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NASA NTRSDate not supplied

PowerCell aboard the EuCROPIS satellite - Results from the first synthetic biology experiment in space

Synthetic biology is the design and construction of new biological parts and systems not found in nature, a technology that we believe will be a game-changing technology for space exploration. The PowerCell concept is an ecology based on photosynthetic microbes taking advantage of in situ materials and energy to generate, on-demand, useful products (food, fuel, cloths, drugs, etc.) that satisfy specific needs of long-term human presence away from Earth. This first PowerCell experiment is testing this concept as well as several key mechanisms of engineering biology in a 48-well plate microfluidics system. NASA's PowerCell experiment was loaded in the fluidics system at NASA Ames in May 2016, and subsequently integrated on the DLR's compact satellite Eu:CROPIS (Euglena Combined Regenerative Organic food Production In Space), as a secondary payload. Launched into Earth’s orbit from the Vandenberg Air Force Base, California, USA, in December 3, 2019, the Eu:CROPIS platform has provided an artificial gravity through rotational acceleration, simulating gravity ranges compatible to those found on Mars and on the Moon, as well as microgravity in the absence of acceleration. Each set of biological experiments performed at Earth's orbit was compared to ground controls. Over 400 days' worth of mission data showed that all hardware and software worked as planned, and microbial growth was observed in all four gravity regimes, including growth on cyanobacterial cell extract in three out of four gravity regimes. The genetic transformation experiment was successful in microgravity and provided valuable long-term reagent stability data, including antibiotic efficacy. The demonstrated ability of hardy bacterial spores to recover from a long-term stasis period will be fundamental for deep human space missions. Lessons learned from hardware design to experimental operations are leading to improved designs and capabilities for future missions.

Ivan Glaucio Paulino Lima↗

NASA NTRS2022

Adaptation to environmental extremes structures functional traits in biological soil crust and hypolithic microbial communities

Biological soil crusts (biocrusts) are widespread in drylands and deserts. At the microhabitat scale, they also host hypolithic communities that live under semi-translucent stones. Both environmental niches experience exposure to extreme conditions such as high UV radiation, desiccation, temperature fluctuations, and resource limitation. However, hypolithic communities are somewhat protected from extremes relative to biocrust communities. Conditions are otherwise similar, so comparing them can answer outstanding questions regarding adaptations to environmental extremes. Using metagenomic sequencing, we assessed the functional potential of dryland soil communities and identified the functional underpinnings of ecological niche differentiation in biocrusts versus hypoliths. We also determined the effect of the anchoring photoautotroph (moss or cyanobacteria). Genes and pathways differing in abundance between biocrusts and hypoliths indicate that biocrust communities adapt to the higher levels of UV radiation, desiccation, and temperature extremes through an increased ability to repair damaged DNA, sense and respond to environmental stimuli, and interact with other community members and the environment. Intracellular competition appears to be crucial to both communities, with biocrust communities waging war using the Type VI Secretion System (T6SS) and hypoliths favoring diversity of antibiotics. The dominant primary producer had a reduced effect on community functional potential compared with niche, but an abundance of genes related to monosaccharide, amino acid, and osmoprotectant uptake in moss-dominated communities indicates reliance on resources provided to heterotrophs by mosses. Our findings indicate that functional traits in dryland communities are driven by adaptations to extremes and we identify strategies that likely enable survival in dryland ecosystems.

Biological soil crusts↗

NASA NTRSDate not supplied

Microbial Monitoring of New Cleanrooms Used to Curate Astrobiologically Relevant Asteroid Samples from Bennu and Ryugu

