1] and therefore it’s feasible that at some point, a commercial space company would bring back materials and either store initially, or permanently, within COTS equipment as a stand-in for custom curation laboratories. While utilizing opportunities to explore this capability at NASA-Johnson Space Center (JSC) during the establishment of other laboratories [2] it was realized that cleanliness and/or other properties could be evaluated for these COTS systems during their installation in advanced research facilities that are not ISO-class rated clean rooms. Several aspects of a COTS-Curation system were explored including various gloves for curation manipulation within a negative pressure glovebox (Fig.1), Balazs organic and inorganic contaminant levels testing prior to glovebox ever being used, mode swapping comparison of recirculation vs. single pass, constant monitoring of oxygen (O2) and moisture (H2O) levels in various conditions, etc. To acquire inorganic and organic compound loads inside the glovebox, Balazs wafer testing and gas sampling were implemented. These are standardized analytical tests provided by Balazs™ NanoAnalysis, a division of Air Liquide USA. Deployment of 8-inch silicon wafer witness plates for 24-hours in an undisturbed environment capture the organic compound load and inorganic trace metal contents which can be obtained by Vapor Phase Decomposition Inductively Coupled Plasma Mass Spectrometry (VPD ICP-MS). Balazs gas sample analysis was also performed for better measurements of volatile organic compounds (VOC) in glovebox air analyzed by Thermal Desorption Gas Chromatography Mass Spectrometry (TD GC-MS). These analytical testings were carried out in a controlled ultra high pure (UHP) gaseous nitrogen (N2)-purged environment where oxygen and moisture contents were continuously monitored at certain temperature and pressure. The preliminary outcomes of these testings are promising. The COTS systems appear to maintain the steady-state controlled environment for days, if not weeks, with uninterrupted gaseous N2-supply which was operated from a standard medium pressure LN2 250L 230L dewar, exchanged as needed. The outgassing load can be maintained by selecting the glove materials that have the least outgassing and particle shedding performances. Further experiments will be considered to validate the preliminary findings. While this project is exploratory, it is not intended as an endorsement by NASA Curation for approved materials or usage for advanced curatorial activities. NASA does not endorse nor promote any one particular product or company. References: [1] McCubbin F. M. et al. (2019) Space Science Reviews 215:A48. [2] Lewis, E.K. et. al (2024) LPSC LV, Abstract #2457.

Curation↗
NASA NTRSDate not supplied

Exploring the Geochemistry of Tholeiitic Basalts and Hyaloclastites Formed in Submarine Volcanoes for Comparison With Venus Lavas

Underwater basaltic lavas exhibit distinct rock textures, mineralogy and morphological differences compared to those formed on land, primarily due to rapid cooling caused by contact with cold seawater instead of air and to high pressures – similar to Venus. The geomorphological similarities between terrestrial submarine volcanoes and some morphological classes of Venusian volcanoes have been investigated, but a comprehensive comparison in geochemical terms has yet to be conducted. Only three missions to Venus (Venera-13, Venera-14, and Vega-2) were able to report bulk composition of Venusian rocks, while other four missions (Venera-8, Venera-9, Venera-10, and Vega-1) provided limited geochemical information. Using X-Ray fluorescent (XRF) analysis, we saw that rocks analyzed on the surface of Venus are tholeiitic and alkalic basalts. This study, as part of the “Analogs for VENus's GEologically Recent Surfaces” (AVENGERS) initiative, aims to examine the possibility of seamounts/submarine volcanoes lavas serving as analogues to Venus lava geochemistry and morphology/formational processes. By sampling and analyzing submarine lavas from three distinct tectonic settings on Earth - hot-spot, convergent margins, and divergent margins - we will assess in which tectonic context the analogy with Venus is most comparable. Fieldwork will be conducted in further detail for submarine volcanoes located in Sicily, Hawaiʻi, and the Southern Pacific (Fiji, Vanuatu, Tonga, Samoa); we will use previously collected samples where these submarine areas are not accessible. Sampling efforts will target areas likely to yield submarine tholeiitic lavas and/or hyaloclastites. Samples collected during fieldwork will undergo detailed whole-rock geochemical analysis, including XRF, X-Ray diffraction (XRD) and Inductively Coupled Plasma Mass Spectrometry (ICPMS). Thin sections will be used to constrain differences in morphology/textures. The obtained geochemical data will then be compared with existing data on Venusian lava geochemistry, allowing for a comprehensive evaluation of potential analogies between submarine lavas from Earth and volcanic rocks on Venus. By elucidating these similarities and pinpointing specific tectonic contexts, our study has the potential to enhance our understanding of Venusian geology and the possibility of ephemeral plate tectonics on the planet.

