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At least 775 records · Page 43

Application of Whole Genome Sequencing and MALDI-TOF to the Identifcation of Bacillus Species Isolated from Cleanrooms at NASA Johnson Space Center

Astromaterial cleanrooms at NASA Johnson Space Center are built environments that hold samples, such as lunar rocks, from different space exploration missions. Bacillus sp. are frequently detected in routine microbial monitoring of these facilities. Since this, and related genera, can form endospores that can withstand harsh conditions, they could contaminate astromaterials. This could confound searches for extraterrestrial life. Whole genome sequencing (WGS) is widely used for identifying bacterial strains and tracking their source; however, WGS is expensive and time consuming. Matrix-assisted laser desorption ionization– time of flight mass spectrometry (MALDI-TOF) shows promise as a low-cost, rapid method of identifying strains of bacteria, but few studies have compared this proteomics method to WGS. To evaluate a high throughput method of tracking the source of contamination of this built environment, WGS and MALDI-TOF was conducted on 18 bacterial strains isolated from surfaces in astromaterials cleanrooms. WGS identified 14 Bacillus, 2 Paenibacillus, 1 Solibacillus and 1 Alcaligenes strains. These isolates showed similarity to strains commonly observed in spacecraft assembly cleanrooms at other facilities. Cluster analysis of mass spectra generated by MALDI-TOF grouped strains together that were greater than 94% similar to each other in terms of amino acid sequences of single copy core genes, as assessed by WGS. This suggests that MALDI-TOF and WGS results are consistent with each other and MALDI-TOF can rapidly identify strains of Bacillus sp. isolated from cleanroom environments with a resolution comparable to WGS. Based on phylogenomic analysis, these results also suggest the presence of a cosmopolitan class of Bacillus sp. that are more likely to be found in cleanrooms and similar built environments than in natural systems.

Farnaz Mazhari↗

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

NASA Discipline Environmental Health↗

Large Plant Growth Chambers: Flying Soon on a Space Station near You!

The International Space Station (ISS) now has platforms for conducting research on horticultural plant species, and those capabilities continue to grow. The Veggie vegetable production system will be deployed to the ISS in Spring of 2014 to act as an applied research platform with goals of studying food production in space, providing the crew with a source of fresh food, allowing behavioral health and plant microbiology experimentation, and being a source of recreation and enjoyment for the crew. Veggie was conceived, designed, and constructed by Orbital Technologies Corporation (ORBITEC, Madison, WI). Veggie is the largest plant growth chamber that NASA has flown to date, and is capable of growing a wide array of horticultural crops. It was designed for low energy usage, low launch mass and stowage volume, and minimal crew time requirements. The Veggie flight hardware consists of a light cap containing red (630 nanometers), blue, (455 nanometers) and green (530 nanometers) light emitting diodes. Interfacing with the light cap is an extendable bellows baseplate secured to the light cap via magnetic closures and stabilized with extensible flexible arms. The baseplate contains vents allowing air from the ISS cabin to be pulled through the plant growth area by a fan in the light cap. The baseplate holds a Veggie root mat reservoir that will supply water to plant pillows attached via elastic cords. Plant pillows are packages of growth media and seeds that will be sent to ISS dry and installed and hydrated on orbit. Pillows can be constructed in various sizes for different plant types. Watering will be via passive wicking from the root mat to the pillows. Science procedures will include photography or videography, plant thinning, pollination, harvesting, microbial sampling, water sampling, etcetera. Veggie is one of the ISS flight options currently available for research investigations on plants. The Plant Habitat (PH) is being designed and constructed through a NASA-ORBITEC collaboration, and is scheduled to fly on ISS around 2016. This large plant chamber will control light quality, level, and timing, temperature, CO2, relative humidity, and irrigation, while scrubbing ethylene. Additional monitoring capabilities include leaf temperature sensing and root zone moisture and oxygen sensing. The PH light cap will have red (630 nanometers), blue (450 nanometers), green (525 nanometers), far red (730 nanometers) and broad spectrum white light emitting diodes. There will be several internal cameras to monitor and record plant growth and operations.