Introduction: NASA has constructed two new cleanrooms to house materials from the OSRIS-REx and Hayabusa2 missions to the asteroids Ryugu (162173) and Bennu (101955), respectively. In accordance with standard astromaterials curation practices, these cleanrooms will be monitored for particulate contamination and maintained to ISO 5 equivalent standards1. Since the samples in these collections are expected to contain prebiotic organic compounds that may help explain the origin of life on Earth, these labs will also be monitored for organic and biological contamination2. Samples from Ryugu arrived on Earth in December, 2020. After basic characterization in Japan, NASA received a subset of these samples at the astromaterials curation facility in Houston in December of 2021. OSIRIS-REx is expected to return samples in September, 2023. Here we present preliminary microbial monitoring results from monthly monitoring of these new labs and the connected microtomy and staging areas that support them, as they are being commissioned. We also compare these results to baseline values for other astromaterials curation labs. We will also briefly describe additional cleaning efforts employed to reduce the bioburden in these new cleanrooms. Methods: Microbial samples were collected from surfaces using a dry macrofoam swab (Puritan Brand 2518051PFRNDFD). Swabs were also opened in the lab but not touched to any surfaces to function as negative controls. Samples and controls were processed inside a class II biosafety cabinet to avoid inadvertent cross contamination. The swabs were suspended in 15 ml of PBS (Phosphate Buffered Saline) and vortexed for 20 seconds to remove cells from the swab surface. The PBS was used to inoculate Petri dishes filled with TSA (Tryptic Soy Agar), Blood Agar, or Reasoners 2 agar to check for microbial growth. Each plate was inoculated with 0.1 ml of PBS. The TSA and blood agar plates were incubated at 35˚C and the Reasoners 2 agar plates were incubated at 25˚C for seven days. Petri dishes filled with Potato dextrose agar, Saboraud dextrose agar, or Saboraud dextrose agar with 0.1 mg/ml of chloramphenicol, an antibiotic, were used to check for fungal growth. These plates were inoculated with 0.3 ml of PBS and incubated at 30˚C. The remaining PBS was frozen at -80 ˚C for DNA sequencing. After incubation, isolates were counted and reisolated for identification. Isolates were identified using the VITEK23 system or by sequencing a portion of the 16S rRNA gene for bacteria or the ribosomal internal transcribed spacer (ITS) for fungi. Sequencing was performed with an ABI 3500 Sanger sequencer. Results: During our initial sampling, six of the seven sites sampled (86%) displayed bacterial or fungal growth. Samples collected from the staging areas and microtomy labs are not included in this calculation since those areas are maintained at a lower ISO 7 equivalent cleanliness standard. A month later, only three of the seven sites (43%) displayed bacterial growth. No fungal growth was detected in the second sampling. Since new equipment had been introduced to the Hayabusa2 lab since the first round of sampling, an additional three sampling sites were included in the second round of sampling. None of these sites displayed microbial growth. These sites will be included in all future sampling efforts. Bacterial isolates have been identified from the following genera at multiple time points: Micrococcus, Staphylococcus, and Bacillus. Isolates from the genera: Microbacterium, Nocardioides, Methylocystis, and Microvirga were identified in the initial sampling, but were not present at later time points. Identification of fungal isolates is in progress. Results are summarized in Table 1. Discussion: The recovery rate or percentage of positive samples4 was initially 86%, which is higher than the median recovery rate for comparable ISO 5 equivalent curation labs like Stardust (33%), Hayabusa (33%), and Cosmic Dust (50%). However, after a month of operation, the recovery rate for these same sites decreased to 43%, which is similar to what we observe in comparable curation cleanrooms with no microbial control requirements. Adding in the new sampling sites further decreases the recovery rate to 30%. With the reduction in recovery rate, we also observed a decrease in microbial diversity. At the first time point, we observed at least 10 different bacterial species and at least two different fungi. This is a higher diversity than the median values for comparable ISO 5 equivalent labs (2-4 isolates per sampling event). After the second sampling, we observed at least 4 bacterial species and no fungi, which is more consistent with comparable labs. We expect the recovery rate and diversity in both labs to continue to decrease as routine operation continues. We will use ultrapure hydrogen peroxide to disinfect equipment and work areas prior to opening any sample containers. Most of the bacterial and fungal isolates were detected on samples from the cleanroom floors. This is consistent with baseline results from other curation labs. Organisms from the genera Bacillus, Staphylococcus, and Micrococcus that were repeatedly detected are common in cleanrooms and on human skin5,6. These organisms are generally thought to be introduced when people enter the cleanroom. Microbacterium, Nocardioides, and Microvirga have also previously been identified in astromaterials cleanrooms, but not as frequently as Bacillus, Staphylococcus, and Micrococcus. Methylocystis is a novel genus in the astromaterials cleanrooms, but it was identified with low accuracy (93% match in the sequenced region of the 16S rRNA gene) and further work is needed to confirm this identification. Microbacterium is a diverse genus with isolates identified from terrestrial and aquatic sediments. Some species of Microbacterium are capable of degrading complex organic compounds found in crude oil. The presence of these bacteria in the OSIRIS REx and Hayabusa2 cleanrooms should be closely monitored. Methylocystis is a genus of methanotrophic bacteria capable of oxidizing methane. If this identification proves to be correct and it is detected again, it should be closely monitored as well. Under nominal operating conditions, samples should not ever encounter the cleanroom floor or other high traffic areas. If we observe an increase in the bioburden in sensitive work areas that appears to be influenced by organism transfer from high traffic areas like the floors, we can employ additional hydrogen peroxide treatments to disinfect high traffic areas. Routine microbial monitoring of these labs will ensure that NASA’s astromaterials collections remain pristine and useful for scientific study. Table 1. Sampling Locations and Colony Counts Bacterial CFUa Fungal CFU Bacterial CFU Fungal CFU Lab - Location 11/2/2021 11/2/2021 12/13/2021 12/13/2021 H2b-Floor 4 8 1 0 H2-staging pass through 3 0 0 0 H2-microtomy pass through TNTCc 0 0 0 H2 Microscope 1 NA NA 0 0 H2 Microscope 2 NA NA 0 0 H2-Table NA NA 0 0 OREXd- microtomy pass through 0 0 6 0 OREX – Anteroom pass through 0 0 0 0 OREX – Floor 1 2 0 0 OREX Witness Foil Table 3 0 1 0 Staging-Floor 16 0 15 0 Microtomy-Floor 3 0 2 0 a: CFU = Colony Forming Unit b: H2 = Hayabusa2 Lab c: TNTC = too numerous to count d: OREX = OSIRIS-REx Lab References: 1. ISO 14644-1:2015 - Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration. 37 (2015). 2. McCubbin, F. M. et al. Space Sci Rev 215, (2019). 3. Pincus, D. H. Encyclopedia of Rapid Microbiological Methods (2005). 4. The United States Pharmacopeial Convention. USP General Chapter <1116> 17, 784–794 (2013). 5. Sheraba, N. S., Yassin, A. S. & Amin, M. BMC Research Notes 3, 278 (2010). 6. Utescher, C. L. de A., Franzolin, M. R., Trabulsi, L. R. & Gambale, V. Brazilian Journal of Microbiology 38, 710–716 (2007).

