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All sourcesNASA NTRSDOE OSTI
NASA NTRS2024

Augmentation of Seed Film Technology With Probiotics in Support of Space Crop Production

This project set out to establish a method for introducing Plant Growth Promoting Microbes to seed film for use in a flight-based crop production system. The inoculation method, demonstrated in this paper, succeeded in this introduction while simultaneously avoiding disrupting the integrity of the seed film material. Future research should continue to explore the idea of growth production and crop protection via intentional introduction of an exophytic microbiome including the verification of effects of varying inoculum concentrations on mature plants. Positive effects are more likely to be noted when multiple organisms are introduced simultaneously. Additional benefit is likely to be seen on an experimental basis when crops are challenged via the introduction of both biotic and abiotic stressors.

Aubrie E Orourke↗
NASA NTRS2023

Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Bailey Marshall↗
NASA NTRSDate not supplied

Ultrashort Pulsed Laser Treatment: A Novel Sterilization Method for Planetary Protection

Introduction: On missions seeking signs of potential life elsewhere in the solar system, we have an obligation not to bring our own Earthly life with us. Reducing the bioburden (number of living microbes) on spacecraft is therefore required by the COSPAR Policy on Planetary Protection to target bodies that are of interest for understanding the origins of life (such as Mars). The method of spacecraft sterilization predominantly used by NASA is Heat Microbial Reduction, which is typically incompatible with heat-sensitive components such as optics and electronics, and is expensive and time-consuming. Here, we present preliminary results on a novel method for spacecraft hardware sterilization: high-intensity ultrashort (femtosecond) pulsed laser illumination. Femtosecond lasers use extremely high photon fluxes (10^29 photons/sec*cm^2, ~0.03 J/cm^2) in extremely short pulses, which can inactivate even stress-tolerant microbial spores with minimal damage to the spacecraft surface. This rapid sterilization technique could be carried out in situ in a spacecraft assembly clean room using high-speed surface scanning, saving critical time and resources. It also can potentially remove or reduce debris from inactivated cells and spores. Methods: To develop optimal laser processing parameters for inactivating planetary protection-relevant organisms on metal surfaces, we inoculated 1-cm^2 mirror-polished aluminum coupons with Bacillus subtilis spores (2x10^5 spores/coupon) and tested the effect of several parameters, including pulse count and fluence. Sterilization effectiveness was measured by recovering spores using a PVA (polyvinyl acetate) peel and conducting serial dilution and plating for colony-forming units (CFUs). Results: Our results show that pulse count and fluence both affect sterilization effectiveness and that within a certain pulse count range, increasing fluence increases effectiveness. We have demonstrated the ability to reduce viable microbial counts by at least 10^-4. Future work will include testing higher abundances and different species of microorganisms, effectiveness on complex surfaces, compatibility with sensitive surfaces, and quantification of cell debris removal. Femtosecond pulsed laser illumination has the potential to provide dramatic savings in both cost and schedule over current methods of bioburden reduction to prevent forward contamination. It may also have the potential for use in surface sterilization of returned samples to prevent back contamination.

Planetary Protection; Lasers; Sterilization; Bacil↗
NASA NTRSDate not supplied

Waste Container Filtration to Minimize Microbial Escape During Long-Term Storage on Mars

In the search for life on Mars, keeping its surface pristine and free of microbes due to human presence is of utmost importance. Astronauts will use emptied logistics containers during their Mars stay to store waste. These containers will be left on the planet’s surface and are a potential source of microorganism contamination. It is desired for these containers to be free from leaks for a minimum of 50 years. If leaks do occur, a likely location will be at any ports. At these locations the mating surfaces will be sealed using a flexible gasket material like a thin metal or a polymer. Eventually these seals will fail. To mitigate these effects, a particulate filter can be employed to equilibrate the pressure between the Martin environment and inside the container. To meet planetary protection requirements, the filter can only allow particulates smaller than 0.2 m diameter to pass. This paper discusses why a particulate filter may be needed and how such a filter could be tested to survive and operate in the harsh Martian environment.

planetary protection↗
NASA NTRSDate not supplied

Silver Foam: A Novel Approach for Long-Term Passive Dosing of Biocide in Spacecraft Potable Water Systems – Update 2024

