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NASA NTRS2001

Comparative Ecology of H2 Cycling in Organotrophic and Phototrophic Ecosystems

The simple biochemistry of H2 is critical to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. The sensitivity of these many processes to H2 can be described quantitatively, at a basic thermodynamic level. This shared dependence on H2 may provide a means for interpreting the ecology and system-level biogeochemistry of widely variant microbial ecosystems on a common (and quantitative) level. Understanding the factors that control H2 itself is a critical prerequisite. Here, we examine two ecosystems that vary widely with respect to H2 cycling. In anoxic, 'organotrophic' sediments from Cape Lookout Bight (North Carolina, USA), H2 partial pressures are strictly maintained at low, steady-state levels by H2-consuming organisms, in a fashion that can be quantitatively predicted by simple thermodynamic calculations. In phototrophic microbial mats from Baja, Mexico, H2 partial pressures are instead controlled by the activity of light-sensitive H2-producing organisms. In consequence, H2 partial pressures within the system fluctuate by orders of magnitude on hour-long time scales. The differences in H2 cycling subsequently impact H2-sensitive microbial processes, such as methanogenesis. For example, the presence of sulfate in the organotrophic system always yielded low levels of H2 that were inhibitory to methanogenesis; however, the elevated levels of H2 in the phototrophic system favored methane production at significant levels, even in the presence of high sulfate concentrations. The myriad of other H2-sensitive microbial processes are expected to exhibit similar behavior.

Hoehler, Tori M.↗
NASA NTRS2002

The Biogeochemistry of Benthic Photosynthetic Communities

Microbial mats are our earliest, and most pervasive evidence of life on Earth. In addition to their importance as the crucible of microbial evolution on Earth, microbial mats may be important in our search for life outside of our solar system. This lecture will introduce various types of microbial mats, and their fossilized counterparts, stromatolites by presenting examples of these communities. Biogeochemical cycles important in microbial mats will be introduced and discussed.

Bebout, Brad↗
NASA NTRS2004

Lunar and Planetary Science XXXV: Astrobiology: Analogs and Applications to the Search for Life

The session "Astrobiology: Analogs and Applications to the Search for Life" included the folowing reports:The Search for Life on Mars Using Macroscopically Visible Microbial Mats (Stromatolites) in 3.5/3.3 Ga Cherts from the Pilbara in Australia and Barberton in South Africa as Analogues; Life in a Mars Analog: Microbial Activity Associated with Carbonate Cemented Lava Breccias from NW Spitsbergen; Groundwater-fed Iron-rich Microbial Mats in a Freshwater Creek: Growth Cycles and Fossilization Potential of Microbial Features; Episodic Fossilization of Microorganisms on an Annual Timescale in an Anthropogenically Modified Natural Environment: Geochemical Controls and Implications for Astrobiology; Proterozoic Microfossils and Their Implications for Recognizing Life on Mars; Microbial Alteration of Volcanic Glass in Modern and Ancient Oceanic Crust as a Proxy for Studies of Extraterrestrial Material ; Olivine Alteration on Earth and Mars; Searching for an Acidic Aquifer in the R!o Tinto Basin. First Geobiology Results of MARTE Project; In-Field Testing of Life Detection Instruments and Protocols in a Mars Analogue Arctic Environment; Habitability of the Shallow Subsurface on Mars: Clues from the Meteorites; Mars Analog Rio Tinto Experiment (MARTE): 2003 Drilling Campaign to Search for a Subsurface Biosphere at Rio Tinto Spain; Characterization of the Organic Matter in an Archean Chert (Warrawoona, Australia); and The Solfatara Crater, Italy: Characterization of Hydrothermal Deposits, Biosignatures and Their Astrobiological Implication.

Lunar and Planetary Institute↗
NASA NTRS2005

The Biogeochemistry of Benthic Photosynthetic Communities

This lecture presentation discusses microbial mats are our earliest, and most pervasive evidence of life on Earth. In addition to their importance as the crucible of microbial evolution on Earth, microbial mats may be important in our search for life outside of our solar system. This lecture will introduce various types of microbial mats, and their fossilized counterparts, stromatolites by presenting examples of these communities. Biogeochemical cycles important in microbial mats will be introduced and discussed.