A B Regberg↗

NASA NTRSDate not supplied

Effects of Spaceflight Relevant Carbon Dioxide Levels on Pathogenesis Related Microbial Characteristics

BACKGROUND Multiple stressors in the spaceflight environment have the potential to alter microbial pathogenesis and virulence characteristics, including microgravity, radiation, and hostile/confined conditions. This combination of factors may lead to a stacking of risks, potentially creating unexpected increase in the risk of infectious disease. One understudied variable is the higher CO2 atmospheric concentration. The current Spaceflight Maximum Allowance Concentration (SMAC) for 24-hour average CO2 is 0.4% (3 mm Hg). These elevated ambient CO2 levels aboard the ISS could potentially influence the diversity and phenotypic responses of the resident microbial communities from both the spacecraft environment (air, surface, water) and crewmembers (gut, nasal, skin microbiomes). METHODS For these studies, we will evaluate the response of Staphylococcus aureus, Streptococcus pneumoniae and Enterobacter aerogenes, which are opportunistic pathogens that are of medical significance and have been or are likely to be found aboard spacecraft. These microorganisms will be cultured in both spaceflight analog and control conditions at CO2levels representing terrestrial atmospheric concentration of 0.04% (0.3 mmHg), ISS elevated concentration of 0.4% (3 mm Hg), and an unexpected elevated concentration of 1 % (7.6 mmHg) to represent a worst case scenario. DISCUSSION Overwhelming evidence from studies performed in food microbiology, marine biology and terrestrial environmental biology suggest altered gene expression, selective bacterial inhibition, increased growth and diversity, and increased antibiotic resistance of bacterial communities individually and in biofilm formation when exposed to increased levels of CO2. While many of these findings are based on levels of CO2 higher than would be found during spaceflight exploration missions, the potential for subtle changes in CO2 levels to exacerbate infectious disease risks warrants an evaluation of pathogen responses to spaceflight CO2 conditions. Our study will provide answers to spaceflight relevant atmospheric conditions, as described in the Risk of Adverse Health Effects Due to Host-Microorganism Interaction (GapMicro 103).