A spacecraft water disinfection system, suitable for extended length space exploration, should prevent or control the growth of microbes, prevent or limit biofilm formation, and prevent microbiologically influenced corrosion. In addition, the system should have minimal maintenance requirements, be chemically compatible with all materials in contact with the water, be safe for human consumption, and be suitable to be shared across international spacecraft platforms and mission architectures. Silver ions are a proven broad-spectrum potable water biocide under investigation for future exploration missions. The competing technology for dosing silver ions in future water systems is based on actively dosing the ions via electrolytic production. Several challenges with this approach have prompted additional investigations into alternative dosing techniques. Control-release technology is an attractive option for developing a high-reliability passive silver dosing device. This paper describes the continued development of a nanoparticle/polyurethane (NP/PU) composite foam for the controlled release of silver ions and is intended to build upon the 2023 International Conference on Environmental Systems (ICES) paper number 251. This paper provides the technical background and performance results for the product variability testing and microbial check valve (MCV) testing of the silver chloride (AgCl) NP/PU composite foams, referred to as AgFoams. The ultimate goal of the project is to develop a stable and reliable passive dosing silver ion release device for use in future spacecraft potable water systems.

Tesia D Irwin↗
NASA NTRSDate not supplied

Microgravity Effect on Bacterial Growth: Further Clarification of the Underlying Mechanism

Gravity interacts with other physical environmental factors to impact the formation of today’s Earth and contribute to biological variations between water and land species. With their simple structures and small sizes, microbes are expected to be less gravity-sensitive than larger species. However, microgravity can significantly impact the mass transfer and interface behavior in the extracellular environment, and various effects of spaceflight on bacterial growth have been observed. Our earlier literature review summarizes the systematic efforts to understand the spaceflight effect on microbial growth through the extracellular mass transfer mechanism. It provides an in-depth literature review to address some discrepancies observed in the literature. This paper analyzes the effects of microgravity on the extracellular environment and their potential impact on bacterial growth to further clarify the underlying mechanism of the microgravity effect on bacterial growth.

Wenyan Li↗
NASA NTRSDate not supplied

Waste Container Filtration to Minimize Microbial Escape During Long-Term Storage on Mars

In the search for life on Mars, keeping its surface pristine and free of microbes due to human presence is of utmost importance. Astronauts will use emptied logistics containers during their Mars stay to store waste. These containers will be left on the planet’s surface and are a potential source of microorganism contamination. It is desired for these containers to be free from leaks for a minimum of 50 years. If leaks do occur, a likely location will be at any ports. At these locations the mating surfaces will be sealed using a flexible gasket material like a thin metal or a polymer. Eventually these seals will fail. To mitigate these effects, a particulate filter can be employed to equilibrate the pressure between the Martin environment and inside the container. To meet planetary protection requirements, the filter can only allow particulates smaller than 0.2 μm diameter to pass. This paper discusses why a particulate filter may be needed and how such a filter could be tested to survive and operate in the harsh Martian environment.

planetary protection↗
NASA NTRS2023

Biogeochemical evolution of ponded meltwater in a High Arctic subglacial tunnel

Subglacial environments comprise ∼10 % of Earth's land surface, host active microbial ecosystems, and are important components of global biogeochemical cycles. However, the broadly inaccessible nature of subglacial systems has left them vastly understudied, and research to date has been limited to laboratory experiments or field measurements using basal ice or subglacial water accessed through boreholes or from the glacier margin. In this study, we extend our understanding of subglacial biogeochemistry and microbiology to include observations of a slushy pond of water that occupied a remnant meltwater channel beneath a polythermal glacier in the Canadian High Arctic over winter. The hydraulics and geochemistry of the system suggest that the pond water originated as late-season, ice-marginal runoff with less than ∼15 % solute contribution from subglacial sources. Over the 8 months of persistent sub-zero regional temperatures, the pond gradually froze, cryo-concentrating solutes in the residual water by up to 7 times. Despite cryo-concentration and the likely influx of some subglacial solute, the pond was depleted in only the most labile and biogeochemically relevant compounds, including ammonium, phosphate, and dissolved organic matter, including a potentially labile tyrosine-like component. DNA amplicon sequencing revealed decreasing microbial diversity with distance into the meltwater channel. The pond at the terminus of the channel hosted a microbial community inherited from late-season meltwater, which was dominated by only six taxa related to known psychrophilic and psychrotolerant heterotrophs that have high metabolic diversity and broad habitat ranges. Collectively, our findings suggest that generalist microbes from the extraglacial or supraglacial environments can become established in subglacial aquatic systems and deplete reservoirs of nutrients and dissolved organic carbon over a period of months. These findings extend our understanding of the microbial and biogeochemical evolution of subglacial aquatic ecosystems and the extent of their habitability.