Space Biology↗

Handling Procedures of Vegetable Crops

The National Aeronautics and Space Administration (NASA) is working towards future long duration manned space flights beyond low earth orbit. The duration of these missions may be as long as 2.5 years and will likely include a stay on a lunar or planetary surface. The primary goal of the Advanced Food System in these long duration exploratory missions is to provide the crew with a palatable, nutritious, and safe food system while minimizing volume, mass, and waste. Vegetable crops can provide the crew with added nutrition and variety. These crops do not require any cooking or food processing prior to consumption. The vegetable crops, unlike prepackaged foods, will provide bright colors, textures (crispy), and fresh aromas. Ten vegetable crops have been identified for possible use in long duration missions. They are lettuce, spinach, carrot, tomato, green onion, radish, bell pepper, strawberries, fresh herbs, and cabbage. Whether these crops are grown on a transit vehicle (e.g., International Space Station) or on the lunar or planetary surface, it will be necessary to determine how to safely handle the vegetables while maintaining acceptability. Since hydrogen peroxide degrades into water and oxygen and is generally recognized as safe (GRAS), hydrogen peroxide has been recommended as the sanitizer. The objective of th is research is to determine the required effective concentration of hydrogen peroxide. In addition, it will be determined whether the use of hydrogen peroxide, although a viable sanitizer, adversely affects the quality of the vegetables. Vegetables will be dipped in 1 % hydrogen peroxide, 3% hydrogen peroxide, or 5% hydrogen peroxide. Treated produce and controls will be stored in plastic bags at 5 C for up to 14 days. Sensory, color, texture, and total plate count will be measured. The effect on several vegetables including lettuce, radish, tomato and strawberries has been completed. Although each vegetable reacts to hydrogen peroxide differently, the data suggest that 5% hydrogen peroxide reduces the shelf life of the vegetable. A dip of either 1 % or 3% hydrogen peroxide helps reduce the microbial total count while not adversely affecting the quality of the vegetable.

Perchonok, Michele↗

The Use of Liquid Isopropyl Alcohol and Hydrogen Peroxide Gas Plasma to Biologically Decontaminate Spacecraft Electronics

Legitimate concern exists regarding sending spacecraft and their associated hardware to solar system bodies where they could possibly contaminate the body's surface with terrestrial microorganisms. The NASA approved guidelines for sterilization as set forth in NPG 8020.12C, which is consistent with the biological contamination control objectives of the Committee on Space Research (COSPAR), recommends subjecting the spacecraft and its associated hardware to dry heat-a dry heat regimen that could potentially employ a temperature of 110(deg)C for up to 200 hours. Such a temperature exposure could prove detrimental to the spacecraft electronics. The stimulated growth of intermetallic compounds (IMCs) in metallic interconnects and/or thermal degradation of organic materials composing much of the hardware could take place over a prolonged temperature regimen. Such detrimental phenomena would almost certainly compromise the integrity and reliability of the electronics. Investigation of sterilization procedures in the medical field suggests that hydrogen peroxide (H202) gas plasma (HPGP) technology can effectively function as an alternative to heat sterilization, especially for heat-sensitive items. Treatment with isopropyl alcohol (IPA) in liquid form prior to exposure of the hardware to HPGP should also prove beneficial. Although IPA is not a sterilant, it is frequently used as a disinfectant because of its bactericidal properties. The use of IPA in electronics cleaning is widely recognized and has been utilized for many years with no adverse affects reported. In addition, IPA is the principal ingredient of the test fluid used in ionic contamination testers to assess the amount of ionic contamination found on the surfaces of printed wiring assemblies. This paper will set forth experimental data confirming the feasibility of the IPA/H202 approach to reach acceptable microbial reduction (MR) levels of spacecraft electronic hardware. In addition, a proposed process flow in which both IPA liquid and HPGP are utilized will be presented in Section 7.0.

microbial reduction↗

On the use of antibiotics to reduce rhizoplane microbial populations in root physiology and ecology investigations