A A Medina-Colorado↗

NASA NTRSDate not supplied

Retrospectives: Intersection of Spaceflight Stressors and Microbial Risk to Crew and Craft

OVERVIEW The spaceflight environment has several unique stressors that affect the health of both the crew and the spacecraft. An area of continued, albeit incomplete, study is the interaction of these stressors on microbial populations inherent to both astronauts and spacecraft surfaces and systems. A primary concern is the potential for the spaceflight environment to perturb the phenotype of these populations towards negative outcomes for crew and craft. In order to effectively mitigate these potential risks, they must first be characterized. We performed a retrospective literature analysis to assess the current state of knowledge regarding the affects of ionizing radiation and elevated CO2 on relevant microbial populations. The results of these retrospectives will guide next steps in the decisions of what (if any) further studies should be pursued and to guide decisions of the need for countermeasures. STRESSORS Ionizing radiation. The health risk involved with increased exposure to cosmic radiation has been studied in crew for 35+ years, with human health and cancer risk being the main focus. However, space radiation could also affect both the resident microorganisms aboard the ISS and the normal, healthy astronaut microbiomes that are of direct concern for crew health. A retrospective review of over 250 publications was accomplished looking at the impact of cumulative ionizing radiation doses lower than 3 Gy (chronic or acute) on microbial populations. Elevated CO2. The health risk involved with elevated atmospheric CO2 in spacecraft, primarily focusing on human toxicological risks, is understudied. The current Spaceflight Maximum Allowance Concentration for 24-hour average CO2 is 0.4% (3 mm Hg), which is significantly higher than terrestrial levels (0.04%). Whether these elevated ambient CO2 levels aboard spacecraft influence the diversity and phenotypic responses of the resident microbial communities from both the spacecraft environment (air, surface, water) and crew members (gut, nasal,skin microbiomes) is not known. A retrospective review was accomplished looking at the impact of chronic CO2 exposure up to 0.7% (5 mm Hg) for up to 6 months and acute exposure up to 2.6% (20 mm Hg) for up to 24 hours. CONCLUSIONS: MICROBIOME OF THE BUILT ENVIRONMENT The microbiome of the built spacecraft environment has been sampled consistently over the course of human spaceflight and significant advancements have been made in identifying microbial populations on the ISS. The dominant source of microbes on spacecraft surfaces are human-derived. Once in the spacecraft built environment,the extreme environment selects for features that enhance survival. While efforts to understand potential antibiotic resistance and pathogenicity of ISS isolates is robust, there is little to no understanding of which spaceflight environmental stressors, to include ionizing radiation or elevated CO2, drive the evolutionary trajectory of spacecraft-associated microbial populations. CONCLUSIONS: MICROBE-HOST INTERACTIONS The host-microbiome field has emerged as an important factor in human health on Earth as well in spaceflight. The field is struggling with the complexity of the system under investigation as there is substantial taxonomic and functional heterogeneity in these communities, making it difficult to establish clear stimulus-response dynamics.Taxonomic characterization is the norm; however, the functional role of each community member is key to linking environmental perturbations to potential dysbiosis. For both ionizing radiation and elevated CO2, the likely target of the perturbation is the host tissue, not the microbes themselves.. Any resulting changes to the microbial community composition and/or function is likely a result of adapting to those changes in the host physiology. RECOMMENDATIONS Emphasize functional characterization as opposed to taxonomic characterization of microbial communities.Increase the number of investigations using chronic, spaceflight-relevant doses of ionizing radiation. MoBE studies should move away from observational studies towards predictive modeling of community dynamics. Continue to develop scale-down models, such as tissues-on-a-chip & defined microbial communities. Focus on the crew response to elevated CO2 over MoBE considerations. Assess how direct contact with the hypercapnic environment affects skin microbiome dynamics.