N Mari↗
NASA NTRSDate not supplied

Bionutrients-1: Utilizing Genomics and Transcriptomics to Assess the Reliability of Microorganisms for In Situ Nutrient Production on Long Duration Missions

The resupply of current long-duration crewed missions to the ISS relies on ground-launched supplies. As NASA looks toward Mars, ground-based resupply will no longer be an option. Critical nutrients, including vitamin C, vitamin K, folate, and thiamin, degrade during long-term storage, and regular consumption of these nutrients is essential for astronaut health. Another challenge of current food systems is the difficulty of consuming sufficient calories when subsisting on the limited flavors of freeze-dried food, which can lead to weight loss. The inclusion of microorganism-based food systems could alleviate both concerns. For example, the fermentation of rehydrated milk into yogurt with microorganisms genetically incorporating genes to produce critical vitamins would allow for both in situ production of nutrients and a fresh food product with additional flavor profiles. In comparison to plant food production, microorganisms require less flight infrastructure. The BioNutrients-1 mission is demonstrating viability of microbial fermentation food production in microgravity and testing the reliability of this approach for long-duration missions lacking resupply. While the BioNutrients-1 mission includes the collection of multiple phenotypic measurements, this status update will focus on the processing of samples for genomics and transcriptomics analyses as well as the planned analysis pipelines. First, the BioNutrients-1 mission seeks to identify microorganisms capable of surviving long-duration storage at ambient temperatures while maintaining genetic fidelity. To achieve this, nine commonly employed microbial species were stored at ambient temperatures in Stasis Packs on the ISS for five years. The viability and mutation rates will be measured at multiple time points for both flown and ground control samples. From an omics perspective, the changes in the bulk rates of point mutations and genetic rearrangements across the Stasis Pack species during the five years of storage will be determined, providing valuable insights into the potential of these microorganisms for long-duration space missions. Second, the BioNutrients-1 mission is characterizing the impact of microgravity on fermentation. Two strains of the yeast Saccharomyces cerevisiae, each encoding antioxidants (β-carotene or zeaxanthin) were flown to ISS for storage and fermentation within simplified bioreactors (Production Packs). The impact of microgravity on the expression of the antioxidant production genes and general metabolic genes will be determined using RNA sequencing. Ultimately, the transcriptome data will be compared to phenotypic measurements, such as the antioxidant yield, end-state biomass, and the production of EtOH, to determine the impacts of microgravity and long-term storage on microbial fermentation. The findings from this research will be instrumental in understanding the challenges and opportunities of microorganism-based food systems in space missions.

BioNutrients↗
DOE OSTI2024

A Framework to Demonstrate a DNP3 Interface With a CIM-Based Data Integration Platform: Preprint

The contemporary electrical grid is characterized by its complexity and abundance of data. A control-rich environment supported by information and communication technologies within an Advanced Distribution Management System (ADMS) presents a viable and cost-effective option for utility companies aiming to implement advanced real-time analytical schemes for monitoring and remotely controlling distribution feeders. Modular platform-based approaches to distribution operations require a structured framework for acquiring field device measurements, performing analytics, converting the setpoint to the correct protocol, and sending it on the appropriate communications network to the field devices. We present the development and deployment of an application service to integrate an open-source standardsbased platform with an ADMS test bed with field devices using the Distributed Network Protocol (DNP3) for data exchange. The step-by-step procedure for establishing the DNP3-Master service on an open-source distribution platform is outlined, comprehensively explaining the Master setup process. Moreover, sample use case results highlight the capabilities of the DNP3- Master service setup. Results demonstrate the scalability and configurability of the DNP3-Master service, making it adaptable for integration with other relevant applications, thus providing potential opportunities for real-world field trials and real-time assessments.