Ashley J Dubnick↗
NASA NTRSDate not supplied

Benchmarking Computational Tools for Calling SNPs and Indels in Complex Microbial Populations

The NASA BioNutrients missions seek to understand the suitability of microorganisms for bioproduction during space flight. One topic of interest is the stability of microbial genomes during long-term ambient storage and subsequent rehydration and growth. To address these questions, samples from 8 species were flown to ISS for 5 years of desiccated storage at ambient temperature (Stasis Packs) and 2 species were packaged along with powdered media inside a bioreactor system to allow hydration and growth in microgravity (Production Packs). For both systems, Whole Genome Sequencing (WGS) of the DNA extracted from the returned samples and paired ground controls will be conducted to identify changes in genome stability due to time, storage conditions and growth in space. Across the technical replicates, ground controls, 10 timepoints, and multiple experimental conditions, ~300 samples have been selected for initial analysis with WGS sequencing to 100x coverage. A flexible and resource efficient mutation calling pipeline is needed to process this large dataset and allow for comparisons between species. Many bioinformatics tools for calling Indels and Single Nucleotide Variants (SNVs) are designed for use with pure isolates, where true variations from the reference genome are expected to dominate the reads aligning to the location of mutation. In contrast, DNA from the Stasis Pack (SP) samples was collected directly after recovery from desiccated storage and the Production Pack (PP) samples were collected after fermentation. In this context, reads with mutations are expected to be less frequent than reads that align with the reference genome, as each sample will include multiple lines of cells. Thus, BioNutrients samples are expected to be similar to samples from cancer cell or “pooled” sequencing approaches. In preparation for the analysis of the BioNutrients samples, we have tested three mutation calling tools (GATK for Microbes, BreSeq and DiscoSNP) designed for complex samples. A challenge of validating mutation identification pipelines is a lack of “Ground Truth” datasets, especially for complex samples. To compare these three tools, we sought to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO as part of the Space Algae-1 mission. Here we present a summary of these tools against the analysis originally conducted using the CRISP tool. Critical metrics are compared such as runtime, the number of SNPs, the number and size of Indels, and patterns of transversion and transitions identified by each tool are reported. By sharing these benchmarking results collected in support of the BioNutrients mission, we aim to guide others seeking to identify SNVs in similarly complex microbial samples.

Biology↗
NASA NTRSDate not supplied

Developing Autonomous Technologies for Biological Missions to Deep Space

In upcoming biological missions beyond low Earth orbit (LEO), the use of autonomous instrumentation will allow scientists to perform a variety of experiments, including the characterization of the response to different space environments (Moon, Mars, interplanetary space) using biological models like microbes, plants, organoids, and tissue chips. BioSentinel is an ongoing deep space mission, currently at over 50 million kilometers from Earth and the first instrument developed to perform biological experiments beyond LEO. Even though the primary objective of this CubeSat mission was to investigate the effects of the deep space radiation environment on budding yeast, the spacecraft bus (i.e., all the subsystems that support the biological payload like power, thermal, data telemetry, navigation, etc.) can accommodate a variety of biological (and physical) experiments and model organisms. LEIA, an upcoming CLPS mission to the lunar surface, uses a microfluidic and optical instrument based on BioSentinel to study the effects of the lunar environment on different cellular processes and on bioproduction of antioxidants. A new series of science mission concepts are being proposed to be accommodated into platforms like BioSentinel. These missions will investigate the response of a variety of organisms to the deep space environment, including but not limited to single-cell eukaryotes, cyanobacteria, plants (including crops), organoids, and tissue chips. In addition to optical absorbance measurements like the ones performed in BioSentinel (and LEIA), we are investigating the use of fluorescence detection, microscopy, sequencing devices, etc. Thus, instruments like the ones proposed here can be adapted to a variety of platforms like free-flyers, deployable payloads, landers, rovers, and the lunar Gateway. These technologies can be used as steppingstones for establishing a sustained human presence on the Moon and in deep space while providing knowledge for the development of potential countermeasures.

Sergio R Santa Maria↗
NASA NTRSDate not supplied

Precision Fermentation: Relieving the Crabtree Effect in S. cerevisiae through Genetic Engineering