Countermeasures↗

NASA NTRSDate not supplied

Machine learning to characterize antimicrobial resistance

Machine learning technique to predict antibiotic resistant microbes on the ISS.

Afshin Beheshti↗

NASA NTRSDate not supplied

Increasing accessibility to deep learning-based analytics for space biology: pretrained models, transfer learning, and analytics platform development

Biological systems react in complex ways to the stressors of spaceflight, and the data capturing these relationships is concomitantly high-dimensional and complex. Deep learning and machine learning approaches are increasingly popular as an analytical approach for space biosciences, due to their ability to model complex relationships in complex data. However, such approaches often require large datasets and extensive computational resources. New approaches that minimize data sizes and computational power needed to leverage machine learning, and resources that make these approaches accessible, are needed to increase accessibility and adoption of machine learning in the space biosciences. Transfer learning, in which a pretrained model of broad utility is trained on a large dataset, and subsequently reused on downstream applications for which data is more limited, is one approach to minimizing data and computational intensity of deep learning applications. This transfer learning approach results in more performant models in high-dimensional, low-sample-size settings such as space biology, as compared to training models on limited data from scratch. This presentation will outline efforts to generate pretrained models for the space biology community, and highlight transfer learning applications modeling microbial antibiotic resistance during spaceflight. Finally, in order to increase accessibility of these models and tools, as well as others, for the broader space biology community, we present a modeling and analysis platform facilitating machine learning applications in space biology. This platform streamlines machine learning training and analysis in a notebook format, facilitates download and use of space biology data from the NASA GeneLab database, and can be utilized on NASA-hosted servers or downloaded and hosted locally. This effort, as part of the AI4LS (Artificial Intelligence for Life in Space) working group, will increase accessibility, feasibility, and performance of machine learning approaches for the space biology community.

Adrienne Hoarfrost↗

NASA NTRSDate not supplied

Characterization of Microbe Resistant Coatings for Use in the ISS Water System

The ISS is a highly controlled environment; however, there are still microbes on it that astronauts bring from their own microbiome. Once within the ISS water treatment system, these microbes not only pose a health risk for astronauts, but they can form biofilms, causing material degradation and system failure. Due to their protective structure, biofilms are notoriously resistant to disinfectants and antibiotics. One proposed solution is applying polymer coatings to the metal parts of the water system to prevent biofilms from forming. Two types of polyampholyte polymers with previously demonstrated nonfouling properties were sent to the ISS in 2021 to test their viability for preventing microbial adhesion in space. However, the only microbial assays performed were microscopy. To enhance this ongoing research project, I developed procedures for culturing, applying, and quantifying Ralstonia pickettii on these polymers. R. pickettii was chosen because it is one of the most prevalent microbes found in the ISS water treatment system. Optical density data was recorded to obtain a growth curve demonstrating a doubling time between 2.83 and 4.88 hours in 30°C, not shaken conditions. Optical density was also correlated to colony forming units in a plating experiment. Another result was design (and pilot testing) of a surface colonization experiment. These results provided vital knowledge about R. pickettii, which will be used for a 2023 payload of antimicrobial polymer coatings on stainless steel, and improved methods for biofilm analysis upon its return to evaluate the efficacy of the coatings.

microbiology↗

NASA NTRSDate not supplied

Effects of Spaceflight Relevant Carbon Dioxide Levels on Pathogenesis Related Microbial Characteristics