Bebout, Brad↗
NASA NTRS2004

Examination of B. subtilis var. niger Spore Killing by Dry Heat Methods

Dry heat microbial reduction is the only NASA approved sterilization method to reduce the microbial bioburden on space-flight hardware prior to launch. Reduction of the microbial bioburden on spacecraft is necessary to meet planetary protection requirements specific for the mission. Microbial bioburden reduction also occurs if a spacecraft enters a planetary atmosphere (e.g., Mars) and is heated due to frictional forces. Temperatures reached during atmospheric entry events (>200 C) are sufficient to damage or destroy flight hardware and also kill microbial spores that reside on the in-bound spacecraft. The goal of this research is to determine the survival rates of bacterial spores when they are subjected to conditions similar to those the spacecraft would encounter (i.e., temperature, pressure, etc.). B. subtilis var. niger spore coupons were exposed to a range of temperatures from 125 C to 200 C in a vacuum oven (at <1 Torr). After the exposures, the spores were removed by sonication, dilutions were made, and the spores were plated using the pour plate method with tryptic soy agar. After 3 days incubation at 32 C, the number of colony-forming units was counted. Lethality rate constants and D-values were calculated at each temperature. The calculated D-values were: 27 minutes (at 125 C), 13 minutes (at 135 C), and <0.1 minutes (at 150 C). The 125 C and 135 C survivor curves appeared as concavedownward curves. The 150 C survivor curve appeared as a straight-line. Due to the prolonged ramp-up time to the exposure conditions, spore killing during the ramp-up resulted in insufficient data to draw curves for exposures at 160 C, 175 C, and 200 C. Exploratory experiments using novel techniques, with short ramp times, for performing high temperature exposures were also examined. Several of these techniques, such as vacuum furnaces, thermal spore exposure vessels, and laser heating of the coupons, will be discussed.

bacillus subtilis var niger↗
NASA NTRS2007

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Ott, C. M.↗
NASA NTRS2011

The Effect of Plant Cultivar, Growth Media, Harvest Method and Post Harvest Treatment on the Microbiology of Edible Crops

Systems for the growth of crops in closed environments are being developed and tested for potential use in space applications to provide a source of fresh food. Plant growth conditions, growth media composition and harvest methods can have an effect on the microbial population of the plant, and therefore should be considered along with the optimization of plant growth and harvest yields to ensure a safe and palatable food crop. This work examines the effect of plant cultivar, growth media, and harvest method on plant microbial populations. Twelve varieties of leafy greens and herbs were grown on a mixture of Fafard #2 and Arcillite in the pillow root containment system currently being considered for the VEGGIE plant growth unit developed by Orbitec. In addition, ,Sierra and Outredgeous lettuce varieties were grown in three different mixtures (Fafard #2, Ardllite, and Perlite/Vermiculite). The plants were analyzed for microbial density. Two harvest methods, "cut and come again" (CACA) and terminal harvest were also compared. In one set ofexpe'riments red leaf lettuce and mizuna were grown in pots in a Biomass Production System for education. Plants were harvested every two weeks by either method. Another set of experiments was performed using the rooting pillows to grow 5 varieties of leafy greens and cut harvesting at different intervals. Radishes were harvested and replanted at two-week intervals. Results indicate up to a 3 IOglO difference in microbial counts between some varieties of plants. Rooting medium resulted in an approximately 2 IOglO lower count in the lettuce grown in arscillite then those grown in the other mixtures. Harvest method and frequency had less impact on microbial counts only showing a significant increase in one variety of plant. Post harvest methods to decrease the bacterial counts on edible crops were investigated in these and other experiments. The effectiveness of PRO-SAN and UV-C radiation is compared.