No straightforward method exists for separating the proportion of ion exchange and respiration due to rhizoplane microbial organisms from that of root ion exchange and respiration. We examined several antibiotics that might be used for the temporary elimination of rhizoplane bacteria from hydroponically grown wheat roots (Triticum aestivum cv. Veery 10). Each antibiotic was tested for herbicidal activity and plate counts were used to enumerate bacteria and evaluate antibiotic kinetics. Only lactam antibiotics (penicillins and cephalosporins) did not reduce wheat growth rates. Aminoglycosides, the pyrimidine trimethoprim, colistin and rifampicin reduced growth rates substantially. Antibiotics acted slowly, with maximum reductions in rhizoplane bacteria occurring after more than 48 h of exposure. Combinations of nonphytotoxic antibiotics reduced platable rhizoplane bacteria by as much as 98%; however, this was generally a reduction from about 10(9) to 10(6) colony forming units per gram of dry root mass, so that many viable bacteria remained on root surfaces. We present evidence which suggests that insufficient bacterial biomass exists on root surfaces of nonstressed plants grown under well-aerated conditions to quantitatively interfere with root nitrogen absorption measurements.

NASA Discipline Life Support Systems↗

VEG-04 Pick-and-Eat Crop Production and Human Research on the International Space Station

Growing fresh, nutritious, palatable produce for crew consumption during spaceflight may provide health-promoting, bioavailable nutrients and enhance the astronaut dietary experience as we move toward longer-duration missions. Tending plants may also serve as a countermeasure for crew psychological stresses associated with spaceflight. However, requirements to support consistent growth of a variety of nutritious crops under spaceflight environmental conditions remain unclear. This study explores the potential to grow crops for consumption on the International Space Station (ISS) using the Veggie vegetable-production system. VEG-04A and B were two flight tests conducted in 2019 with the leafy green crop Mizuna mustard. Mizuna was grown in two Veggie chambers simultaneously, with the chambers set to different red-to-blue light formulations; one Veggie was programmed as “red-rich” and the second as “blue-rich.” Light quality is known to impact plant growth, nutrition, microbiology, and organoleptic characteristics on Earth, and the Veggie flight tests examined how these impacts might differ in microgravity. VEG-04A was a 35-day growth test with a single harvest. VEG-04B, a 58-day test with harvests at four, six, and eight weeks from the same plants, assessed sustained productivity. Challenges with the watering program occurred early during VEG-04A, and several plants failed to survive in both the flight and ground control operations. Thus, prior to VEG-04B, an extra test was conducted to tailor water timing and volumes. This test allowed a fine tuning of methods for VEG-04B, and generally plant survival was better in that experiment. At each harvest, the astronauts froze half of the edible plant tissue to return to Earth and weighed the remaining half using the ISS Mass Measurement Device (MMD). Weighed samples were then cleaned with produce-sanitizing wipes, and consenting crew members participated in organoleptic evaluation of the fresh produce. Organoleptic evaluations were conducted on the Mizuna crops grown in both light treatments, and data from these tests are compared to ground data collected at JSC. The remaining sanitized produce was available for crew consumption as desired. Frozen flight samples were returned for microbial and chemical analyses to assess food safety and nutritional quality. Microbial assessments included culturing and identifying aerobic bacteria, yeasts, and molds, and screening for specific human pathogens. Chemical nutrient analyses included assessing elements, antioxidants, and phenolics in plant tissues. Crew members involved in plant operations completed behavioral health surveys pre-flight, in-flight associated with plant growth and harvest activities, and post-flight. Surveys captured the amount of time spent on different plant-related operations, enjoyment of the different activities, engagement with Veggie, experience interacting with Veggie, and sensory stimulation associated with growing plants in Veggie.