Multiple stressors in the spaceflight environment, including microgravity and radiation, have the potential to alter microbial pathogenesis and virulence characteristics. Understanding how these and other factors may exacerbate the risk of infectious disease for astronauts is important to ensure the success of the mission. One understudied question in our assessment of infectious disease risk is the contribution carbon dioxide (CO2) at atmospheric concentrations higher than would be observed in terrestrial settings. Previous terrestrial studies have shown altered gene expression, selective bacterial inhibition, increased growth and diversity, and increased antibiotic resistance of bacterial communities individually and in biofilms when exposed to increased levels of CO2. While many of these findings are based on levels of CO2 higher than would be found during spaceflight exploration missions, the potential for subtle changes in CO2 levels to exacerbate infectious disease risks warrants an evaluation of pathogen responses to spaceflight CO2 conditions. To address this question, we are evaluating the effects of elevated atmospheric CO2 on microbial concentration, diversity, and pathogenesis-related characteristics using medically significant microorganisms cultured at CO2 levels representing terrestrial atmospheric concentration (≈0.04 %), ISS elevated concentration (0.4 %), and an unexpectedly elevated concentration (1 %) to represent a worst-case scenario. To determine if microbial responses to these CO2 concentrations may be exacerbated in response to the spaceflight environment, microorganisms are being cultured under static conditions and both spaceflight analogue and control conditions using the Rotating Wall Vessel (RWV) bioreactor (Synthecon, Houston, TX). For these studies, we will evaluate the response of Staphylococcus aureus, Streptococcus pneumoniae, and Enterobacter aerogenes, which are opportunistic bacterial pathogens that are of medical significance and have been or are likely to be found aboard human-inhabited spacecraft. Bacteria will be evaluated individually and together as a consortium. The following Specific Aims are be investigated: Aim 1. Characterize the growth, diversity, and pathogenesis-related stress responses of medically significant microorganisms grown under multiple CO2 concentrations under static growth in a liquid medium. Stress responses include acid stress, oxidative stress, and thermal stress. Aim 2. Characterize the growth, diversity, and pathogenesis-related stress responses of medically significant microorganisms grown under multiple CO2 concentrations in both spaceflight analogue and control conditions. Stress responses include acid stress, oxidative stress, and thermal stress.

S G Thornhill↗

NASA NTRSDate not supplied

Spaceflight-induced Changes in Microbial Virulence and the Impact to the Host Immune Response

INTRODUCTION Over the past 50 years, many microorganisms have displayed unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in stress resistance, biofilm production, antibiotic sensitivity, final cell concentration, gene expression, enhanced host-pathogen interaction, and virulence. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system; consisting of altered leukocyte distribution, reductions in T and NK cell function, altered cytokine profiles, and reactivation of latent herpesviruses. Further, astronauts have some degree of clinical incidence, primarily infectious disease episodes and atopic dermatitis. The impact of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. SPECIFIC AIMS In this study, the following Specific Aims are being investigated, using the microbial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli (EHEC). Aim 1: Characterize the effect of spaceflight analogue culture on microbial pathogenesis-related stress responses and in vitro host-pathogen interactions. Analyses include microbial stress responses as well as colonization and viability following pathogen challenge of three-dimensional (3-D) tissue co-culture models containing immune cells. We specifically selected obligate and opportunistic pathogens that are medically important and have been or are likely to be found aboard spacecraft. Aim 2: Characterize the effect of spaceflight analogue culture on the virulence potential of pathogenic microorganisms. Virulence will be assessed using a mouse model of infection. SIGNIFICANCE The goal of this study is to use spaceflight analogue conditions to gain insight into the breadth of medically-significant International Space Station microorganisms that have altered virulence and the impact of those changes on the immune response of the host. This information will provide critical understanding into the impact of microgravity on potential alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗

NASA NTRSDate not supplied

Boric Acid Dispersion of Biofilm (BAD-B)

Biofilms in spacecraft water recycling systems (WRS) can cause crew illness, occlude water lines, and induce corrosion on WRS materials. The treatment of biofilms in spacecraft water systems has historically been limited by a lack of suitable methods to overcome biofilm-associated antimicrobial tolerance (AMT). One way to get around AMT is to induce biofilm dispersion. To address AMT in spacecraft WRS, I evaluated the use of boric acid (BA) as an agent to disperse biofilms formed on common WRS materials, including 316L stainless steel, PFTE Teflon, and Ti6Al4V titanium alloy. Recent studies have shown that BA can be effective at dispersing biofilms of a variety of bacterial species. BA has also been shown to interact synergistically with antibiotics and disinfectants.