Hummerick, Mary P.↗
NASA NTRS2012

Surface, Water, and Air Biocharacterization (SWAB) Flight Experiment

The determination of risk from infectious disease during spaceflight missions is composed of several factors including both the concentration and characteristics of the microorganisms to which the crew are exposed. Thus, having a good understanding of the microbial ecology aboard spacecraft provides the necessary information to mitigate health risks to the crew. While preventive measures are taken to minimize the presence of pathogens on spacecraft, medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a specific culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. To address this bias in our understanding of the ISS environment, the Surface, Water, and Air Biocharacterization (SWAB) Flight Experiment was designed to investigate and develop monitoring technology to provide better microbial characterization. For the SWAB flight experiment, we hypothesized that environmental analysis using non-culture-based technologies would reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. Key findings during this experiment included: a) Generally, advanced molecular techniques were able to reveal a few organisms not recovered using culture-based methods; however, there is no indication that current monitoring is "missing" any medically significant bacteria or fungi. b) Molecular techniques have tremendous potential for microbial monitoring, however, sample preparation and data analysis present challenges for spaceflight hardware. c) Analytical results indicate that some molecular techniques, such as denaturing gradient gel electrophoresis (DGGE), can be much less sensitive than culture-based methods. d) More sensitive molecular techniques, such as quantitative polymerase chain reaction (QPCR), were able to identify viral DNA from ISS environments, suggesting potential transfer of the organism between crewmembers. In addition, the hardware selected for this experiment represented advances for next-generation sample collection. The advanced nature of this collection hardware was noted, when the Sartorius MD8 Air Port air sampler from the SWAB experiment remained on board ISS at the request of JAXA investigators, who intend to use it in completion of their microbial ecology experiment.

Castro, V. A.↗
NASA NTRS2014

Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

Microbial control in the spacecraft environment is a daunting task, especially in the presence of human crew members. Currently, assessing the potential crew health risk associated with a microbial contamination event requires return of representative environmental samples that are analyzed in a ground-based laboratory. It is therefore not currently possible to quickly identify microbes during spaceflight. This project addresses the unmet need for spaceflight-compatible microbial identification technology. The electrochemical detection and identification platform is expected to provide a sensitive, specific, and rapid sample-to-answer capability for in-flight microbial monitoring that can distinguish between related microorganisms (pathogens and non-pathogens) as well as chemical contaminants. This will dramatically enhance our ability to monitor the spacecraft environment and the health risk to the crew. Further, the project is expected to eliminate the need for sample return while significantly reducing crew time required for detection of multiple targets. Initial work will focus on the optimization of bacterial detection and identification. The platform is designed to release nucleic acids (DNA and RNA) from microorganisms without the use of harmful chemicals. Bacterial DNA or RNA is captured by bacteria-specific probe molecules that are bound to a microelectrode, and that capture event can generate a small change in the electrical current (Lam, et al. 2012. Anal. Chem. 84(1): 21-5.). This current is measured, and a determination is made whether a given microbe is present in the sample analyzed. Chemical detection can be accomplished by directly applying a sample to the microelectrode and measuring the resulting current change. This rapid microbial and chemical detection device is designed to be a low-cost, low-power platform anticipated to be operated independently of an external power source, characteristics optimal for manned spaceflight and areas where power and computing resources are scarce.

Pierson, Duane↗
NASA NTRS2014

Spaceflight Microbiology: Benefits for Long Duration Spaceflight and Our Understanding of Microorganisms on Earth

Spaceflight microbiology is composed of both operational and experimental components that complement each other in our understanding of microbial interactions and their responses in the microgravity of spaceflight. Operationally, efforts to mitigate microbiological risk to the crew and the spacecraft have historically focused on minimizing the number of detectable organisms, relying heavily on preventative measures, including appropriate vehicle design, crew quarantine prior to flight, and stringent microbial monitoring. Preflight monitoring targets have included the astronauts, spaceflight foods, potable water systems, the vehicle air and surfaces, and the cargo carried aboard the spacecraft. This approach has been very successful for earlier missions; however, the construction and long-term habitation of the International Space Station (ISS) has created the need for additional inflight monitoring of the environment and potable water systems using hardware designed for both in-flight microbial enumeration and sample collection and return to Earth. In addition to operational activities, the ISS is providing a research platform to advance our understanding of microbiomes in the built environment. Adding to the research possibilities of this system are multiple reports of unique changes in microbial gene expression and phenotypic responses, including virulence and biofilm formation, in response to spaceflight culture. The tremendous potential of the ISS research platform led the National Research Council to recommend that NASA utilize the ISS as a microbial observatory. Collectively, the findings from operational and research activities on the ISS are expected to both enable future space exploration and translate to basic and applied research on Earth.