Veggie↗

VEG-04 PICK-AND-EAT CROP PRODUCTION AND HUMAN RESEARCH ON THE INTERNATIONAL SPACE STATION

Growing fresh, nutritious, palatable produce for crew consumption during spaceflight may provide health-promoting, bioavailable nutrients and enhance the astronaut dietary experience as we move toward longer-duration missions. Tending plants may also serve as a countermeasure for crew psychological stresses associated with spaceflight. However, requirements to support consistent growth of a variety of nutritious crops under spaceflight environmental conditions remain unclear. This study explores the potential to grow crops for consumption on the International Space Station (ISS) using the Veggie vegetable-production system. VEG-04A and B were two flight tests conducted in 2019 with the leafy green crop Mizuna mustard. Mizuna was grown in two Veggie chambers simultaneously, with the chambers set to different red-to-blue light formulations; one Veggie was programmed as “red-rich” and the second as “blue-rich.” Light quality is known to impact plant growth, nutrition, microbiology, and organoleptic characteristics on Earth, and the Veggie flight tests examined how these impacts might differ in microgravity. VEG-04A was a 35-day growth test with a single harvest. VEG-04B, a 58-day test with harvests at four, six, and eight weeks from the same plants, assessed sustained productivity. Challenges with the watering program occurred early during VEG-04A, and several plants failed to survive in both the flight and ground control operations. Thus, prior to VEG-04B, an extra test was conducted to tailor water timing and volumes. This test allowed a fine tuning of methods for VEG-04B, and generally plant survival was better in that experiment. At each harvest, the astronauts froze half of the edible plant tissue to return to Earth and weighed the remaining half using the ISS Mass Measurement Device (MMD). Weighed samples were then cleaned with produce-sanitizing wipes, and consenting crew members participated in organoleptic evaluation of the fresh produce. Organoleptic evaluations were conducted on the Mizuna crops grown in both light treatments, and data from these tests are compared to ground data collected at JSC. The remaining sanitized produce was available for crew consumption as desired. Frozen flight samples were returned for microbial and chemical analyses to assess food safety and nutritional quality. Microbial assessments included culturing and identifying aerobic bacteria, yeasts, and molds, and screening for specific human pathogens. Chemical nutrient analyses included assessing elements, antioxidants, and phenolics in plant tissues. Crew members involved in plant operations completed behavioral health surveys pre-flight, in-flight associated with plant growth and harvest activities, and post-flight. Surveys captured the amount of time spent on different plant-related operations, enjoyment of the different activities, engagement with Veggie, experience interacting with Veggie, and sensory stimulation associated with growing plants in Veggie. Within each study, plant growth did not differ across light treatment or location (flight versus ground). In general, more biomass was produced in most treatments during the longer study, but growth of this crop declined over time with the repeated harvests. On average, bacterial and fungal counts were significantly lower on ground control samples than flight samples, and microbial counts increased with repeated harvests. Light treatment did not influence any elements in tissues tested; however, the growth duration did impact levels of several elements. Organoleptic scores were generally higher in flight, and ground tasters considered samples more bitter. Amount of interaction and responses to Veggie varied widely by individual. Enjoyable tasks had higher impact than non-enjoyable tasks and interacting with Veggie was generally viewed as positive. These tests on ISS are helping to mitigate the risk of an inadequate food supply for long-duration missions by adding fresh vegetables and key nutrients to the crew diet, and indicating which plant care activities are providing behavioral health benefits for the crew. This research was co-funded by the Human Research Program and Space Biology (MTL#1075) in the ILSRA 2015 NRA call. Videos due Jan. 25th.

Veggie↗

EDL Simulation Results for the Mars 2020 Landing Site Safety Assessment

The Mars 2020 rover is NASA’s next flagship mission, set to explore Mars in search of scientific evidence of past microbial life. Importantly, the rover will also, for the first time, have the ability to collect and cache rock and soil samples for retrieval and return to laboratories here on Earth. A key step in the development of the Mars 2020 mission is the selection of a suitable landing site with the largest likelihood of meeting scientific goals. This decision is a complex and critical one that requires close interaction between the scientific and engineering communities. The chosen landing site must be both scientifically interesting — providing the project with the greatest possible chance of gathering credible and defendable scientific evidence — and also safe enough to attempt a landing in the first place. Thus, arguably one of the most important undertakings of the Entry, Descent, and Landing (EDL) team, is to effectively enumerate, quantify, and communicate the landing risks to all of the stakeholders. The culmination of this effort is the Landing Site Safety Assessment, which is a review commissioned by the project, presided over by the EDL Standing Review Board, and attended by management and science stakeholders, in which the EDL team communicates their assessment of the associated landing risks and the statistical probability of a successful landing at each of the final candidate landing sites. This paper summarizes the results of high-fidelity computer simulations of the Mars 2020 EDL sequence used in this assessment. From an EDL performance perspective, all four candidates offer similar level of robustness, which is in-family with Mars Science Laboratory (MSL). However, two new features of the Mars 2020 EDL sequence – range trigger and Terrain-Relative Navigation (TRN) – dramatically enhance the capability of the EDL system to safely land at landing sites with much more rugged terrain than ever before considered. This has allowed the landing site selection for Mars 2020 to proceed in a manner that has been unprecedentedly weighted more heavily toward scientific interest and less heavily on engineering constraints. With TRN, the overall probability of success is predicted to be approximately 99% for all of the candidates.