microbiology↗

NASA NTRSDate not supplied

Spaceflight Autonomous Multigenerational Microbial Sequencer in Support of Plant-Growth Systems

The CubeSat platform has proven successful in obtaining meaningful life science information when biological payloads are incorporated. Examples include: 1) the first-ever CubeSat with a biological payload, GeneSat-1, which demonstrated decreased growth rates for flight samples of Escherichia coli in low Earth orbit (Parra et al. 2008); 2) PharmaSat, demonstrated that Saccharomyces cerevisiae in the microgravity environment exhibits a significant level of metabolic activity even at high doses of applied antifungal (Ricco et al. 2011); 3) O/OREOS, which used Bacillus subtilis(bacteria) to demonstrate for the first time that microorganisms can be loaded in a dried, dormant form and then rehydrated and grown in orbit months after launch (Nicholson et al. 2011; Ehrenfreund et al. 2014; 4) the SporeSat payload, which investigated Ceratopteris richardii(fern spores) using lab-on-a-chip devices (BioCDs) and minicentrifuges to produce artificial gravitational forces in ground studies (Park et al. 2017), with demonstration of the BioCD and minicentrifuge in space; 5) EcAMSat, the first CubeSat to be directly deployed from the ISS for an experiment assessing antibiotic resistance of E. coli in the microgravity environment (Padgen et al. 2020); 6) BioSentinel, exposed a culture of yeast to galactic cosmic radiation (GCR) and solar particle events while in heliocentric orbit to measure the rate of double-strand-break repair using DNA-repair-deficient mutants. This effort measures the metabolic parameters of yeast in a deep-space environment compared to Earth ambient conditions using a 3-color LED detection system (Ricco et al. 2020; Padgen et al. 2021). We aim to expand this list to include a Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS). SAMMS will allow for the genome level understanding of changes in growth and metabolic activity for any organism. While microbes are suitable for early studies in our proposed platform because of their small size, small and relatively less-complicated genomes, fast generation times, and relevance to life support systems; multicellular organisms can similarly be evaluated for their genetic response to the spaceflight environment. The Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS) will enable autonomous sequencing of biological samples in plant production units, cislunar orbit and on the lunar surface to examine spaceflight effects (ie. radiation, altered gravity, reduced pressures) on plant and microbial genomes.On this team a Kennedy Space Center (KSC) space crop production and water systems microbiologist/molecular biologist works with a Johnson Space Center (JSC) International Space Station (ISS) microbial sequencing expert and an Ames Research Center (ARC) CubeSat Engineering team to convert an automated Oxford Nanopore librarypreparation and sequencing method to a fluidic CubeSat payload system. The Oxford Nanopore MinION sequencing platform has proven successful in the spaceflight environment onboard the ISS (Stahl-Rommel et al. 2021). Further long-duration spaceflight and exposure to high levels of radiation will cause genotypic effects in biological organisms that may affect their function. Monitoring the adaption of a population to the spaceflight environment and any subsequent beneficial mutations will allow for the harnessing of organisms best suited for use in life support systems. This will ensure that the selected life support-essential microorganisms maintain their intended specified function over generations of culturing in the relevant spaceflight environment without becoming hazardous to crew or spacecraft systems.

Aubrie E Orourke↗

NASA NTRSDate not supplied

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogen shave repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in space flight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. Herein, we present data from one of these pathogens, the foodborne bacterium, S. enterica Enteritidis, which is closely related to S. enterica Typhimurium. Phenotypes evaluated included growth profiles, environmental stress responses(acid, oxidative, bile, and thermal stresses),and in vitro colonization of3-D biomimetic cultures of human intestinal tissue containing immune cells. Transcriptomic profiling and virulence studies are ongoing. We show that S. Enteritidis exhibited key alterations in pathogenic responses to LSMMG culture that suggest increased infection risk, including several responses which were different from those observed in the closely related pathovar S. Typhimurium. This information will provide critical mechanistic insight into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗

NASA NTRS2024

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

C.M. Ott↗

NASA NTRSDate not supplied

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogens have repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in spaceflight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. The information to date is providing a better understanding into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗

NASA NTRSDate not supplied

The Need for Earth-Based Experiments to Inform Microbial Evolution on Planetary Surfaces