Ott, C. Mark↗
NASA NTRS2018

EVA Swab Tool to Support Planetary Protection and Astrobiology Evaluations

When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed systems meet planetary protection requirements-and to protect our science from human contamination-we'll need to assess whether microorganisms may be leaking or venting from our spacecraft. Microbial sample collection outside of a pressurized spacecraft is complicated by temperature extremes, low pressures that preclude the use of laboratory standard (wetted) swabs, and operation either in bulky spacesuits or with robotic assistance. Engineers at the National Aeronautics and Space Administration (NASA) recently developed a swab kit for use in collecting microbial samples from the external surfaces of crewed spacecraft, including spacesuits. The Extravehicular Activity (EVA) Swab Kit consists of a single swab tool handle and an eight-canister sample caddy. The design team minimized development cost by re-purposing a heritage Space Shuttle tile repair handle that was designed to quickly snap into different tool attachments by engaging a mating device in each attachment. This allowed the tool handle to snap onto a fresh swab attachment much like popular shaving razor handles can snap onto a disposable blade cartridge. To disengage the handle from a swab, the user performs two independent functions, which can be done with a single hand. This dual operation mitigates the risk that a swab will be inadvertently released and lost in microgravity. Each swab attachment is fitted with commercially available foam swab tips, vendor-certified to be sterile for Deoxyribonucleic Acid (DNA). A microbial filter installed in the bottom of each sample container allows the container to outgas and repressurize without introducing microbial contaminants to internal void spaces. Extensive ground testing, post-test handling, and sample analysis confirmed the design is able to maintain sterile conditions as the canister moves between various pressure environments. To further minimize cost, the design team acquired extensive ground test experience in a relevant flight environment by piggy-backing onto suited crew training runs. These training runs allowed the project to validate tool interfaces with pressurized EVA gloves and collect user feedback on the tool design and function, as well as characterize baseline microbial data for different types of spacesuits. In general, test subjects found the EVA Swab Kit relatively straightforward to operate, but identified a number of design improvements that will be incorporated into the final design. Although originally intended to help characterize human forward contaminants, this tool has other potential applications, such as for collecting and preserving space-exposed materials to support astrobiology experiments.

Rucker, Michelle A.↗
NASA NTRS2018

EVA Swab Tool to Support Planetary Protection and Astrobiology Evaluations

When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed systems meet planetary protection requirements-and to protect our science from human contamination-we'll need to assess whether microorganisms may be leaking or venting from our spacecraft. Microbial sample collection outside of a pressurized spacecraft is complicated by temperature extremes, low pressures that preclude the use of laboratory standard (wetted) swabs, and operation either in bulky spacesuits or with robotic assistance. A team at the National Aeronautics and Space Administration (NASA) recently developed a swab kit for use in collecting microbial samples from the external surfaces of crewed spacecraft, including spacesuits. The Extravehicular Activity (EVA) Swab Kit consists of a single swab tool handle and an eight-canister sample caddy. The design team minimized development cost by re-purposing a heritage Space Shuttle tile repair handle that was designed to quickly snap into different tool attachments by engaging a mating device in each end effector. This allowed the tool handle to snap onto a fresh swab end effector much like popular shaving razor handles can snap onto a disposable blade cartridge. To disengage the handle from a swab, the user performs two independent functions, which can be done with a single hand. This dual operation mitigates the risk that a swab will be inadvertently released and lost in microgravity. Each swab end effector is fitted with commercially available foam swab tips, vendor-certified to be sterile for Deoxyribonucleic Acid (DNA). A microbial filter installed in the bottom of each sample container allows the container to outgas and re-pressurize without introducing microbial contaminants to internal void spaces. Extensive ground testing, post-test handling, and sample analysis confirmed the design is able to maintain sterile conditions as the canister moves between various pressure environments. To further minimize cost, the design team acquired extensive ground test experience in a relevant flight environment by piggy-backing onto suited crew training runs. These training runs allowed the project to validate tool interfaces with pressurized EVA gloves and collect user feedback on the tool design and function, as well as characterize baseline microbial data for different types of spacesuits. In general, test subjects found the EVA Swab Kit relatively straightforward to operate, but identified a number of design improvements that will be incorporated into the final design. Although originally intended to help characterize human forward contaminants, this tool has other potential applications, such as for collecting and preserving space-exposed materials to support astrobiology experiments.