David Way↗

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.↗

Congruence of Clusters Defined By Whole Genome Sequencing and MALDI-TOF for Bacteria Isolated From Cleanrooms

Introduction: Oligotrophic conditions can render cleanrooms inhospitable to microbes. Despite these constraints, fungi and bacteria are frequently isolated from surfaces in astromaterials cleanrooms at the Johnson Space Center. Bacillus species are of particular concern because endospores belonging to this genus are resilient and can affect astromaterials. Current monitoring programs rely on 16S rRNA sequencing and the VITEK2 Compact system. These methods have limited power to resolve Bacillus species. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to the industry gold standard, whole genome sequencing (WGS). Methods: Based on 16S rRNA classification, we selected 14 isolates for analysis with MALDI-TOF and WGS. Mass spectra were generated with MALDI-TOF MS and processed with custom scripts to identify clusters of closely related isolates and calculate a matrix of pairwise cosine similarity scores. Hybrid Illumina and Nanopore sequencing were used to generate draft genomes. Pairwise similarity scores were calculated from these genomes based on the average amino acid identity (AAI) predicted from single copy core genes. Congruence of clustering between these methods, was assessed by calculating adjusted Rand and Wallace coefficients. Results: Clusters of species generated from MALDI-TOF MS showed good agreement of phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other in terms of predicted amino acid sequences consistently showed cosine similarities of mass spectra > 0.65 and, of the 9 clusters identified with WGS, 8 were identical with MALDI-TOF. This corresponds to an adjusted Rand index of 0.95 and a 95% confidence interval of 0.80 – 1.00 for adjusted Wallace coefficients. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.84) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. Conclusion: This study shows that MALDI-TOF and WGS exhibit a similar ability to delineate Bacillus species isolated from cleanrooms and taxonomic units described by these two methods are consistent with one another. Since MALDI-TOF MS is low in cost and high in throughput, this approach appears to be an ideal option for routine microbial monitoring and identifying Bacillus species.

Farnaz Mazhari↗

Stable isotopic biogeochemistry of carbon and nitrogen in a perennially ice-covered Antarctic lake

Lake Hoare (77 degrees 38' S, 162 degrees 53' E) is an amictic, oligotrophic, 34-m-deep, closed-basin lake in Taylor Valley, Antarctica. Its perennial ice cover minimizes wind-generated currents and reduces light penetration, as well as restricts sediment deposition into the lake and the exchange of atmospheric gases between the water column and the atmosphere. The biological community of Lake Hoare consists solely of microorganisms -- both planktonic populations and benthic microbial mats. Lake Hoare is one of several perennially ice-covered lakes in the McMurdo Dry Valleys that represent the end-member conditions of cold desert and saline lakes. The dry valley lakes provide a unique opportunity to examine lacustrine processes that operate at all latitudes, but under an extreme set of environmental conditions. The dry valley lakes may also offer a valuable record of catchment and global changes in the past and present. Furthermore, these lakes are modern-day equivalents of periglacial lakes that are likely to have been common during periods of glacial maxima at temperate latitudes. We have analyzed the dissolved inorganic carbon (DIC) of Lake Hoare for delta 13C and the organic matter of the sediments and sediment-trap material for delta 13C and delta 15N. The delta 13C of the DIC indicates that 12C is differentially removed in the shallow, oxic portions of the lake via photosynthesis. In the anoxic portions of the lake (27-34 m) a net addition of 12C to the DIC pool occurs via organic matter decomposition. The dissolution of CaCO3 at depth also contributes to the DIC pool. Except near the Canada Glacier where a substantial amount of allochthonous organic matter enters the lake, the organic carbon being deposited on the lake bottom at different sites is isotopically similar, suggesting an autochthonous source for the organic carbon. Preliminary inorganic carbon flux calculations suggest that a high percentage of the organic carbon fixed in the water column is remineralized as it falls through the water column. At nearby Lake Fryxell, the substantial (relative to Lake Hoare) glacial meltstream input overprints Fryxell's shallow-water biological delta 13C signal with delta 13C-depleted DIC. In contrast, Lake Hoare is not significantly affected by surface-water input and mixing, and therefore the delta 13C patterns observed arise primarily from biological dynamics within the lake. Organic matter in Lake Hoare is depleted in 15N, which we suggest is partially the result of the addition of relatively light inorganic nitrogen into the lake system from terrestrial sources.