ADMS↗
DOE OSTI2025

Novel Preprocessing Techniques for Enhancing Flowability of Miscanthus

Purpose-grown energy crops possess significant potential as promising feedstocks for biofuel and biochemical production. However, similar to other biomass feedstocks, their utilization has been hindered by handling and feeding challenges such as clogging and segregation, which prevent biorefineries from operating at full capacity. These challenges arise primarily due to particle attributes including irregular shape, high flexibility, and high compressibility, leading to interlocking, high compaction, and significant friction under stress consolidation. To enhance operational reliability, it is crucial to focus on preprocessing techniques to address these issues, alongside accurately characterizing the mechanical and physical properties and flow performance of these feedstocks. This study investigates novel preprocessing techniques, including pelletization and torrefaction, to fundamentally alter these particle attributes and improve flowability. Miscanthus, an herbaceous energy crop, was the primary focus of this study. Samples were processed using a Forest Concepts Crumbler rotary shear system and an oscillating multi-stack screen bed to achieve two nominal (baseline) particle sizes: 6mm and 2mm. The study evaluated the efficiency and energy consumption of size reduction, revealing that while 2mm particles required more energy, they exhibited better flowability and higher yield strength compared to 6mm particles. Shear and compression tests assessed the material's shear strength, internal friction, and elastic modulus, while wedge-shaped hopper tests measured the critical arching distance and mass flow rate. The results provide valuable insights into the mechanical properties and processing efficiency of Miscanthus, contributing to the development of a preprocessing framework that enhances energy efficiency and material flowability in bioenergy production. However, further preprocessing and development are required to optimize this framework fully.

09 - BIOMASS FUELS↗
NASA NTRS2010

Methods for producing silicon carbide architectural preforms

Methods are disclosed for producing architectural preforms and high-temperature composite structures containing high-strength ceramic fibers with reduced preforming stresses within each fiber, with an in-situ grown coating on each fiber surface, with reduced boron within the bulk of each fiber, and with improved tensile creep and rupture resistance properties for each fiber. The methods include the steps of preparing an original sample of a preform formed from a pre-selected high-strength silicon carbide ceramic fiber type, placing the original sample in a processing furnace under a pre-selected preforming stress state and thermally treating the sample in the processing furnace at a pre-selected processing temperature and hold time in a processing gas having a pre-selected composition, pressure, and flow rate. For the high-temperature composite structures, the method includes additional steps of depositing a thin interphase coating on the surface of each fiber and forming a ceramic or carbon-based matrix within the sample.

DiCarlo, James A.↗
NASA NTRS2014

Methods for Producing High-Performance Silicon Carbide Fibers, Architectural Preforms, and High-Temperature Composite Structures

Methods are disclosed for producing architectural preforms and high-temperature composite structures containing high-strength ceramic fibers with reduced preforming stresses within each fiber, with an in-situ grown coating on each fiber surface, with reduced boron within the bulk of each fiber, and with improved tensile creep and rupture resistance properties tier each fiber. The methods include the steps of preparing an original sample of a preform formed from a pre-selected high-strength silicon carbide ceramic fiber type, placing the original sample in a processing furnace under a pre-selected preforming stress state and thermally treating the sample in the processing furnace at a pre-selected processing temperature and hold time in a processing gas having a pre-selected composition, pressure, and flow rate. For the high-temperature composite structures, the method includes additional steps of depositing a thin interphase coating on the surface of each fiber and forming a ceramic or carbon-based matrix within the sample.