Rucker, Michelle A.↗
NASA NTRS2018

EVA Swab Tool to Support Planetary Protection and Astrobiology Evaluations

When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed systems meet planetary protection requirements?and to protect our science from human contamination?we'll need to assess whether microorganisms may be leaking or venting from our spacecraft. Microbial sample collection outside of a pressurized spacecraft is complicated by temperature extremes, low pressures that preclude the use of laboratory standard (wetted) swabs, and operation either in bulky spacesuits or with robotic assistance. A team at the National Aeronautics and Space Administration (NASA) recently developed a swab kit for use in collecting microbial samples from the external surfaces of crewed spacecraft, including spacesuits. The Extravehicular Activity (EVA) Swab Kit consists of a single swab tool handle and an eight-canister sample caddy. The design team minimized development cost by re-purposing a heritage Space Shuttle tile repair handle that was designed to quickly snap into different tool attachments by engaging a mating device in each end effector. This allowed the tool handle to snap onto a fresh swab end effector much like popular shaving razor handles can snap onto a disposable blade cartridge. To disengage the handle from a swab, the user performs two independent functions, which can be done with a single hand. This dual operation mitigates the risk that a swab will be inadvertently released and lost in microgravity. Each swab end effector is fitted with commercially available foam swab tips, vendor-certified to be sterile for Deoxyribonucleic Acid (DNA). A microbial filter installed in the bottom of each sample container allows the container to outgas and re-pressurize without introducing microbial contaminants to internal void spaces. Extensive ground testing, post-test handling, and sample analysis confirmed the design is able to maintain sterile conditions as the canister moves between various pressure environments. To further minimize cost, the design team acquired extensive ground test experience in a relevant flight environment by piggy-backing onto suited crew training runs. These training runs allowed the project to validate tool interfaces with pressurized EVA gloves and collect user feedback on the tool design and function, as well as characterize baseline microbial data for different types of spacesuits. In general, test subjects found the EVA Swab Kit relatively straightforward to operate, but identified a number of design improvements that will be incorporated into the final design. Although originally intended to help characterize human forward contaminants, this tool has other potential applications, such as for collecting and preserving space-exposed materials to support astrobiology experiments.

planetary protection↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
NASA NTRSDate not supplied

Responses of Microbes to Modeled Space Radiation

The built environment of spaceships is host to a microbial community that affects crew and craft alike. While the static composition of this community has been characterized and its temporal dynamics examined, the mechanisms controlling its make-up and evolutionary trajectory are not understood. Systematic analyses of microbial diversity show consistent patterns in community composition and function. Understanding these patterns' ecological origins remains a significant challenge, as it requires connecting processes at varying temporal and spatial scales. However, it is clear that the state and trajectories of microbial communities are in-part determined by their physical environment. In this regard, the spaceflight environment includes numerous interacting factors that differentiate it from Earth environments, including an altered atmospheric composition, reduced gravity (and thus altered fluid dynamics), and increased ionizing radiation. These factors impart selective pressures on microbial communities that affect their evolutionary trajectories and thus the risks and benefits these communities represent to crew and craft. The radiation environment of space leads to chronic exposure to low doses and is difficult to mimic on Earth. Thus, little is known about how microbial communities in spacecraft will respond and evolve. Therefore, given the limitations of existing studies, we aim to empirically determine how exposure to low doses of ionizing radiation for thousands of cell divisions affects rates of mutation accumulation in bacteria and the trajectory of their evolution. In this way, we will provide a critical set of data for designing safe and robust space missions. Here we discuss our progress towards this aim, including the construction of exposure facilities, our culturing and analysis approach, and preliminary data.

radiation↗
NASA NTRSDate not supplied

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗
NASA NTRSDate not supplied