NASA Program Exobiology↗

Health significance and occurrence of injured bacteria in drinking water

Enteropathogenic and indicator bacteria become injured in drinking water with exposure to sublethal levels of various biological, chemical and physical factors. One manifestation of this injury is the inability to grow and form colonies on selective media containing surfactants. The resulting underestimation of indicator bacteria can lead to a false estimation of water potability. m-T7 medium was developed specifically for the recovery of injured coliforms (both "total" and fecal) in drinking water. The m-T7 method was used to survey operating drinking water treatment and distribution systems for the presence of injured coliforms that were undetected with currently used media. The mean recovery with m-Endo LES medium was less than 1/100 ml while it ranged between 6 and 68/100ml with m-T7 agar. The majority of samples giving positive results with m-T7 medium yielded no detectable coliforms with m-Endo LES agar. Over 95% of the coliform bacteria in these samples were injured. Laboratory experiments were also done to ascribe the virulence of injured waterborne pathogens. Enteropathogens including Salmonella typhimurium, Yersinia enterocolitica and Shigella spp. required up to 20 times the chlorine levels to produce the same injury in enterotoxigenic Escherichia coli (ETEC) and nonpathogenic coliforms. Similar results were seen with Y. enterocolitica exposed to copper. The recovery of ETEC was followed by delayed enterotoxin production, both in vitro and in the gut of experimental animals. This indicates that injured waterborne enteropathogenic bacteria can be virulent.

NASA Discipline Number 13-40↗

Establishing and Monitoring an Aseptic Workspace

When are aseptic operations necessary? In order to meet certain bioburden requirements, some components must undergo dry heat microbial reduction (DHMR) or other sterilizing procedures. If sensitive surfaces must be re-exposed after DHMR, this could compromise the bioburden levels. Recontaminating sterilized surfaces could be costly both in time by requiring repeated DHMR and risk to the hardware, which may not be compatible with repeated high temperature bakes. In order to prevent recontamination of the sensitive surfaces, an aseptic environment and sterile technique must be employed. Aseptic environments mean working in a space with almost no detectable bioburden in the air or on surfaces. Ideally, DHMR happens as late as possible to avoid requiring aseptic operations, as it can be considered a high-risk operations. Preparing the cleanroom for aseptic operations Establishing an ISO (International Organization for Standardization) class 5 space to minimize airborne particles. Maintain low bioburden in the cleanroom by using biocidal cleaners. Using multiple biocidal techniques decreases the likelihood of selecting for resistant microorganisms. 70% Isopropyl Alcohol (IPA) denatures the proteins in a microorganism (note: 70% IPA is better at killing microorganisms than 100% IPA) 7% hydrogen peroxide: damages DNA and proteins through oxygen radical damage. Ultraviolet-C (UV-C) lamps: causes crosslinking in DNA which prevents replication. Monitor cleanroom regularly for bioburden trending: Standard bioassay: Swab or wipe samples of cleanroom surfaces processed for colony forming unity (viable or spore selected); Rapid bioassay: Adenosine triphosphate (ATP) or Limulus amebocyte lysate (LAL) for a bioburden snapshot. High levels can signal an immediate re-cleaning before standard bioassay samples are taken. Airborne monitoring: Active (pulling air through a filter) or passive (particle fallout) for bioburden. Verify bioburden levels just before aseptic operation. Test hardware and cleanroom surfaces and air 3 days before the planned aseptic operation. Rapid bioburden just before aseptic operation to ensure room was not re-contaminated. Preparing personnel and tools: Personnel training. Everyone in the cleanroom: Standard cleanroom certification Everyone on the team: 1 day Planetary Protection overview. Aseptic operators only: Half-day aseptic operations training. Covers sterile garmenting/gloves, Sterile handling with a focus on contact transfer risk, tool/GSE preparation, and two-operator system for opening sterilized tools/components. Tool sterilization: All tools to be used during an aseptic operation need to be identified. Compatible tools are sterilized by DHMR or Autoclave. Double wrapped so that the exterior bag can be handled by a non-sterile operator, and the sterile. Tools that are not compatible with high heat do not come in contact with sensitive surfaces: either substitutes are found, or tools are isolated by wrapping in sterile foil. During an aseptic operation. Pre-task to make sure everyone understands the operations, who is handling what, and when the most critical surfaces will be exposed. Monitoring during the operation. Bioburden: active and passive airborne bioburden sampling, glove-tip dabs onto a plate after completion of operation (3 days for results). Particles: real time particle counter constantly running, with alarm for exceeding ISO 5 conditions.