DiCarlo, James A.↗
NASA NTRS1995

(abstract) Microwave Induced Combustion Synthesis of Ceramic and Ceramic-Metal Composites

We have used microwaves to induce self-propagating high temperature synthesis (SHS) in the reaction 3TiO(sub 2)+3C+(4+x)Al ---> 3TiC+2Al(sub 2)O(sub 3)+xAl. The SHS process was studied for x = 0 and 4, with and without the application of uniaxial pressure. A TE102 microwave cavity mode internally ignited the samples using less than 50 watts. The resultant microstructure of the microwave processed samples were compared to SHS products obtained using a conventional hot wire ignition technique. Uniaxial stresses along the vertical axis of approximately 1400 and 200 psi were applied simultaneously as the SHS reactions were initiated in some of the conventional and microwave processed samples, respectively. SEM photomicrographs will be presented that show the different microstructural features of the products using the microwave and hot wire techniques.

ceramic microwave ceramic-metal composites microwa↗
NASA NTRS2021

Sample Return Containerization and Planetary Protection Using Brazing for Breaking the Chain of Contact to Mars

Planetary protection of Mars samples returned to Earth in a future NASA mission is a critical part of preventing uncontrolled biological materials being released from the samples. The planetary protection process requires addressing the potential risks and would involve “breaking the chain of contact (BTC)”, where any returned material reaching Earth for further analysis would have to be sealed inside a container with extremely high confidence. The sterilization process would require destroying any potential biological materials that may contaminate the external surface of the container. A novel process for containing the returned samples has been conceived and developed at JPL. The process consists of using induction heated brazing to synchronously sterilize, separate, seam and seal the container. In this case. the braze material is heated to the level of melting at temperatures higher than 500°C to assure sterilizing exposed areas since carbon bonds are broken above this temperature. The container that encloses the samples consists of double walls with interstitial space that is Earth-clean. The process consists of two-steps, where one was intended to be prepared on Earth and the second potentially executed on orbit around Mars. The latest results of this study that established the needed capability are described and discussed in this manuscript.

Shirajian, Nareg↗
NASA NTRS2021

Verifying Mars 2020 Sampling and Caching Robotic Functions with Position Budgeting Process and Tool

The Mars 2020 Perseverance Rover was launched on July 30th, 2020 with one of the most complex robotic systems ever implemented on an interplanetary mission. Much of this robotic complexity resides in the Rover Sampling and Caching Subsystem (SCS) to enable collection of Martian samples for eventual return to earth and preparation of surfaces for close-up surface science observations. Two robotic arms are used by SCS: the large Robotic Arm (RA) positions the coring drill and science instruments mounted to the Turret for surface interactions, and the smaller Sample Handling Assembly (SHA) manipulates Sample Tube Assemblies (STA) within the Adaptive Caching Assembly (ACA) to prepare them for sample collection, processing, and hermetic sealing. The robotic arms interact with the Martian surface and other SCS components in many ways and in a variety of configurations, with positioning accuracy requirements ranging from tens of millimeters for some surface interactions down to sub-millimeter accuracy for some ACA interactions.This paper describes the process and tool used to calculate the SCS robotic interaction positioning budgets and verify the as-built hardware when delivered. To ensure that these robotic systems are able to perform their tasks, each robotic interaction with another element was broken down into its composite functions. To calculate a positioning budget margin for each function, an allowable was defined and then compared to the list of error sources that contribute to misalignment. Across the subsystem, over 250 functions were identified to be assessed, with almost 500 error sources feeding into their budget calculations. In addition to using as-built values in the budgets for SCS Verification and Validation (V&V) after the hardware was complete, these budgets were populated with design data during the design phase to identify areas of concern and guide hardware design to ensure positive position budget margins.Because of the size of the SCS team that had inputs to the positioning budgets and the sheer number of items in the budgets that needed to be created, updated, and verified, having a tool that would allow for simultaneous access and robust data integrity and processing was imperative. To accomplish this, a web-based MySQL database was created that allowed users to create function position budgets, link individual errors and allowables to them, and view function position budget margin reports. Each error and allowable records data for lateral, normal, angular, and clocking errors, with the ability to add as-built data for up to four different hardware builds. Margin reports can be generated for either design values alone or replacing design data with as-built data when available. These as-built reports are used for final verification of the SCS positioning requirements. Ultimately, the SCS positioning budget process and database tool led to successful interactions during test and an SCS robotic system that is ready to perform sample acquisition and caching on the surface of Mars.