VEG-05 Tomato Crop Testing on the International Space Station

Production of fresh, nutritious, and tasty produce for astronauts during spaceflight may provide health-promoting, bioavailable nutrients, enhance the dietary experience, and reduce menu fatigue as we move into longer-duration missions. Growing and caring for plants may also reduce the psychological stresses associated with spaceflight and enhance connection to Earth. A diversity of crops will be required to provide nutrition, variety, and resiliency, however requirements for consistent growth under spaceflight environmental conditions remain unclear. VEG-05 is part of a series of experiments with pick-and-eat salad crops to better define best practices for crop production in space. VEG-05 and predecessor experiments VEG-04A and VEG-04B, grew salad crops in the Veggie vegetable production facilities on the International Space Station using different lighting treatments. In VEG-05 we grew ‘Red Robin’ dwarf cherry tomatoes under two different red: blue lighting spectra. Light can impact the growth habit, yield, nutritional composition, microbial levels, and even flavor attributes within crops, and our goal was to assess these characteristics in ‘Red Robin’ tomatoes during VEG-05. Considerable pre-flight verification testing was performed prior to launch in Nov. 2022. Prior to the flight and ground experiments, lighting in both Veggie units on ISS was measured and lights were standardized between flight and ground hardware. VEG-05 flight operations ran between December 2022 and March 2023, with a ground control initially 48 hours delayed. Due to environmental challenges of very low humidity during the first week of the experiment, seed germination was low for both flight and ground plants. The flight experiment ultimately had 4 - 5 plants per treatment out of the planned 6 plants, but the initial ground control had only three plants in one treatment and none in the other, so this ground control was restarted at the beginning of Feb. 2023 and ran through May, with successful growth of all 12 plants. Both flight and ground control ran 100 days, with harvests of fruit at day 83, day 90, and day 100. Flight plants had uneven growth, and following the early drying events, excess water was frequently observed, which led to a variety of plant stress responses including uneven plant growth, excess adventitious root formation, flower and fruit abortion, and visible microbial growth. In total, from the five surviving red-rich lighted plants, only 5 ripe fruit were produced, and from the four surviving blue-rich lighted plants, 10 fruit were produced with only 6 of these ripe by day 100. Because of the small fruit number and the unsatisfactory growth, crew members were not allowed to consume the tomatoes, and all fruit, as well as large branches with leaves, samples of the adventitious roots, two plant rooting pillows from each treatment, microbial sampling swabs, and some water samples were returned for analysis. Because of the small sample sizes and factors affecting growth on the ISS, objectives of assessing light quality effect (red: blue light treatments) will not be achieved. Revised objectives of this study include to compare stressed flight plants with normal ground plants to determine the impact of plant overwatering stress in space on food safety and the plant microbial community, to determine nutrient content changes in fruit and leaves from stressed plants, and to evaluate stress metabolism changes in returned tissue by transcriptomic analysis. Postflight analysis is underway with the following analyses being conducted: A. culturable microbiology and food safety as well as molecular microbial community analysis of 1. ripe fruit, 2. leaves, stems, and adventitious roots, 3. pillow components (roots, wicks, and substrates), 4. swabs, and 5. water samples from root mats before and after growth. B. transcriptomics of leaf tissue and adventitious roots, and C. elemental analysis of leaf tissue. If sufficient tissue remains elemental analyses will also be conducted on fruit. While not generating the desired information on spaceflight growth responses of healthy crops, our team is hopeful that these analyses will shed light on tomato responses to stress in this environment as plant overwatering stress is a mission-relevant condition that could occur in future space crop growth systems. This research was co-funded by the Human Research Program and Space Biology (MTL#1075) in the ILSRA 2015 NRA call.

Gioia D. Massa↗
DOE OSTI2022

Devices and methods for generating electrical current from methane

Methods, microbial fuel cells and microbial consortia for generating electrical current are provided according to the present invention which include providing a microbial consortium to an anode chamber of a microbial fuel cell, wherein the microbial consortium includes: 1) an engineered methanogen that contains a heterologous nucleic acid sequence encoding methyl-coenzyme M reductase derived from an anaerobic methane oxidizer, 2) an exoelectrogen microbe that produces electrically-conductive appendages and/or one or more types of electron carrier, and 3) a sludge, methane-acclimated sludge, a sludge isolate component, a methane-acclimated sludge isolate component chosen from Paracoccus spp., Geotoga spp., Geobacter spp., Methanosarcina spp., Garciella spp., humic acids; or a combination of any two or more thereof.

Wood, Thomas K.↗

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