aseptic processing↗

The effect of simulated microgravity on bacteria from the Mir space station

The effects of simulated microgravity on two bacterial isolates, Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water systems aboard the Mir space station were examined. These bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method of enumeration and medium. In contrast, for R. pickettii (that was non-motile and lacked flagella), there were significantly higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity depended on the strain used (e.g., the motile strain exhibited no response to microgravity, while the non-motile strain did), the method of enumeration, and the nutrient concentration of the medium. Under oligotrophic conditions, non-motile cells may remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the cell may be sufficient so that high growth is observed until nutrients becoming limiting.

Non-NASA Center↗

Artificial Intelligence Medical Support for Long-Duration Space Missions

We envision an artificial intelligence (AI) based system that will provide support and recommendations to the crew medical officer (CMO) and ground flight surgeon during long-duration space missions. Such a system would be pretrained on the knowledgebase of clinical knowledge on Earth, minimizing the amount of Earth data that needs to be transferred into space. Then during deployment, the system would be constantly refined through active learning from diverse streams of data from sensors in the spacecraft, data collected daily from individual astronauts, and human-in-the-loop feedback from the crew. The model could be interrogated for predictions and recommendations on personalized crew health based on the overall status of the spacecraft, medicinal stores, and status of other crew members. Adaptation techniques would be used to incorporate spaceflight data that have very different distributions from the training data due to the extreme environment. Edge computing and the most advanced neuromorphic processing would enable computation in scenarios with low power and bandwidth, while dimensionality reduction would be employed to ensure that the input data streams from spaceflight are as small as possible. In order to realize this long-term vision, several hardware and software aspects need to be developed and assembled. First, models pretrained on Earth biomedical data would need to be evaluated for predictive accuracy, and the best one selected. That model would need to be adapted to learn from diverse, sparse, and inconsistently measured data streams, as well as human-in-the-loop feedback. A data integration, standardization, and dimensionality reduction methodology would need to be developed to handle all data types and feed them into the model. Once the software and data infrastructure is developed, it would need to be integrated with small footprint compute processors and tested in high-radiation, high-vibration, unregulated temperature situations. As a short-term goal, we recommend to focus on the development of the data and model software structure. Several large language models (LLM) already exist that have been trained on Earth biomedical and clinical knowledgebases, including BioMedLLM, Med-PaLM, SPOKE LLM, and Foresight. These models need to be evaluated for accuracy and the best one chosen for a proof-of-concept structure, while maintaining awareness of the accelerating AI field and incorporating any newly improved model architectures as needed. Then, we recommend to develop a database of synthetic data types to mimic the diverse data streams that are expected in a long-duration space mission. This should include environmental and microbial data from the spacecraft, non-invasive data from wearables and point-of-care devices employed by astronauts, and more invasive molecular and physiological monitoring of clinical and biomarker data from astronauts. The data standardization methodology should be developed, and these data streams used to refine the clinical LLM. Several scenarios should be developed that could plausibly come up in a long-duration space mission, and changes or aberrations introduced to the data at specific times to mimic these scenarios. Then, question and answer tasks should be designed to interrogate the model for predictions and recommendations, with acceptable answers already identified.