Williams, Jeffrey↗
NASA NTRS1978

Miniregoliths. I - Dusty lunar rocks and lunar soil layers

A detailed Monte-Carlo model for rock surface evolution shows that erosion processes alone cannot account for the shapes of the solar flare particle track profiles generally observed at depths of about 100 microns and less in rocks. The observed profiles are easily explained by a steady accumulation of fine dust at a rate of 0.3 to 3 mm per m.y., depending on the micrometeoroid impact rate which controls the dust cover and results in maximum dust thicknesses on the order of 100 microns to 1 mm. The commonly used lunar soil track parameters are derived in terms of parameters characterizing the exposure of soil grains in the few-millimeter-thick surface mixing and maturation zone which is one form of miniregolith. Correlation plots permit determining the degree of mixing in soil samples and the amount of processing (maturation) in surface miniregoliths. It is shown that the sampling process often artificially mixes together finer distinct layers, and that ancient miniregolith layers on the order of a millimeter thick are probably common in the lunar soil.

Comstock, G. M.↗
NASA NTRS2002

Technology Development and Advanced Planning for Curation of Returned Mars Samples

NASA Johnson Space Center (JSC) curates extraterrestrial samples, providing the international science community with lunar rock and soil returned by the Apollo astronauts, meteorites collected in Antarctica, cosmic dust collected in the stratosphere, and hardware exposed to the space environment. Curation comprises initial characterization of new samples, preparation and allocation of samples for research, and clean, secure long-term storage. The foundations of this effort are the specialized cleanrooms (class 10 to 10,000) for each of the four types of materials, the supporting facilities, and the people, many of whom have been doing detailed work in clean environments for decades. JSC is also preparing to curate the next generation of extraterrestrial samples. These include samples collected from the solar wind, a comet, and an asteroid. Early planning and R\&D are underway to support post-mission sample handling and curation of samples returned from Mars. One of the strong scientific reasons for returning samples from Mars is to search for evidence of current or past life in the samples. Because of the remote possibility that the samples may contain life forms that are hazardous to the terrestrial biosphere, the National Research Council has recommended that all samples returned from Mars be kept under strict biological containment until tests show that they can safely be released to other laboratories. It is possible that Mars samples may contain only scarce or subtle traces of life or prebiotic chemistry that could readily be overwhelmed by terrestrial contamination . Thus, the facilities used to contain, process, and analyze samples from Mars must have a combination of high-level biocontainment and organic / inorganic chemical cleanliness that is unprecedented. JSC has been conducting feasibility studies and developing designs for a sample receiving facility that would offer biocontainment at least the equivalent of current maximum containment BSL-4 (BioSafety Level 4) laboratories, while simultaneously maintaining cleanliness levels equaling those of state-of-the-art cleanrooms. Unique requirements for the processing of Mars samples have inspired a program to develop handling techniques that are much more precise and reliable than the approach (currently used for lunar samples) of employing gloved human hands in nitrogen-filled gloveboxes. Individual samples from Mars are expected to be much smaller than lunar samples, the total mass of samples returned by each mission being 0.5- 1 kg, compared with many tens of kg of lunar samples returned by each of the six Apollo missions. Smaller samples require much more of the processing to be done under microscopic observation. In addition, the requirements for cleanliness and high-level containment would be difficult to satisfy while using traditional gloveboxes. JSC has constructed a laboratory to test concepts and technologies important to future sample curation. The Advanced Curation Laboratory includes a new-generation glovebox equipped with a robotic arm to evaluate the usability of robotic and teleoperated systems to perform curatorial tasks. The laboratory also contains equipment for precision cleaning and the measurement of trace organic contamination.