Nutrient degradation in the current NASA pre-packaged food system poses significant challenges to crew health in missions beyond low earth orbit as certain vitamins and nutrients have been shown to degrade during extended storage, and resupply and fresh food items cannot be readily provided. BioNutrients(BN) uses synthetic biology in an effort to supplement the NASA food system by delivering in situ production of nutrients and therapeutics produced by genetically engineered microbes. One yeast S. cerevisiae strain utilized in BN is genetically modified to produce two types of antioxidants: zeaxanthin and β-carotene. Zeaxanthin is a non-provitamin A carotenoid and has been implicated in eye health and vision performance, and β-carotene is a provitamin A carotenoid. Microbial production of high-value nutrients can aid our understanding and address the issue of nutrient degradation and loss in the NASA food system. Although S. cerevisiae is an excellent candidate for a long shelf life, on-demand nutrient production system, its space applications are limited by the Crabtree effect where respiration is shut down in favor of fermentative metabolism when glucose is present in high concentrations, causing the release of unwanted ethanol. Current and likely future life support systems are negatively impacted by ethanol and ethanol release is strictly limited.To improve feasibility of using S. cerevisiae, we explored ways to reduce the Crabtree effect, thereby limiting its ethanol production. Specifically, we used CRISPR-cas9 to genetically modify various genes in this pathway

E Zaroff↗
NASA NTRSDate not supplied

The Lunar Explorer Instrument for Space Biology Applications (LEIA) Pre-Flight Tests: Mechanical Considerations​

The BioSensor payload on the upcoming LEIA platform aboard the CLPS (CP-22) lander will carry yeast to the Moon's south pole to study the effects of lunar radiation and gravity. From Earth, BioSensor will transport desiccated yeast within 16 fluidic cards, each containing 16 fluidic wells of 100 microliters volume. At the start of the experiment, these microbes will be autonomously rehydrated on the Moon, and optical absorbance measurements at three wavelengths will be performed across all 256 wells. Absorbance measurements in fluidic wells are susceptible to noise and artifacts due to bubble formation during well rehydration and microbial growth. BioSensor, as a biofluidics system, faces significant data anomalies caused by payload vibrations, particularly from the operation of the co-located PROSPECT’s ProSEED drill, to assess lunar resources by penetrating the subsurface. To evaluate the impact of drill vibrations, fully assembled and filled fluidic cards were subjected to sine sweep signals in the x, y, z planes, ranging from 5 - 2560 Hz at g-loads of 0.25 and 0.5, with on-board accelerometer for localized vibration measurement. Wells with various fill conditions, including partial fills with different bubble sizes and completely filled wells, were tested. Results indicated that small bubbles adhered to well walls at high frequencies but were dislodged at low frequencies, causing data anomalies. Medium and large bubbles exhibited higher interference with optical data due to lower surface tension and variable bubble surface flexion. Overall, it was found that irrespective of the fill type, low-frequency vibrations were most detrimental to optical readings. Understanding the drill schedule and correlating it with BioSensor timestamps will help identify periods when BioSensor data is unreliable, a crucial step for accurately interpreting the microbial response data and ensuring the validity of the BioSensor's scientific measurements on the lunar surface.

Chinmayee Govinda Raj↗
NASA NTRSDate not supplied

Ultrashort Pulse Laser Surface Processing Techniques for Sterilization of Metal Surfaces for Planetary Protection

To prevent forward contamination from microbes aboard spacecraft intended to search for extraterrestrial life, there is a need for effective sterilization methods. However, current techniques are both time-consuming and expensive. For example, dry heat sterilization requires removal from the assembly site and several days of treatment. Furthermore, some components such as optics and electronics are not compatible with current sterilization techniques. Here, we report the latest results in our development of a novel femtosecond laser processing technique for the rapid sterilization of spacecraft hardware. Femtosecond lasers produce extremely high photon fluxes (10^29 photons/sec*cm^2, ~0.03 J/cm^2) in extremely short pulses, which can inactivate even stress-tolerant microbial spores with minimal damage to the spacecraft surface. Aluminum coupons were inoculated with specific densities of Bacillus subtilis bacterial endospores. These coupons were treated with various laser illumination parameters. Afterward, metal coupon samples were assayed for viable spores using a polyvinyl alcohol (PVA) peel, serial dilution, and plating for colony-forming units (CFU). Results indicate that with high enough energy density and pulse counts, most bacterial spores are inactivated with minimal damage to the metal. The sterilization is dependent on both the fluence and pulse count. In addition, femtosecond pulses are more effective than longer pulses for inactivation. These experiments have consistently achieved 4-log reduction in viable spores. Sterilization has been achieved on both flat metal coupons and non-flat surfaces with microchannels, with a slight reduction in sterilization efficiency on the uneven surface. The application of air flow during laser processing was also investigated as a way to remove spores that are dislodged from the surface by the laser illumination, which would contribute to the reduction of spacecraft bioburden. With laser processing technology rapidly evolving, our results support the possibility of an extremely rapid, in-situ surface sterilization method for use in spacecraft assembly clean rooms.

Kaleb McQuillan↗