Artificial Intelligence↗

Pick-and-Eat Space Crop Production Flight Testing on the International Space Station

Growing fresh, nutritious, palatable produce for crew consumption during spaceflight may provide health-promoting, bioavailable nutrients and enhance the astronaut dietary experience as we move toward longer-duration missions. However, requirements to support consistent growth of a variety of high-quality crops under spaceflight environmental conditions remain unclear. This study explores the potential to grow crops for consumption on the International Space Station (ISS) using Veggie, NASA’s vegetable production chamber system. VEG-04A and VEG-04B were two flight tests with ground components conducted with the leafy green crop mizuna mustard. In each location, mizuna was grown in two Veggie units simultaneously, with the chambers set to different red-to-blue-to-green light formulations. Preflight verification testing with various lighting treatments was conducted to down-select two treatments that contributed to the best desirable growth and sensory qualities in mizuna mustard. For the flight tests, one Veggie was programmed as “red-rich” with an average of 270 μmol m-2 s-1 of 630 nm red light, 30 μmol m-2 s-1 of 455 nm blue light, and 30 μmol m-2 s-1 of 530 nm green light. The second Veggie was “blue-rich” with an average of 150 µmol m-2 s-1 of 630 nm red light, 150 µmol m-2 s-1 of 455 nm blue light, and 30 µmol m-2 s-1 of 530 nm green light. Light quality is known to impact plant growth, nutrition, microbiology, and sensory characteristics on Earth, and the Veggie flight tests examined how these impacts might differ in microgravity. VEG-04A, a 35-day growth test with a single harvest, was initiated in June and harvested in July 2019. VEG-04B, a 56-day test with three harvests from the same plants, assessed sustained productivity. Preflight testing for VEG-04B was conducted after the VEG-04A flight test to improve the operations, approaches, and watering requirements, which resulted in better crop establishment in the VEG-04B flight test. VEG-04B was initiated in October 2019 with harvests at four, six, and eight weeks after initiation. At all of the harvests, the astronauts froze half of the edible plant tissue to return to Earth and weighed the remaining half using the Mass Measurement Device (MMD). Weighed samples were then cleaned with produce-sanitizing wipes, and consenting crew members participated in sensory evaluations of the fresh produce. The remaining sanitized produce was available for crew consumption as desired. Frozen flight samples were returned to Earth for chemical and microbial analyses to assess nutritional quality and food safety. Flight-grown mizuna was generally more acceptable to the crew and had higher nutrient levels, although mizuna grown in the ground control performed better in terms of higher yield and lower microbial load. This presentation will focus on results from the nutritional and sensory analyses, including how nutrients identified as key for supplementing the crew diet varied across lighting treatments, harvest approaches, and spaceflight versus ground conditions. It is our hope that these tests on the ISS will help mitigate the risk of an inadequate food supply for long-duration missions by adding fresh vegetables to the crew diet. This study was supported by NASA’s Human Research and Space Biology Programs through the HERO NNJ13ZSA002N-ILSRA grant solicitation.

Jess Bunchek↗

Development of Storage Methods for Saccharomyces Strains to be Utilized for In Situ Nutrient Production in Long-Duration Space Missions

Long-duration space missions will benefit from closed-loop life support technologies that minimize mass, volume, and power as well as decrease reliance on Earth-based resupply. A system for In situ production of essential vitamins and nutrients can address the documented problem of degradation of stored food and supplements. Research has shown that the edible yeast Saccharomyces cerevisiae can be used as an on-demand system for the production of various compounds that are beneficial to human health. A critical objective in the development of this approach for long-duration space missions is the effective storage of the selected microorganisms. This research investigates the effects of different storage methods on survival rates of the non-sporulating probiotic S. boulardii, and S. cerevisiae spores and vegetative cells. Dehydration has been shown to increase long-term yeast viability, which also allows increased shelf-life and reduction in mass and volume. The process of dehydration causes detrimental effects on vegetative cells, including oxidative damage and membrane disruption. To maximize cell viability, various dehydration methods are tested here, including lyophilization (freeze-drying), air drying, and dehydration by vacuum. As a potential solution to damage caused by lyophilization, the efficacy of various cryoprotectants was tested. Furthermore, in an attempt to maintain higher survival rates, the effect of temperature during long-term storage was investigated. Data show spores of the wild-type strain to be more resilient to dehydration-related stressors than vegetative cells of either strain, and maintain high viability rates even after one year at room temperature. In the event that engineering the organism to produce targeted nutrient compounds interferes with effective sporulation of S. cerevisiae, a more robust method for improving vegetative cell storage is being sought. Therefore, anhydrobiotic engineering of S. cerevisiae and S. boulardii is being conducted.

life support↗