Lindstrom, David J.↗
NASA NTRS1985

Flat-plate solar array project process development area process research of non-CZ silicon material

Three sets of samples were laser processed and then cell processed. The laser processing was carried out on P-type and N-type web at laser power levels from 0.5 joule/sq cm to 2.5 joule/sq cm. Six different liquid dopants were tested (3 phosphorus dopants, 2 boron dopants, 1 aluminum dopant). The laser processed web strips were fabricated into solar cells immediately after laser processing and after various annealing cycles. Spreading resistance measurements made on a number of these samples indicate that the N(+)P (phosphorus doped) junction is approx. 0.2 micrometers deep and suitable for solar cells. However, the P(+)N (or P(+)P) junction is very shallow ( 0.1 micrometers) with a low surface concentration and resulting high resistance. Due to this effect, the fabricated cells are of low efficiency. The maximum efficiency attained was 9.6% on P-type web after a 700 C anneal. The main reason for the low efficiency was a high series resistance in the cell due to a high resistance back contact.

Source record↗
NASA NTRS1999

Coarsening in Solid-Liquid Mixtures Studied on the Space Shuttle

Ostwald ripening, or coarsening, is a process in which large particles in a two-phase mixture grow at the expense of small particles. It is a ubiquitous natural phenomena occurring in the late stages of virtually all phase separation processes. In addition, a large number of commercially important alloys undergo coarsening because they are composed of particles embedded in a matrix. Many of them, such as high-temperature superalloys used for turbine blade materials and low-temperature aluminum alloys, coarsen in the solid state. In addition, many alloys, such as the tungsten-heavy metal systems, coarsen in the solid-liquid state during liquid phase sintering. Numerous theories have been proposed that predict the rate at which the coarsening process occurs and the shape of the particle size distribution. Unfortunately, these theories have never been tested using a system that satisfies all the assumptions of the theory. In an effort to test these theories, NASA studied the coarsening process in a solid-liquid mixture composed of solid tin particles in a liquid lead-tin matrix. On Earth, the solid tin particles float to the surface of the sample, like ice in water. In contrast, in a microgravity environment this does not occur. The microstructures in the ground- and space-processed samples (see the photos) show clearly the effects of gravity on the coarsening process. The STS-83-processed sample (right image) shows nearly spherical uniformly dispersed solid tin particles. In contrast, the identically processed, ground-based sample (left image) shows significant density-driven, nonspherical particles, and because of the higher effective solid volume fraction, a larger particle size after the same coarsening time. The "Coarsening in Solid-Liquid Mixtures" (CSLM) experiment was conducted in the Middeck Glovebox facility (MGBX) flown aboard the shuttle in the Microgravity Science Laboratory (MSL-1/1R) on STS-83/94. The primary objective of CSLM is to measure the temporal evolution of the solid particles during coarsening.

Caruso, John J.↗
NASA NTRS2006

Towards a Theory of Sampled-Data Piecewise-Deterministic Markov Processes

The analysis and design of practical control systems requires that stochastic models be employed. Analysis and design tools have been developed, for example, for Markovian jump linear continuous and discrete-time systems, piecewise-deterministic processes (PDP's), and general stochastic hybrid systems (GSHS's). These model classes have been used in many applications, including fault tolerant control and networked control systems. This paper presents initial results on the analysis of a sampled-data PDP representation of a nonlinear sampled-data system with a jump linear controller. In particular, it is shown that the state of the sampled-data PDP satisfies the strong Markov property. In addition, a relation between the invariant measures of a sampled-data system driven by a stochastic process and its associated discrete-time representation are presented. As an application, when the plant is linear with no external input, a sufficient testable condition for the convergence in distribution to the invariant delta Dirac measure is given.

Herencia-Zapana, Heber↗
NASA NTRS2012

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Benardini, James N.↗