Introduction: Historically, the focus of planetary protection at NASA has been on unmanned, robotic missions. Such missions have paved the way for understanding how to implement planetary protection in a feasible and cost-sensitive way. However, with the introduction of crewed missions to Mars in the not-sodistant future, there is a need to better define and understand how to implement planetary protection under new circumstances, as well as understand the risk of contaminating Mars. One unavoidable fact is that microbes will go where humans go. Therefore, it is critical to understand how these microbes may (and will) impact our ability to conduct meaningful, reliable astrobiological science. Microorganisms have spent millions of years evolving to survive in extreme environments here on Earth. Already there are indications that microbes aboard the International Space Station evolve and adapt to life in low earth orbit. The microbes that are eventually taken to Mars with humans will also adapt, potentially causing harmful effects to crew and/or the planetary or astrobiological science conducted. Therefore, it is of critical interest that we evaluate and characterize the potential risks of microbial evolution on Mars. It is expected that microbes carried by humans will begin to evolve to new environments even before landing on Mars, during the several month cruise phase. Once landed, microbes will encounter different stressors within the crew habitats on Mars. During extravehicular activities, venting, or other release events, microbes will find their way out onto the Martian surface. The induced environments around crewed systems will create potentially-favorable conditions for microbes to continue evolving on Mars. Eventually, microbes may find their way beyond the close confines of the crewed area and continue evolving so as to fill new or distant niches on the Martian surface. It is challenging to replicate Martian environments here on Earth, making it nearly impossible to predict the evolutionary changes that microbes would undergo on Mars. But this work is critical. Serial passaging experiments performed by Richard Lenski on E. coli show the dramatic changes microbes can undergo even within a laboratory setting. Furthermore, experiments performed by Michael Baym also demonstrate the power of single mutations in microbial development of antibiotic resistance [3]. Long duration experiments should be performed on a suite of microbes exposed to environments likely to be experienced on the Martian surface. While simulating space environments can be challenging, facilities exist that can achieve individual and combinatorial environmental conditions to simulate space and planetary conditions. Such chambers should be employed for microbial studies. Currently, at the Marshall Space Flight Center, we have used various stressors like drying, vacuum, proton radiation, and ultraviolet light both separately and in combination, to evaluate the survival of cleanroom microbes. Shockingly, several non-spore forming isolates have demonstrated the ability to survive many extreme conditions (manuscript in preparation). These short duration exposures must be augmented with larger and more gradual studies to replicate what microbes might experience in the transition from cruise, to surface habitats, to induced surface environments, and finally true Martian environments. While no Earth-based experiment can perfectly replicate the Martian environment, nor could we test every possible microbe in simulation experimental regimes, efforts should be made to examine the evolutionary potential of the “usual suspects” seen on the ISS or in other crewed environments to begin to fill this important knowledge gap.

Chelsi D. Cassilly↗

NASA NTRS2000

Relief from glucose interference in microcin B17 biosynthesis by growth in a rotating-wall bioreactor

Glucose interference in production of microcin B17 by Escherichia coli ZK650 was decreased sevenfold by growth in a ground-based rotating-wall bioreactor operated in the simulated microgravity mode as compared with growth in flasks. When cells were grown in the bioreactor in the normal gravity mode, relief from glucose interference was even more dramatic, amounting to a decrease in glucose interference of over 100-fold.

NASA Discipline Environmental Health↗

NASA NTRS1997

Gramicidin S production by Bacillus brevis in simulated microgravity

In a continuing study of microbial secondary metabolism in simulated microgravity, we have examined gramicidin S (GS) production by Bacillus brevis strain Nagano in NASA High Aspect Rotating Vessels (HARVs), which are designed to simulate some aspects of microgravity. Growth and GS production were found to occur under simulated microgravity. When performance under simulated microgravity was compared with that under normal gravity conditions in the bioreactors, GS production was found to be unaffected by simulated microgravity. The repressive effect of glycerol in flask fermentations was not observed in the HARV. Thus the negative effect of glycerol on specific GS formation is dependent on shear and/or vessel geometry, not gravity.

NASA Discipline Number 04-10↗